Evaluating the Accuracy of HIV-1 Viral Load Testing Using near point of care Xpert HIV- 1 System at Solwezi General Hospital, Zambia.

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Keembe Siakantu, David Chisompola, Aaron Tembo Konzani This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5723442/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Jul, 2025 Read the published version in BMC Research Notes → Version 1 posted 8 You are reading this latest preprint version Abstract Objective HIV-1 Viral Load Testing plays a crucial role in the management of HIV/AIDS patients by quantifying the presence of HIV-1 RNA in the blood, which directly relates to viral replication and disease progression. Monitoring viral load enables healthcare professionals to evaluate the effectiveness of Anti-Retroviral Therapy, adapt treatment strategies, and assess the overall health status of individuals with HIV. This was a cross-sectional study to evaluate analytical performance (accuracy, precision and linearity of the near point of care Xpert HIV-1 viral load. Results The analytical performance of the GeneXpert assay to the conventional Hologic Panther system was strong. Specifically, the GeneXpert demonstrated a sensitivity, specificity, and accuracy of 87.6%, 100%, and 98.5%, respectively, when compared to the Hologic Panther. A strong correlation between the two assays was evident (r = 0.97, p < 0.0001). Additionally, precision, as indicated by the coefficient of variation (CV), for high viral load was 1.38% for GeneXpert while for low viral load, it was 2.59%. In the linearity analysis, all data points remained within acceptable ranges, yielding correlation coefficients ≥ 0.99 for both assays. Conclusion The Xpert HIV-1 viral load assay demonstrates high efficacy in accurately identifying and quantifying HIV-1 viral loads, making it a valuable tool for HIV diagnosis and monitoring. This reliability makes it particularly suitable for critical populations requiring timely and accurate viral load monitoring. Diagnosis Accuracy GeneXpert Hologic Panther Solwezi Zambia Figures Figure 1 Figure 2 Introduction Background The Human Immunodeficiency Viruses (HIV) has a significant global impact, and it has been estimated that 38 million people worldwide are living with HIV/AIDS in 2020 [1]. In Africa, particularly in Zambia, the burden of HIV remains alarmingly high. As of 2020, approximately 1.2 million people in the country were receiving treatment for HIV, highlighting the ongoing public health challenge and the critical need for sustained intervention efforts [2]. Zambia has been significantly impacted by the HIV-1 epidemic, with approximately 11.0% of the population aged 15 and older living with HIV in 2021. In Northwestern Province, 6.8% of the province's population is affected by the virus [3]. This indicates a considerable number of individuals in Solwezi who require effective management and monitoring of the HIV infection. Viral load (VL) testing using the near point-of-care (POC) GeneXpert platform (Cepheid, Sunnyvale, CA, USA) with the Xpert HIV-1 quantitative assay offers the advantage of being used closer to patients. It is easy to use, gives quick results, is cost-effective, and has the potential to serve as an alternative to centralized viral load testing platforms [4]. POC has been introduced for rural populations to expand access to clinical laboratory testing by eliminating the need for sample transportation, laboratory and data management infrastructure, and highly trained staff [5]. Therefore, ensuring access to reliable and accurate viral load testing is vital in improving patient outcomes and reducing HIV transmission rates in Solwezi and similar high-burden areas [4]. The GeneXpert system was recently adopted to reduce the turnaround time for the urgent population such as pregnant and breastfeeding women and children from 0 to 14 years in point of care facilities [6]. Thereby, adding the Xpert HIV-1 VL to the employed methods for HIV-1 viral load testing in Zambia [6]. However, the evaluation of Xpert HIV-1 VL systems' performance and accuracy remains unclear. Differences in detection methodology, sample handling, operator skill level, and other factors may result in discrepancies between the conventional systems' and point of care measurements. Such discrepancies might not only affect individual patient care but could also have broader implications for public health, especially in settings where the HIV prevalence is high [7]. Therefore, it is critical to understand and address this knowledge gap to optimize HIV-1 viral load testing and thus improve patient outcomes and public health strategies. This study aimed to evaluate analytical performance and accuracy of HIV-1 viral load measurements of the GeneXpert system in comparison to the Hologic Panther system. Methods Study design and setting We conducted a cross-sectional study at Solwezi General Hospital Laboratory in Solwezi District, the main referral centre for HIV Viral load monitoring in the North-western Province of Zambia. Sample Size A total of 159 EDTA plasma remnant samples collected from individuals living with HIV who were receiving antiretroviral therapy (ART) and undergoing routine viral load monitoring at Solwezi General Hospital using the Hologic Panther [ 8 ]. A total of 117 samples were used to assess sensitivity and specificity, while 32 samples were analysed for the correlation coefficient. Precision testing was conducted using five low HIV viral load samples and five high HIV viral load samples, each processed in five replicates over five consecutive days. Low viral load was defined as ≤ 999 copies/mL, while high viral load was defined as ≥ 100,000 copies/mL. For linearity assessment, five pooled samples were diluted and processed to evaluate the Pearson correlation coefficient. All results were converted to log10 copies/mL for statistical analysis. Sample collection and storage Plasma was extracted from whole-blood samples collected in EDTA-containing tubes and separated by centrifugation at 1,500 × g for 10 minutes. An initial 0.6 mL aliquot was tested using the Aptima HIV-1 assay as part of routine HIV-1 viral load monitoring. Additional plasma aliquots were stored at − 80°C in volumes designated for the Xpert HIV-1 assay, undergoing a single freeze-thaw cycle before analysis. On the day of testing, the aliquots were thawed, vortex-mixed, and analysed for viral load quantification. Test Method The Xpert HIV-1 Viral Load is a cartridge-based, total nucleic acid real-time polymerase chain reaction (RT-PCR) test for the detection and quantification of Human Immunodeficiency Virus type 1 (HIV-1) RNA in human plasma from HIV-1 infected