Should WHO-prequalified antigen-antibody fourth-generation rapid diagnostic tests be used to detect acute HIV infection? A systematic review and meta-analysis of diagnostic performance

Should WHO-prequalified antigen-antibody fourth-generation rapid diagnostic tests be used to detect acute HIV infection? A systematic review and meta-analysis of diagnostic performance | medRxiv Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search Should WHO-prequalified antigen-antibody fourth-generation rapid diagnostic tests be used to detect acute HIV infection? A systematic review and meta-analysis of diagnostic performance View ORCID Profile Margot Neveux , View ORCID Profile Runa Hylin , View ORCID Profile Veronica Ruiz Gonzalez , View ORCID Profile Abril Patricia Lopez Parra , View ORCID Profile Ali Onoja , View ORCID Profile Sunee Sirivichayakul , View ORCID Profile Akanmu A Sulaimon , View ORCID Profile Gallican Rwibasira , View ORCID Profile Missiani Ochwoto doi: https://doi.org/10.64898/2026.03.19.26347008 Margot Neveux 1 Independent researcher Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Margot Neveux Runa Hylin 1 Independent researcher Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Runa Hylin Veronica Ruiz Gonzalez 2 Clinica Especializada Condesa , Mexico Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Veronica Ruiz Gonzalez For correspondence: vruizgo{at}gmail.com Abril Patricia Lopez Parra 2 Clinica Especializada Condesa , Mexico Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Abril Patricia Lopez Parra Ali Onoja 3 HIV Testing Services , FMoH, Nigeria Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ali Onoja Sunee Sirivichayakul 4 Chulalongkorn University , Thailand Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sunee Sirivichayakul Akanmu A Sulaimon 5 Department of Haematology and Blood Transfusion, College of Medicine University of Lagos Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Akanmu A Sulaimon Gallican Rwibasira 6 Biomedical Center Division , Rwanda Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gallican Rwibasira Missiani Ochwoto 7 K enya Medical Research Institute (KEMRI) Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Missiani Ochwoto Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract Introduction Diagnostics have become the fundamental backbone of HIV prevention, treatment and long-term retention in care, and are critical to achieving the 95-95-95 UNAIDS targets. To effectively reach underserved and remote populations, diagnostic technologies must be cost-effective, robust, user-friendly and suitable for settings with limited infrastructure. Among available testing modalities, rapid diagnostic tests (RDTs) play a central role in expanding HIV testing coverage. Earlier generations of RDTs were limited by their inability to detect acute HIV, with limited ability to detect p24 antigen (Ag), an early marker of HIV infection, which is expected to shorten the diagnostic window to two-to-three weeks. The introduction of fourth-generation RDTs, which detects both chronic and acute HIV infection through p24 Ag detection, was designed to ensure that the traditional diagnostic window of two-to-three months is shortened to approximately two-to-three weeks. However, integrating these assays into existing testing algorithms requires clear evidence that they meet high standards of quality and performance. This systematic review aims to assess the performance of WHO-prequalified fourth-generation Ag/Ab RDTs. Methods We performed a systematic search across six databases to identify studies evaluating Ag/Ab RDTs against laboratory reference standards in individuals aged 12 years and older, spanning 1 January 2010 to 31 December 2025. Outcomes were limited to measures of diagnostic accuracy. A meta-analysis focusing exclusively on WHO-prequalified fourth-generation RDTs was performed using a bivariate random-effect model. Results 1,932 records were screened, of which 31 diagnostic accuracy studies from 19 countries were included. 