Zahreddine Monica, Parra Beatriz, Pierce Laura, de Oliveira Danielle Ferreira, Carabali Mabel, Charland Katia, Abreu Kellyanne, Ridde Valéry, Lima Danielle Malta, Zinszer Kate
Centre de recherche en santé publique, Université de Montréal, Montréal, Québec, Canada.
Microbiology Department, Universidad del Valle, Cali, Colombia.
PLoS Negl Trop Dis. 2025 Feb 28;19(2):e0012880. doi: 10.1371/journal.pntd.0012880. eCollection 2025 Feb.
Dengue virus (DENV) seroprevalence studies often rely on Enzyme-Linked Immunosorbent Assay (ELISA) testing of serum samples, but ELISA testing of dried blood spot (DBS) samples offer several advantages for field-based research in resource-limited settings. However, DBS' limited sample volume can be challenging for test sensitivity, requiring validation studies with standard methods (e.g., analysis of serum through ELISAs or Plaque Reduction Neutralization Tests (PRNTs)). In preparation for a large cluster randomized controlled trial, we conducted a pilot study in 2019 to validate the use of DBS compared to serum samples for DENV IgG testing. We aimed to identify the optimal DBS dilution for IgG detection and to estimate the correlation, magnitude of agreement, and sensitivity and specificity of IgG detection in DBS versus serum samples.
METHODOLOGY/PRINCIPAL FINDINGS: We conducted this pilot validation study among 119 healthy participants in Fortaleza, Brazil to evaluate and optimize the detection of DENV IgG from DBS compared to serum. Each participant provided paired DBS and venous blood samples, which were evaluated for DENV IgG using the Panbio Dengue IgG indirect ELISA. DBS elution diluted 1:4 was optimal compared with serum results, with high correlation (r= 0.98) and near-perfect agreement (kappa = 0.95). At this dilution, DBS had a sensitivity of 100%, a specificity of 92.3%, a 97.9% positive predictive value, and a 100% negative predictive value compared with serum.
CONCLUSIONS/SIGNIFICANCE: These results validate using DBS instead of serum for detection of prior dengue infection among similar populations in endemic regions, without sacrificing test sensitivity and specificity. The validity of using DBS for ELISA to detect prior dengue infection could have important implications for field-based research. A limitation to this study was that the potential for misclassification due to cross-reactivity (e.g., with Zika virus, Yellow Fever vaccine) was not assessed.
登革病毒（DENV）血清流行率研究通常依赖于对血清样本进行酶联免疫吸附测定（ELISA）检测，但对干血斑（DBS）样本进行ELISA检测在资源有限环境下的现场研究中具有多个优势。然而，DBS样本量有限可能对检测灵敏度构成挑战，需要采用标准方法进行验证研究（例如，通过ELISA或蚀斑减少中和试验（PRNT）分析血清）。为筹备一项大型整群随机对照试验，我们于2019年开展了一项试点研究，以验证与血清样本相比，DBS用于DENV IgG检测的情况。我们旨在确定用于IgG检测的最佳DBS稀释度，并估计DBS相对于血清样本中IgG检测的相关性、一致性程度以及灵敏度和特异性。
方法/主要发现：我们在巴西福塔雷萨的119名健康参与者中开展了这项试点验证研究，以评估和优化与血清相比从DBS中检测DENV IgG的情况。每位参与者提供了配对的DBS和静脉血样本，使用Panbio登革热IgG间接ELISA对其进行DENV IgG评估。与血清结果相比，1:4稀释的DBS洗脱液最为理想，具有高度相关性（r = 0.98）和近乎完美的一致性（kappa = 0.95）。在此稀释度下，与血清相比，DBS的灵敏度为100%，特异性为92.3%，阳性预测值为97.9%，阴性预测值为100%。
结论/意义：这些结果验证了在流行地区的类似人群中使用DBS而非血清来检测既往登革热感染，且不牺牲检测灵敏度和特异性。使用DBS进行ELISA检测既往登革热感染的有效性可能对现场研究具有重要意义。本研究的一个局限性在于未评估因交叉反应（例如，与寨卡病毒、黄热病疫苗）导致错误分类的可能性。