individuals, using the automated GeneXpert Instrument Systems (Cepheid, Sunnyvale, CA, USA) [ 9 ]. It is a near point of care test that can quantify HIV-1 RNA over the range of 40 to 10,000,000 copies/mL. The Xpert HIV-1 VL assay targets the Long Terminal Repeats (LTR) gene of the HIV-1 RNA [ 10 ]. A volume of 1000 µl of plasma specimen is needed to perform the assay (if using the transfer pipette included in the kit, a minimum of 1.2 mL of plasma is required) [ 9 ]. This assay necessitates additional laboratory equipment for specimen preparation, such as a centrifuge and a refrigerator (if specimen storage is required), but it can still be conducted in laboratories with minimal resources. The equipment relies on a consistent power supply [ 9 ]. The Hologic Aptima HIV-1 Quant Dx viral load assay was conducted following the manufacturer's guidelines and analysed using the fully automated Panther instrument system. The Aptima assays utilize real-time transcription-mediated amplification (RT-TMA) for the detection and quantification of HIV-1 RNA [ 11 ]. The Aptima HIV-1 Quant Dx assay targets the polymerase (pol) and LTR regions, has a minimal required sample volume of 0.7 mL of specimen, and reports quantitative HIV-1 results in a range of 30 to 10,000.000 copies/mL [ 11 ]. The predefined thresholds for evaluating the acceptability of results were set as follows: Sensitivity ≥ 94.1%, Specificity ≥ 98.5%, Correlation coefficient (R-value) ≥ 0.941, precision with a coefficient of variation (CV) ≤ 3%, and Linearity with an R² ≥ 0.99 across serial dilutions [ 9 ]. Statistical Analysis Data collection was carried out using an Excel spreadsheet, which was later exported to IBM SPSS Statistics 22 and R Studio for statistical analysis. The precision of the Xpert HIV-1 VL platform was assessed using the Coefficient of Variation (CV) calculated through the one-way ANOVA method. The accuracy of the Xpert HIV-1 VL platform was determined using the Pearson correlation coefficient (R 2 ), while its linearity was evaluated through the correlation coefficient (r). Ethical clearance Ethical approval for the study was granted by the Mulungushi University School of Medicine Ethics Committee, and authorization to conduct the research was obtained from the management of Solwezi General Hospital. Results Sensitivity and Specificity A total number of 159 samples that were initially processed using the Aptima-HIV-1 assays were compared to the Xpert-HIV-1 VL samples. By using the quantification thresholds of the two assays, the VL results were classified as “Target not detected” or “Quantified”. The Xpert HIV-1 sensitivity and specificity using Aptima HIV-1 as reference method was was 87.62% [95% CI, 79.01% – 93.17%], and 100.00% [95% CI, 79.95% – 100.00%] respectively, as shown below in Table 1 . Table 1 Sensitivity and Specificity Estimated value (%) (n = 117) 95% Confidence interval Lower limit Upper limit Sensitivity 87.62 74.59 89.00 Specificity 100 79.96 100 Correlation between Xpert-HIV-1 VL and Hologic Panther The mean VL obtained by the Xpert-HIV-1 VL method was 2.96 log10 cp/mL with a Standard Deviation (SD) of 1.65 log10 cp/mL, while the mean VL obtained by the Hologic Panther was 3.06 log10 cp/mL (SD: 1.60 log10 cp/mL). A correlation between the two assays was observed (r = 0.97, p < 0.0001) as shown in Fig. 1 . Precision results The repeatability and within-laboratory mean results for samples with high and low concentrations demonstrated coefficient of variation (CV) values were 1.38 and 2.59, as highlighted in Table 2 and Table 3 . Table 2 Precision for High positive viral load Analyte Test Type Precision High Positive Acceptability Mean CV (%) Claim Study CV (%) HIV Viral Load High Positive Repeatability 5.18 3.0 0.71 Acceptable Within Lab precision 3.0 1.38 Acceptable Table 3 Precision for Low positive viral load Analyte Test Type Precision Low Positive Acceptability Mean CV (%) Claim Study CV (%) HIV Viral Load Low Positive Repeatability 2.81 3.0 1.85 Acceptable Within Lab precision 3.0 2.59 Acceptable Linearity Results All data points fell within the acceptable range, as demonstrated in Fig. 2 . Discussion The Xpert HIV-1 VL assay demonstrated an excellent analytical performance when compared to the conventional Hologic Panther system. Our evaluation confirms that the Xpert HIV-1 VL assay satisfies the performance criteria essential for clinical diagnosis. The Xpert HIV-1 VL assay achieved a sensitivity of 87.6% and a specificity of 100%, highlighting its reliability in distinguishing true positives and negatives. A strong correlation between the two assays was observed (r = 0.97, p < 0.0001). Reproducibility, assessed through the coefficient of variation (CV), was excellent, with a CV of 1.38% for high viral loads and 2.59% for low viral loads. Linearity analysis further confirmed the assay's accuracy, with all data points falling within acceptable ranges and correlation coefficients (R²) consistently ≥ 0.992. To our knowledge, this study is the first to evaluate the analytical performance of the Xpert HIV-1 assay compared to the Aptima HIV-1 assay. To support this analysis, we referenced the World Health Organisation (WHO) prequalification report and previous studies that assessed the Xpert HIV-1 assay against other analysers, such as the Roche Cobas assays. Our study demonstrated a lower sensitivity compared to the findings of Kone et al. (2020) and Wesolowski et al. (2020), who reported sensitivities of 93.10% and 97.9%, respectively, when evaluating the Xpert HIV-1 VL assay against the Roche Cobas system [ 7 , 10 ]. This discrepancy may be attributed to differences in sample handling. Our study utilized stored remnant plasma, which could have undergone RNA degradation, potentially affecting detection rates. Despite this, our study achieved a perfect specificity than what was reported by Kone et al. (2020) and by WHO prequalification report, highlighting the validity of our assay in correctly identifying true negatives [ 7 , 9 ]. Our findings on precision outperformed those reported by Woo et al. (2024), as our CV for precision was consistently below 5% [ 12 ]. This is a notable improvement over Smita et al. (2017), who reported CVs of 4.15% and 3.52% [ 13 ]. Furthermore, Gous et al. (2016), in their comparison of the Xpert HIV-1 VL assay with the Roche Cobas TaqMan v2 and Abbott HIV-1 assays, reported CVs of 1.5% and 0.9% [ 14 ]. Our findings aligned with these values for high-positive samples, although our CV for low-positive samples was higher than Gous et al, indicating potential areas for further optimization. Our CV for the low positive was still lower than what the WHO Prequalification of In Vitro Diagnostics report