15 studies used US-only approved products, 12 used WHO-prequalified products and four used commercially discontinued products. The pooled sensitivity of WHO-prequalified Ag/Ab RDTs for acute HIV infection (AHI) was 94% (95% CI: 86%-99%). An RNA threshold of ≥ 1,000,000 copies/mL was used as a proxy for high viraemia and used as a cut-off for the following analyses. The cut-off based analysis is considered more suited to decision-making, as it focuses on cases most likely to be associated with higher viraemia and greater potential for detection during the p24 Ag window. When using enzyme immunoassay (EIA) as the reference standard, the pooled p24 Ag sensitivity was 76% (95% CI: 62%-88%), and the pooled p24 Ag sensitivity when using nucleic acid amplification test (NAAT) as the reference standard was 75% (95% CI: 41%-97%). In the general population, the pooled sensitivity for p24 antigen detection was 77% (95% CI: 60%-92%). Amongst risk populations, only three studies had available raw data, and the pooled sensitivity was 62% (95% CI: 10%-97%). In plasma and serum specimens, pooled p24 Ag sensitivity was 74% (95% CI: 57%-88). Discussion Collectively, these findings indicate that WHO-prequalified fourth-generation Ag/Ab RDTs can function as a scalable frontline screening tool, particularly in low- and middle-income countries, while offering incremental holistic detection through p24 Ag. Their effective deployment, however, depends on maintaining standard algorithm safeguards, including repeat testing and targeted laboratory referral when acute infection is suspected. Conclusions Results from this meta-analysis support the use of WHO-prequalified fourth-generation Ag/Ab RDTs for general population screening. From a programmatic perspective, the added value of WHO-prequalified fourth-generation RDTs lies in their ability to combine rapid, decentralized access to testing, with incremental yet impactful improvements in holistic detection. Competing Interest Statement The authors have declared no competing interest. Funding Statement Funding for this review was provided by Abbott Laboratories. Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Footnotes Email addresses of authors: Margot Neveux: margotrneveux{at}gmail.com , Runa Hylin: runa.hylin{at}gmail.com , Veronica Ruiz Gonzalez: vruizgo{at}gmail.com , Abril P. Lopez Parra: abril.biol{at}gmail.com , Ali Onoja: onojaali{at}yahoo.com , Sunee Sirivichayakul: Sunee.S{at}chula.ac.th , Akanmu Alani Sulaimon: asulaimona{at}gmail.com , Gallican Rwibasira: gallican.rwibasira{at}rbc.gov.rw , Missiani Ochwoto: omissiani{at}gmail.com Data Availability Introduction: Diagnostics have become the fundamental backbone of HIV prevention, treatment and long-term retention in care, and are critical to achieving the 95-95-95 UNAIDS targets. To effectively reach underserved and remote populations, diagnostic technologies must be cost-effective, robust, user-friendly and suitable for settings with limited infrastructure. Among available testing modalities, rapid diagnostic tests (RDTs) play a central role in expanding HIV testing coverage. Earlier generations of RDTs were limited by their inability to detect acute HIV, with limited ability to detect p24 antigen (Ag), an early marker of HIV infection, which is expected to shorten the diagnostic window to two-to-three weeks. The introduction of fourth-generation RDTs, which detects both chronic and acute HIV infection through p24 Ag detection, was designed to ensure that the traditional diagnostic window of two-to-three months is shortened to approximately two-to-three weeks. However, integrating these assays into existing testing algorithms requires clear evidence that they meet high standards of quality and performance. This systematic review aims to assess the performance of WHO-prequalified fourth-generation Ag/Ab RDTs. Methods: We performed a systematic search across six databases to identify studies evaluating Ag/Ab RDTs against laboratory reference standards in individuals aged 12 years and older, spanning 1 January 2010 to 31 December 2025. Outcomes were limited to measures of diagnostic accuracy. A meta-analysis focusing exclusively on WHO-prequalified fourth-generation RDTs was performed using a bivariate random-effect model. Results: 1,932 records were screened, of which 31 diagnostic accuracy studies from 19 countries were included. 