B-215 Evaluating RT-qPCR for Dengue Virus Detection: Outcomes of a Brazilian External Quality Assessment Program

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Abstract Background Dengue virus (DENV), responsible for a prevalent arthropod-borne viral illness, poses a substantial public health challenge worldwide. Clinically, DENV's manifestation often resembles that of other arboviruses, resulting in frequent healthcare visits and complicating the early diagnosis, which is critical for patient management. While virus isolation is a viable method, RT-qPCR assays yield significantly quicker results. Rapid immunochromatographic tests are available; yet, RT-qPCR not only matches their sensitivity but also provides distinct advantages in clinical laboratory settings. These include higher throughput and integrated, traceable quality control within each routine, offering less subjective and more dependable results. Maintaining high diagnostic standards for DENV is imperative. External quality assessment programs (EQAP) play a key role in enhancing laboratory diagnostic capabilities by monitoring and assessing diagnostic methods. This study evaluates the diagnostic efficacy of RT-qPCR for DENV using data from an EQAP conducted by a Brazilian provider, accredited by ABNT NBR ISO/IEC 17043:2011. Methods The quality control samples comprised lyophilized suspensions containing viable DENV particles, grown on vero cells (BCRJ 0245/ATCC CCL-81) under BSL-3 conditions. From February 2017 to November 2022, twenty-four EQAP survey rounds were carried out, each encompassing three samples, culminating in 1,153 data points. The evaluation metrics included participant numbers, adequacy percentage, sensitivity, specificity, and the incidence of false positives and negatives. The Mann-Kendall test assessed trends in these metrics over the survey rounds. The RT-qPCR kits employed by participants were classified either as in-vitro diagnostic (IVD) or as laboratory-developed tests (LDT). Kits that demonstrated high performance in terms of adequacy, sensitivity, and specificity were identified and listed. Results The number of laboratories participating has increased. Starting with four in 2017, the program expanded to 28 by 2022. Laboratory accuracy was high during the evaluation period, with a median of 99% (97.8% in 2017 and 99.2% in 2022). The Mann-Kendall test indicated a significant trend of increased accuracy (p<0.001). Specificity remained stable at 98.8%, while sensitivity improved, with a median of 99.4%—rising from 93.3% in 2017 to 100% in 2022 (p<0.001). Only three false negatives were reported over three separate rounds, and six false positives were reported across four rounds. Both LDT and IVD RT-qPCR kits demonstrated high accuracy (99.4% and 98.4%, respectively). Biomanguinhos ZDC, Viasure ZDC, and ZDC Multiplex kits all showed 100% adequacy, sensitivity, and specificity. Conclusions This study highlights the increasing adoption of RT-qPCR for DENV detection, as evidenced by the growing number of laboratories in the EQAP program and the consistently high adequacy rates from 2017 to 2022. Nonetheless, the number of participating laboratories remains relatively small for a vast tropical country like Brazil. The high sensitivity and specificity, along with the infrequency of false results, underscore its effectiveness. The superior performance of certain kits suggests possible advancements in DENV diagnosis, contributing to global public health initiatives against this arboviral disease.