for 2023 for the evaluation of Xpert HIV-1 VL [ 9 ]. Our study also demonstrated a stronger correlation (r > 0.97) between the evaluated assays than the findings of Smita et al. (2017), who reported a correlation coefficient of 0.886 when comparing the GeneXpert assay with the Abbott assay [ 13 ]. Similarly, our findings showed superior performance when comparing the Xpert HIV-1 assay to the Hologic Panther system, outperforming the correlation reported by Mor et al. (2015) (r > 0.89) [ 15 ]. The linearity of the Xpert HIV-1 VL assay in our study was consistent with Wesolowski et al. (2020) and by WHO prequalification, who reported a correlation coefficient (R²) > 0.99 when comparing the assay with the Roche Cobas Ampliprep/Cobas TaqMan system [ 9 , 10 ]. Lastly, our study reported mean viral load estimations for the Xpert HIV-1, with 2.81 log copies/mL for the low viral load and 5.18 log copies/mL for the high viral load, respectively, whereas our results demonstrated improved precision and reliability in viral load quantification. The Xpert HIV-1 VL assay offers exceptional accuracy and a significantly reduced turnaround time, making it an ideal choice for clinical laboratories serving critical populations. Moreover, its single-cartridge design minimizes additional costs and manpower typically required from conducting VL testing and reduces the need for extensive infrastructure required by conventional molecular platforms. The differences in detection thresholds between the Xpert HIV-1 VL (40 copies/mL) and Hologic Panther (30 copies/mL) assays may partially account for the observed sensitivity of 87.6%. Samples with viral loads near the lower limit of detection (30–40 copies/mL) could be quantified by the Panther but classified as "target not detected" by the Xpert HIV-1, leading to a reduced apparent sensitivity of the latter. This aligns with prior studies reporting sensitivity variations when comparing assays with various thresholds [ 9 , 10 ]. Clinically, this difference is unlikely to compromise patient management, as both thresholds are well below the WHO-defined virologic failure threshold (≥ 1,000 copies/mL). However, in research settings requiring ultra-sensitive detection (e.g., HIV cure studies), the Panther’s lower threshold may offer an advantage. Future evaluations should include stratification by viral load ranges to clarify the Xpert’s performance near its detection limit. Conclusion The GeneXpert HIV-1 viral load assay demonstrates high efficacy in accurately identifying and quantifying HIV-1 viral loads, making it a valuable tool for HIV diagnosis and monitoring. Its strong analytical performance, combined with its compatibility with conventional equipment systems, positions it as a flexible and dependable solution for resource-limited settings such as Zambia. This reliability makes it particularly suitable for critical populations requiring timely and accurate viral load monitoring. Future research should focus on optimizing assay performance and addressing any limitations to further enhance its utility in diverse healthcare settings. Limitations This study had several limitations. First, the use of stored specimen remnants may have introduced the potential for specimen degradation, which could have contributed to the lower sensitivity observed. Second, the study was limited to a single comparator assay, the Hologic Panther VL system, which, while robust, may be less accurate than other widely used platforms such as the Roche Cobas TaqMan HIV-1 assay. Lastly, the limited availability of studies comparing the performance of the Xpert HIV-1 VL assay to the Hologic Panther system restricts the broader contextualization of our findings. Future research incorporating fresh specimens and multiple comparator assays is recommended to validate and expand upon these results. Declarations Ethics approval and consent to participate This study obtained ethical approval from the Mulungushi University School of Medicine Research Committee (IRB: 00012281, FWA: 0002888) under reference number SMHS-MU1-2024-28. Permission to conduct the study was granted by Solwezi General Hospital Management. Informed consent was waived for this study. To ensure confidentiality and anonymity, participants were assigned unique project identification numbers exclusively for research purposes. Availability of data and material The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors have no conflicts of interest to declare. Funding The authors did not receive any external funding for this study. All reagents and materials required were provided by Solwezi General Hospital. Author’s contributions KS conceived the study. DC and ATK conducted data collection. DC conducted data analysis and writing of the manuscript. KS and ATK reviewed. All authors read and approved the final manuscript. Consent for publication Not Applicable Acknowledgements We extend our heartfelt gratitude to the Laboratory personnel, the research team, and the Office of the Senior Medical Superintendent at Solwezi General Hospital for their unwavering support and dedication to advancing research initiatives. Your enthusiasm and collaboration have been invaluable to the success of this project. References UNAIDS. (2020) Data 2020. Program HIV/AIDS 1–436. World Health Organization (WHO). African Region (2023) Country Disease Outlook. 1–146. Ministry of Health Zambia. Zambia Population-based HIV/AID Impact Assessment. Minist. Heal. Zambia; 2021. Ochodo EA, Olwanda EE, Deeks JJ, Mallett S. Point-of-care viral load tests to detect high HIV viral load in people living with HIV/AIDS attending health facilities. Cochrane database Syst Rev. 2022;3:CD013208. Mashamba-Thompson TP, Jama NA, Sartorius B, Drain PK, Thompson RM. (2017) Implementation of Point-of-Care Diagnostics in Rural Primary Healthcare Clinics in South Africa: Perspectives of Key Stakeholders. Diagnostics (Basel, Switzerland). https://doi.org/10.3390/diagnostics7010003 Girdwood S, Pandey M, Machila T, et al. The integration of tuberculosis and HIV testing on GeneXpert can substantially improve access and same-day diagnosis and benefit tuberculosis programmes: A diagnostic network optimization analysis in Zambia. PLOS Glob public Heal. 2023;3:e0001179. Kone B, Goita D, Dolo O et al. (2020) Performance Comparison of Xpert HIV-1 Viral Load Assay and Roche Taqman and Abbott M2000 RT in Bamako, Mali. J AIDS Clin Res 11. Rossetti R, Smith T, Luo W, Masciotra S. Performance evaluation of the MedMira reveal G4 LAB S/P and POC HIV antibody rapid screening tests using plasma and whole blood specimens. J Clin Virol. 