15 studies used US-only approved products, 12 used WHO-prequalified products and four used commercially discontinued products. The pooled sensitivity of WHO-prequalified Ag/Ab RDTs for acute HIV infection (AHI) was 94% (95% CI: 86%-99%). An RNA threshold of ≥ 1,000,000 copies/mL was used as a proxy for high viraemia and used as a cut-off for the following analyses. The cut-off based analysis is considered more suited to decision-making, as it focuses on cases most likely to be associated with higher viraemia and greater potential for detection during the p24 Ag window. When using enzyme immunoassay (EIA) as the reference standard, the pooled p24 Ag sensitivity was 76% (95% CI: 62%-88%), and the pooled p24 Ag sensitivity when using nucleic acid amplification test (NAAT) as the reference standard was 75% (95% CI: 41%-97%). In the general population, the pooled sensitivity for p24 antigen detection was 77% (95% CI: 60%-92%). Amongst risk populations, only three studies had available raw data, and the pooled sensitivity was 62% (95% CI: 10%-97%). In plasma and serum specimens, pooled p24 Ag sensitivity was 74% (95% CI: 57%-88). Discussion: Collectively, these findings indicate that WHO-prequalified fourth-generation Ag/Ab RDTs can function as a scalable frontline screening tool, particularly in low- and middle-income countries, while offering incremental holistic detection through p24 Ag. Their effective deployment, however, depends on maintaining standard algorithm safeguards, including repeat testing and targeted laboratory referral when acute infection is suspected. Conclusions: Results from this meta-analysis support the use of WHO-prequalified fourth-generation Ag/Ab RDTs for general population screening. From a programmatic perspective, the added value of WHO-prequalified fourth-generation RDTs lies in their ability to combine rapid, decentralized access to testing, with incremental yet impactful improvements in holistic detection. Copyright The copyright holder has placed this preprint in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, remix, or adapt this material for any purpose without crediting the original authors. Back to top Previous Next Posted March 19, 2026. Download PDF Supplementary Material Data/Code Email Thank you for your interest in spreading the word about medRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Should WHO-prequalified antigen-antibody fourth-generation rapid diagnostic tests be used to detect acute HIV infection? A systematic review and meta-analysis of diagnostic performance Message Subject (Your Name) has forwarded a page to you from medRxiv Message Body (Your Name) thought you would like to see this page from the medRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Should WHO-prequalified antigen-antibody fourth-generation rapid diagnostic tests be used to detect acute HIV infection? A systematic review and meta-analysis of diagnostic performance Margot Neveux , Runa Hylin , Veronica Ruiz Gonzalez , Abril Patricia Lopez Parra , Ali Onoja , Sunee Sirivichayakul , Akanmu A Sulaimon , Gallican Rwibasira , Missiani Ochwoto medRxiv 2026.03.19.26347008; doi: https://doi.org/10.64898/2026.03.19.26347008 Share This Article: Copy Citation Tools Should WHO-prequalified antigen-antibody fourth-generation rapid diagnostic tests be used to detect acute HIV infection? A systematic review and meta-analysis of diagnostic performance Margot Neveux , Runa Hylin , Veronica Ruiz Gonzalez , Abril Patricia Lopez Parra , Ali Onoja , Sunee Sirivichayakul , Akanmu A Sulaimon , Gallican Rwibasira , Missiani Ochwoto medRxiv 2026.03.19.26347008; doi: https://doi.org/10.64898/2026.03.19.26347008 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area HIV/AIDS Subject Areas All Articles Addiction Medicine (638) Allergy and Immunology (918) Anesthesia (351) Cardiovascular Medicine (5007) Dentistry and Oral Medicine (496) Dermatology (428) Emergency Medicine (676) Endocrinology (including Diabetes Mellitus and Metabolic Disease) (1693) Epidemiology (16267) Forensic Medicine (36) Gastroenterology (1241) Genetic and Genomic Medicine (7280) Geriatric Medicine (751) Health Economics (1093) Health Informatics (5291) Health Policy (1467) Health Systems and Quality Improvement (1832) Hematology (599) HIV/AIDS (1382) Infectious Diseases (except HIV/AIDS) (16492) Intensive Care and Critical Care Medicine (1203) Medical Education (700) Medical Ethics (156) Nephrology (752) Neurology (7598) Nursing (378) Nutrition (1130) Obstetrics and Gynecology (1296) Occupational and Environmental Health (1040) Oncology (3746) Ophthalmology (1093) Orthopedics (409) Otolaryngology (465) Pain Medicine (501) Palliative Medicine (143) Pathology (721) Pediatrics (1858) Pharmacology and Therapeutics (756) Primary Care Research (786) Psychiatry and Clinical Psychology (6133) Public and Global Health (9994) Radiology and Imaging (2532) Rehabilitation Medicine and Physical Therapy (1520) Respiratory Medicine (1270) Rheumatology (661) Sexual and Reproductive Health (804) Sports Medicine (597) Surgery (803) Toxicology (109) Transplantation (319) Urology (308)

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