2020;127:104344. Pqdx WHO, Report P, Hiv- X, Load V, Dx G, Infinity- G, Infinity- G, Ab C. (2020) WHO Prequalification of In Vitro Diagnostics PUBLIC REPORT Product: Xpert HIV-1 Viral Load with GeneXpert Dx, GeneXpert Infinity-48s and GeneXpert Infinity-80 Summary of WHO prequalification assessment for Xpert HIV-1 Viral Load. Wesolowski L, Fowler W, Luo W, et al. Evaluation of the performance of the Cepheid Xpert HIV-1 Viral Load Assay for quantitative and diagnostic uses. J Clin Virol. 2020;122:104214. Lewis S, Morehead E, Cheung H, Mostafa HH. Clinical performance evaluation of the Aptima viral assays for the quantitation of HIV-1, HCV, and HBV in plasma samples. Diagn Microbiol Infect Dis. 2023;106:115951. Woo K-S, Kwak M-S, Han J-Y. Performance Evaluation of Hologic Panther Aptima System to Detect HBV, HCV, and HIV-1 Infections: A Comparison with Abbott Alinity m System. J Lab Med Qual Assur. 2024;46:96–102. Kulkarni S, Jadhav S, Khopkar P, et al. GeneXpert HIV-1 quant assay, a new tool for scale up of viral load monitoring in the success of ART programme in India. BMC Infect Dis. 2017;17:506. Gous N, Scott L, Berrie L, Stevens W. Options to Expand HIV Viral Load Testing in South Africa: Evaluation of the GeneXpert® HIV-1 Viral Load Assay. PLoS ONE. 2016;11:e0168244. Mor O, Gozlan Y, Wax M, Mileguir F, Rakovsky A, Noy B, Mendelson E, Levy I. Evaluation of the RealTime HIV-1, Xpert HIV-1, and Aptima HIV-1 Quant Dx Assays in Comparison to the NucliSens EasyQ HIV-1 v2.0 Assay for Quantification of HIV-1 Viral Load. J Clin Microbiol. 2015;53:3458–65. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 21 Jul, 2025 Read the published version in BMC Research Notes → Version 1 posted Editorial decision: Revision requested 08 Jul, 2025 Reviews received at journal 17 Apr, 2025 Reviews received at journal 09 Apr, 2025 Reviewers agreed at journal 07 Apr, 2025 Reviewers agreed at journal 04 Apr, 2025 Reviewers invited by journal 04 Apr, 2025 Submission checks completed at journal 04 Apr, 2025 First submitted to journal 03 Apr, 2025 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. 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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-5723442","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":438439447,"identity":"941e605b-879d-4451-993d-050ac44dea0f","order_by":0,"name":"Keembe Siakantu","email":"","orcid":"","institution":"Solwezi General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Keembe","middleName":"","lastName":"Siakantu","suffix":""},{"id":438439448,"identity":"e6450847-c259-447b-adaf-a92bbacc9f33","order_by":1,"name":"David Chisompola","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYDCCAwxsDAlgFnMDwwcgxcZOvBbGBsYZIC3MxGhhgGph5gHbRkAH3wHmZw8e/LGJNmdvbPxs82ubPB8zA+OHjzm4tUgeYDM3SGxLy93Zc7BZOrfvtmEbMwOz5MxtuLUYHOBhk0hsOJy74UZig3Ruz21GoBY2Zl5CWhL+gLU0/7bsuW1PpBY2sJY2aYYftxMJapE8zGYmAfVLm2Vvw+3kNmbGZrx+4Tve/Ezyxx+b3O3szYdv/Phz23Z+e/PBDx/xaIHHggGIYGwDkw141CN7Ckz+IU7xKBgFo2AUjCwAALFtVB8kziUyAAAAAElFTkSuQmCC","orcid":"","institution":"Arthur Davison Children's Hospital","correspondingAuthor":true,"prefix":"","firstName":"David","middleName":"","lastName":"Chisompola","suffix":""},{"id":438439449,"identity":"8e1efc6d-0fd2-4cd7-9e1d-6bf3e5b5a310","order_by":2,"name":"Aaron Tembo Konzani","email":"","orcid":"","institution":"Pathology Laboratory Dept, Tropical Gastroenterology and Nutrition group, Lusaka, Zambia","correspondingAuthor":false,"prefix":"","firstName":"Aaron","middleName":"Tembo","lastName":"Konzani","suffix":""}],"badges":[],"createdAt":"2024-12-27 20:08:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5723442/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5723442/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13104-025-07387-w","type":"published","date":"2025-07-21T15:57:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80042906,"identity":"cc2349a5-503e-436d-8f4b-5fe05a74048f","added_by":"auto","created_at":"2025-04-07 09:31:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":64310,"visible":true,"origin":"","legend":"\u003cp\u003eXpert-HIV-1 VL and Hologic Panther correlation chart as shown here\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5723442/v1/aa0d367f38a0e8e7ba37060f.png"},{"id":80046036,"identity":"63999807-e332-4e5f-bb75-98bc8a5b15b7","added_by":"auto","created_at":"2025-04-07 09:47:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":60617,"visible":true,"origin":"","legend":"\u003cp\u003eXpert HIV-1 VL Linearity\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5723442/v1/55b330ff42813c406bd27f34.png"},{"id":87756685,"identity":"bae306aa-539a-4129-a0fa-e45ffbc1de14","added_by":"auto","created_at":"2025-07-28 16:07:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":671228,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5723442/v1/432c5e4a-4d3c-46ff-9449-c1c389ebb06d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluating the Accuracy of HIV-1 Viral Load Testing Using near point of care Xpert HIV- 1 System at Solwezi General Hospital, Zambia.","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Human Immunodeficiency Viruses (HIV) has a significant global impact, and it has been estimated that 38 million people worldwide are living with HIV/AIDS in 2020 [1]. In Africa, particularly in Zambia, the burden of HIV remains alarmingly high. As of 2020, approximately 1.2 million people in the country were receiving treatment for HIV, highlighting the ongoing public health challenge and the critical need for sustained intervention efforts [2].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eZambia has been significantly impacted by the HIV-1 epidemic, with approximately 11.0% of the population aged 15 and older living with HIV in 2021. In Northwestern Province, 6.8% of the province\u0026apos;s population is affected by the virus\u0026nbsp;[3]. This indicates a considerable number of individuals in Solwezi who require effective management and monitoring of the HIV infection.\u003c/p\u003e\n\u003cp\u003eViral load (VL)\u0026nbsp;testing using the near point-of-care (POC) GeneXpert platform (Cepheid, Sunnyvale, CA, USA) with the Xpert HIV-1 quantitative assay offers the advantage of being used closer to patients. It is easy to use, gives quick results, is cost-effective, and has the potential to serve as an alternative to centralized viral load testing platforms [4]. POC has been introduced for rural populations to expand access to clinical laboratory testing by eliminating the need for sample transportation, laboratory and data management infrastructure, and highly trained staff [5]. Therefore, ensuring access to reliable and accurate viral load testing is vital in improving patient outcomes and reducing HIV transmission rates in Solwezi and similar high-burden areas [4].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe GeneXpert system was recently adopted to reduce the turnaround time for the urgent population such as pregnant and breastfeeding women and children from 0 to 14 years in point of care facilities [6]. Thereby, adding the Xpert HIV-1 VL to the employed methods for HIV-1 viral load testing in Zambia [6]. However, the evaluation of Xpert HIV-1 VL systems\u0026apos; performance and accuracy remains unclear. Differences in detection methodology, sample handling, operator skill level, and other factors may result in discrepancies between the conventional systems\u0026apos; and point of care measurements. Such discrepancies might not only affect individual patient care but could also have broader implications for public health, especially in settings where the HIV prevalence is high [7]. Therefore, it is critical to understand and address this knowledge gap to optimize HIV-1 viral load testing and thus improve patient outcomes and public health strategies. This study aimed to evaluate analytical performance and accuracy of HIV-1 viral load measurements of the GeneXpert system in comparison to the Hologic Panther system.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eStudy design and setting\u003c/h2\u003e \u003cp\u003eWe conducted a cross-sectional study at Solwezi General Hospital Laboratory in Solwezi District, the main referral centre for HIV Viral load monitoring in the North-western Province of Zambia.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eSample Size\u003c/h3\u003e\n\u003cp\u003eA total of 159 EDTA plasma remnant samples collected from individuals living with HIV who were receiving antiretroviral therapy (ART) and undergoing routine viral load monitoring at Solwezi General Hospital using the Hologic Panther [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. A total of 117 samples were used to assess sensitivity and specificity, while 32 samples were analysed for the correlation coefficient. Precision testing was conducted using five low HIV viral load samples and five high HIV viral load samples, each processed in five replicates over five consecutive days. Low viral load was defined as \u0026le;\u0026thinsp;999 copies/mL, while high viral load was defined as \u0026ge;\u0026thinsp;100,000 copies/mL. For linearity assessment, five pooled samples were diluted and processed to evaluate the Pearson correlation coefficient. All results were converted to log10 copies/mL for statistical analysis.\u003c/p\u003e\n\u003ch3\u003eSample collection and storage\u003c/h3\u003e\n\u003cp\u003ePlasma was extracted from whole-blood samples collected in EDTA-containing tubes and separated by centrifugation at 1,500 \u0026times; g for 10 minutes. An initial 0.6 mL aliquot was tested using the Aptima HIV-1 assay as part of routine HIV-1 viral load monitoring. Additional plasma aliquots were stored at \u0026minus;\u0026thinsp;80\u0026deg;C in volumes designated for the Xpert HIV-1 assay, undergoing a single freeze-thaw cycle before analysis. On the day of testing, the aliquots were thawed, vortex-mixed, and analysed for viral load quantification.\u003c/p\u003e\n\u003ch3\u003eTest Method\u003c/h3\u003e\n\u003cp\u003eThe Xpert HIV-1 Viral Load is a cartridge-based, total nucleic acid real-time polymerase chain reaction (RT-PCR) test for the detection and quantification of Human Immunodeficiency Virus type 1 (HIV-1) RNA in human plasma from HIV-1 infected individuals, using the automated GeneXpert Instrument Systems (Cepheid, Sunnyvale, CA, USA) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It is a near point of care test that can quantify HIV-1 RNA over the range of 40 to 10,000,000 copies/mL. The Xpert HIV-1 VL assay targets the Long Terminal Repeats (LTR) gene of the HIV-1 RNA [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. A volume of 1000 \u0026micro;l of plasma specimen is needed to perform the assay (if using the transfer pipette included in the kit, a minimum of 1.2 mL of plasma is required) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This assay necessitates additional laboratory equipment for specimen preparation, such as a centrifuge and a refrigerator (if specimen storage is required), but it can still be conducted in laboratories with minimal resources. The equipment relies on a consistent power supply [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe Hologic Aptima HIV-1 Quant Dx viral load assay was conducted following the manufacturer's guidelines and analysed using the fully automated Panther instrument system. The Aptima assays utilize real-time transcription-mediated amplification (RT-TMA) for the detection and quantification of HIV-1 RNA [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The Aptima HIV-1 Quant Dx assay targets the polymerase (pol) and LTR regions, has a minimal required sample volume of 0.7 mL of specimen, and reports quantitative HIV-1 results in a range of 30 to 10,000.000 copies/mL [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe predefined thresholds for evaluating the acceptability of results were set as follows: Sensitivity\u0026thinsp;\u0026ge;\u0026thinsp;94.1%, Specificity\u0026thinsp;\u0026ge;\u0026thinsp;98.5%, Correlation coefficient (R-value)\u0026thinsp;\u0026ge;\u0026thinsp;0.941, precision with a coefficient of variation (CV)\u0026thinsp;\u0026le;\u0026thinsp;3%, and Linearity with an R\u0026sup2; \u0026ge; 0.99 across serial dilutions [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData collection was carried out using an Excel spreadsheet, which was later exported to IBM SPSS Statistics 22 and R Studio for statistical analysis. The precision of the Xpert HIV-1 VL platform was assessed using the Coefficient of Variation (CV) calculated through the one-way ANOVA method. The accuracy of the Xpert HIV-1 VL platform was determined using the Pearson correlation coefficient (R\u003csup\u003e2\u003c/sup\u003e), while its linearity was evaluated through the correlation coefficient (r).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEthical clearance\u003c/h3\u003e\n\u003cp\u003eEthical approval for the study was granted by the Mulungushi University School of Medicine Ethics Committee, and authorization to conduct the research was obtained from the management of Solwezi General Hospital.\u003c/p\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSensitivity and Specificity\u003c/h2\u003e \u003cp\u003eA total number of 159 samples that were initially processed using the Aptima-HIV-1 assays were compared to the Xpert-HIV-1 VL samples. By using the quantification thresholds of the two assays, the VL results were classified as \u0026ldquo;Target not detected\u0026rdquo; or \u0026ldquo;Quantified\u0026rdquo;. The Xpert HIV-1 sensitivity and specificity using Aptima HIV-1 as reference method was was 87.62% [95% CI, 79.01% \u0026ndash; 93.17%], and 100.00% [95% CI, 79.95% \u0026ndash; 100.00%] respectively, as shown below in Table\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\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\u003eSensitivity and Specificity\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=\"char\" char=\".\" 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\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEstimated value\u003c/p\u003e \u003cp\u003e(%) (n\u0026thinsp;=\u0026thinsp;117)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e95% Confidence interval\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLower limit\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUpper limit\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSensitivity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e87.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e74.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e89.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecificity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e79.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCorrelation between Xpert-HIV-1 VL and Hologic Panther\u003c/h2\u003e \u003cp\u003eThe mean VL obtained by the Xpert-HIV-1 VL method was 2.96 log10 cp/mL with a Standard Deviation (SD) of 1.65 log10 cp/mL, while the mean VL obtained by the Hologic Panther was 3.06 log10 cp/mL (SD: 1.60 log10 cp/mL). A correlation between the two assays was observed (r\u0026thinsp;=\u0026thinsp;0.97, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePrecision results\u003c/h2\u003e \u003cp\u003eThe repeatability and within-laboratory mean results for samples with high and low concentrations demonstrated coefficient of variation (CV) values were 1.38 and 2.59, as highlighted in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\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\u003ePrecision for High positive viral load\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAnalyte\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTest Type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003ePrecision High Positive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAcceptability\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCV (%) Claim\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eStudy CV (%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eHIV Viral Load High Positive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRepeatability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e5.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAcceptable\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWithin Lab precision\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAcceptable\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\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\u003ePrecision for Low positive viral load\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAnalyte\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTest Type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003ePrecision Low Positive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAcceptability\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCV (%) Claim\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eStudy CV (%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eHIV Viral Load Low Positive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRepeatability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e2.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAcceptable\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWithin Lab precision\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAcceptable\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLinearity Results\u003c/h2\u003e \u003cp\u003eAll data points fell within the acceptable range, as demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe Xpert HIV-1 VL assay demonstrated an excellent analytical performance when compared to the conventional Hologic Panther system. Our evaluation confirms that the Xpert HIV-1 VL assay satisfies the performance criteria essential for clinical diagnosis. The Xpert HIV-1 VL assay achieved a sensitivity of 87.6% and a specificity of 100%, highlighting its reliability in distinguishing true positives and negatives. A strong correlation between the two assays was observed (r\u0026thinsp;=\u0026thinsp;0.97, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Reproducibility, assessed through the coefficient of variation (CV), was excellent, with a CV of 1.38% for high viral loads and 2.59% for low viral loads. Linearity analysis further confirmed the assay's accuracy, with all data points falling within acceptable ranges and correlation coefficients (R\u0026sup2;) consistently\u0026thinsp;\u0026ge;\u0026thinsp;0.992.\u003c/p\u003e \u003cp\u003eTo our knowledge, this study is the first to evaluate the analytical performance of the Xpert HIV-1 assay compared to the Aptima HIV-1 assay. To support this analysis, we referenced the World Health Organisation (WHO) prequalification report and previous studies that assessed the Xpert HIV-1 assay against other analysers, such as the Roche Cobas assays.\u003c/p\u003e \u003cp\u003eOur study demonstrated a lower sensitivity compared to the findings of Kone et al. (2020) and Wesolowski et al. (2020), who reported sensitivities of 93.10% and 97.9%, respectively, when evaluating the Xpert HIV-1 VL assay against the Roche Cobas system [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This discrepancy may be attributed to differences in sample handling. Our study utilized stored remnant plasma, which could have undergone RNA degradation, potentially affecting detection rates.\u003c/p\u003e \u003cp\u003eDespite this, our study achieved a perfect specificity than what was reported by Kone et al. (2020) and by WHO prequalification report, highlighting the validity of our assay in correctly identifying true negatives [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur findings on precision outperformed those reported by Woo et al. (2024), as our CV for precision was consistently below 5% [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This is a notable improvement over Smita et al. (2017), who reported CVs of 4.15% and 3.52% [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, Gous et al. (2016), in their comparison of the Xpert HIV-1 VL assay with the Roche Cobas TaqMan v2 and Abbott HIV-1 assays, reported CVs of 1.5% and 0.9% [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Our findings aligned with these values for high-positive samples, although our CV for low-positive samples was higher than Gous et al, indicating potential areas for further optimization. Our CV for the low positive was still lower than what the WHO Prequalification of In Vitro Diagnostics report for 2023 for the evaluation of Xpert HIV-1 VL [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur study also demonstrated a stronger correlation (r\u0026thinsp;\u0026gt;\u0026thinsp;0.97) between the evaluated assays than the findings of Smita et al. (2017), who reported a correlation coefficient of 0.886 when comparing the GeneXpert assay with the Abbott assay [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Similarly, our findings showed superior performance when comparing the Xpert HIV-1 assay to the Hologic Panther system, outperforming the correlation reported by Mor et al. (2015) (r\u0026thinsp;\u0026gt;\u0026thinsp;0.89) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe linearity of the Xpert HIV-1 VL assay in our study was consistent with Wesolowski et al. (2020) and by WHO prequalification, who reported a correlation coefficient (R\u0026sup2;)\u0026thinsp;\u0026gt;\u0026thinsp;0.99 when comparing the assay with the Roche Cobas Ampliprep/Cobas TaqMan system [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLastly, our study reported mean viral load estimations for the Xpert HIV-1, with 2.81 log copies/mL for the low viral load and 5.18 log copies/mL for the high viral load, respectively, whereas our results demonstrated improved precision and reliability in viral load quantification.\u003c/p\u003e \u003cp\u003eThe Xpert HIV-1 VL assay offers exceptional accuracy and a significantly reduced turnaround time, making it an ideal choice for clinical laboratories serving critical populations. Moreover, its single-cartridge design minimizes additional costs and manpower typically required from conducting VL testing and reduces the need for extensive infrastructure required by conventional molecular platforms.\u003c/p\u003e \u003cp\u003eThe differences in detection thresholds between the Xpert HIV-1 VL (40 copies/mL) and Hologic Panther (30 copies/mL) assays may partially account for the observed sensitivity of 87.6%. Samples with viral loads near the lower limit of detection (30\u0026ndash;40 copies/mL) could be quantified by the Panther but classified as \"target not detected\" by the Xpert HIV-1, leading to a reduced apparent sensitivity of the latter. This aligns with prior studies reporting sensitivity variations when comparing assays with various thresholds [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Clinically, this difference is unlikely to compromise patient management, as both thresholds are well below the WHO-defined virologic failure threshold (\u0026ge;\u0026thinsp;1,000 copies/mL). However, in research settings requiring ultra-sensitive detection (e.g., HIV cure studies), the Panther\u0026rsquo;s lower threshold may offer an advantage. Future evaluations should include stratification by viral load ranges to clarify the Xpert\u0026rsquo;s performance near its detection limit.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe GeneXpert HIV-1 viral load assay demonstrates high efficacy in accurately identifying and quantifying HIV-1 viral loads, making it a valuable tool for HIV diagnosis and monitoring. Its strong analytical performance, combined with its compatibility with conventional equipment systems, positions it as a flexible and dependable solution for resource-limited settings such as Zambia. This reliability makes it particularly suitable for critical populations requiring timely and accurate viral load monitoring. Future research should focus on optimizing assay performance and addressing any limitations to further enhance its utility in diverse healthcare settings.\u003c/p\u003e "},{"header":"Limitations","content":"\u003cp\u003eThis study had several limitations. First, the use of stored specimen remnants may have introduced the potential for specimen degradation, which could have contributed to the lower sensitivity observed. Second, the study was limited to a single comparator assay, the Hologic Panther VL system, which, while robust, may be less accurate than other widely used platforms such as the Roche Cobas TaqMan HIV-1 assay. Lastly, the limited availability of studies comparing the performance of the Xpert HIV-1 VL assay to the Hologic Panther system restricts the broader contextualization of our findings. Future research incorporating fresh specimens and multiple comparator assays is recommended to validate and expand upon these results.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study obtained ethical approval from the Mulungushi University School of Medicine Research Committee (IRB: 00012281, FWA: 0002888) under reference number SMHS-MU1-2024-28. Permission to conduct the study was granted by Solwezi General Hospital Management. Informed consent was waived for this study. To ensure confidentiality and anonymity, participants were assigned unique project identification numbers exclusively for research purposes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors did not receive any external funding for this study. All reagents and materials required were provided by Solwezi General Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKS conceived the study. DC and ATK conducted data collection. DC conducted data analysis and writing of the manuscript. KS and ATK reviewed. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our heartfelt gratitude to the Laboratory personnel, the research team, and the Office of the Senior Medical Superintendent at Solwezi General Hospital for their unwavering support and dedication to advancing research initiatives. Your enthusiasm and collaboration have been invaluable to the success of this project.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eUNAIDS. (2020) Data 2020. Program HIV/AIDS 1\u0026ndash;436.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWorld Health Organization (WHO). African Region (2023) Country Disease Outlook. 1\u0026ndash;146.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMinistry of Health Zambia. Zambia Population-based HIV/AID Impact Assessment. Minist. Heal. Zambia; 2021.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOchodo EA, Olwanda EE, Deeks JJ, Mallett S. Point-of-care viral load tests to detect high HIV viral load in people living with HIV/AIDS attending health facilities. Cochrane database Syst Rev. 2022;3:CD013208.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMashamba-Thompson TP, Jama NA, Sartorius B, Drain PK, Thompson RM. (2017) Implementation of Point-of-Care Diagnostics in Rural Primary Healthcare Clinics in South Africa: Perspectives of Key Stakeholders. Diagnostics (Basel, Switzerland). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/diagnostics7010003\u003c/span\u003e\u003cspan address=\"10.3390/diagnostics7010003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGirdwood S, Pandey M, Machila T, et al. The integration of tuberculosis and HIV testing on GeneXpert can substantially improve access and same-day diagnosis and benefit tuberculosis programmes: A diagnostic network optimization analysis in Zambia. PLOS Glob public Heal. 2023;3:e0001179.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKone B, Goita D, Dolo O et al. (2020) Performance Comparison of Xpert HIV-1 Viral Load Assay and Roche Taqman and Abbott M2000 RT in Bamako, Mali. J AIDS Clin Res 11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRossetti R, Smith T, Luo W, Masciotra S. Performance evaluation of the MedMira reveal G4 LAB S/P and POC HIV antibody rapid screening tests using plasma and whole blood specimens. J Clin Virol. 2020;127:104344.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePqdx WHO, Report P, Hiv- X, Load V, Dx G, Infinity- G, Infinity- G, Ab C. (2020) WHO Prequalification of In Vitro Diagnostics PUBLIC REPORT Product: Xpert HIV-1 Viral Load with GeneXpert Dx, GeneXpert Infinity-48s and GeneXpert Infinity-80 Summary of WHO prequalification assessment for Xpert HIV-1 Viral Load.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWesolowski L, Fowler W, Luo W, et al. Evaluation of the performance of the Cepheid Xpert HIV-1 Viral Load Assay for quantitative and diagnostic uses. J Clin Virol. 2020;122:104214.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLewis S, Morehead E, Cheung H, Mostafa HH. Clinical performance evaluation of the Aptima viral assays for the quantitation of HIV-1, HCV, and HBV in plasma samples. Diagn Microbiol Infect Dis. 2023;106:115951.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWoo K-S, Kwak M-S, Han J-Y. Performance Evaluation of Hologic Panther Aptima System to Detect HBV, HCV, and HIV-1 Infections: A Comparison with Abbott Alinity m System. J Lab Med Qual Assur. 2024;46:96\u0026ndash;102.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKulkarni S, Jadhav S, Khopkar P, et al. GeneXpert HIV-1 quant assay, a new tool for scale up of viral load monitoring in the success of ART programme in India. BMC Infect Dis. 2017;17:506.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGous N, Scott L, Berrie L, Stevens W. Options to Expand HIV Viral Load Testing in South Africa: Evaluation of the GeneXpert\u0026reg; HIV-1 Viral Load Assay. PLoS ONE. 2016;11:e0168244.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMor O, Gozlan Y, Wax M, Mileguir F, Rakovsky A, Noy B, Mendelson E, Levy I. Evaluation of the RealTime HIV-1, Xpert HIV-1, and Aptima HIV-1 Quant Dx Assays in Comparison to the NucliSens EasyQ HIV-1 v2.0 Assay for Quantification of HIV-1 Viral Load. J Clin Microbiol. 2015;53:3458\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Diagnosis, Accuracy, GeneXpert, Hologic Panther, Solwezi, Zambia","lastPublishedDoi":"10.21203/rs.3.rs-5723442/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5723442/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eHIV-1 Viral Load Testing plays a crucial role in the management of HIV/AIDS patients by quantifying the presence of HIV-1 RNA in the blood, which directly relates to viral replication and disease progression. Monitoring viral load enables healthcare professionals to evaluate the effectiveness of Anti-Retroviral Therapy, adapt treatment strategies, and assess the overall health status of individuals with HIV. This was a cross-sectional study to evaluate analytical performance (accuracy, precision and linearity of the near point of care Xpert HIV-1 viral load.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe analytical performance of the GeneXpert assay to the conventional Hologic Panther system was strong. Specifically, the GeneXpert demonstrated a sensitivity, specificity, and accuracy of 87.6%, 100%, and 98.5%, respectively, when compared to the Hologic Panther. A strong correlation between the two assays was evident (r\u0026thinsp;=\u0026thinsp;0.97, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Additionally, precision, as indicated by the coefficient of variation (CV), for high viral load was 1.38% for GeneXpert while for low viral load, it was 2.59%. In the linearity analysis, all data points remained within acceptable ranges, yielding correlation coefficients\u0026thinsp;\u0026ge;\u0026thinsp;0.99 for both assays.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe Xpert HIV-1 viral load assay demonstrates high efficacy in accurately identifying and quantifying HIV-1 viral loads, making it a valuable tool for HIV diagnosis and monitoring. This reliability makes it particularly suitable for critical populations requiring timely and accurate viral load monitoring.\u003c/p\u003e","manuscriptTitle":"Evaluating the Accuracy of HIV-1 Viral Load Testing Using near point of care Xpert HIV- 1 System at Solwezi General Hospital, Zambia.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-07 09:31:44","doi":"10.21203/rs.3.rs-5723442/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-08T19:23:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-17T21:14:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-09T07:07:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"259604188762588736773701248690831177488","date":"2025-04-07T15:42:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"181738848682054677530863646879006989066","date":"2025-04-04T11:46:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-04T10:54:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-04T06:11:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Research Notes","date":"2025-04-03T06:56:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b2db0d8d-9952-48f3-86f0-04e409c2118b","owner":[],"postedDate":"April 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-28T16:00:25+00:00","versionOfRecord":{"articleIdentity":"rs-5723442","link":"https://doi.org/10.1186/s13104-025-07387-w","journal":{"identity":"bmc-research-notes","isVorOnly":false,"title":"BMC Research Notes"},"publishedOn":"2025-07-21 15:57:17","publishedOnDateReadable":"July 21st, 2025"},"versionCreatedAt":"2025-04-07 09:31:44","video":"","vorDoi":"10.1186/s13104-025-07387-w","vorDoiUrl":"https://doi.org/10.1186/s13104-025-07387-w","workflowStages":[]},"version":"v1","identity":"rs-5723442","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5723442","identity":"rs-5723442","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}