Using Rapid Diagnostic Tests as a Source of Viral RNA for Dengue Serotyping by RT-PCR - A Novel Epidemiological Tool

Abstract

BackgroundDengue virus infection causes major public health problems in tropical and subtropical areas. In many endemic areas, including the Lao PDR, inadequate access to laboratory facilities is a major obstacle to surveillance and study of dengue epidemiology. Filter paper is widely used for blood collection for subsequent laboratory testing for antibody and nucleic acid detection. For the first time, we demonstrate that dengue viral RNA can be extracted from dengue rapid diagnostic tests (RDT) and then submitted to real-time RT-PCR for serotyping.Methodology/Principal FindingsWe evaluated the Standard Diagnostics (SD) Bioline Dengue Duo RDT, a commonly used test in dengue endemic areas. First, using the QIAamp RNA kit, dengue RNA was purified from the sample pad of the NS1 RDT loaded with virus isolates of the four serotypes, then quantified by RT-PCR. We observed greater recovery of virus, with a mean of 27 times more RNA recovered from RDT, than from filter paper. Second, we evaluated dengue NS1 RDTs from patients at Mahosot Hospital, Vientiane, (99 patients) and from rural Salavan Provincial Hospital (362 patients). There was good agreement between dengue RT-PCR from NS1 RDT with RT-PCR performed on RNA extracted from patient sera, either using RDT loaded with blood (82.8% and 91.4%, in Vientiane and Salavan, respectively) or serum (91.9% and 93.9%). There was 100% concordance between RDT and serum RT-PCR of infecting dengue serotype.Conclusions/SignificanceTherefore, the collection of NS1 positive RDTs, which do not require cold storage, may be a novel approach for dengue serotyping by RT-PCR and offers promising prospects for the collection of epidemiological data from previously inaccessible tropical areas to aid surveillance and public health interventions.

Isolation and molecular characterization of dengue virus clinical isolates from pediatric patients in New Delhi

“…Our report confirms previous observations where viral detection was reported to be more efficient with whole blood as compared to plasma (Klungthong et al, 2007). Dengue serotyping using rapid diagnostic tests have also demonstrated the convenience and utility of using whole blood for isolation of viral RNA (Vongsouvath et al, 2016). Another study which used cellular components of whole blood, serum, clot specimens and plasma from blood bank donors who were DENV RNA positive also demonstrated that the detection of viral RNA by RT-PCR was more consistent with cellular components of blood (Añez et al, 2016).…”

Rapid Diagnostic Tests as a Source of Dengue Virus RNA for Envelope Gene Amplification: A Proof of Concept

“…In addition, these specialized laboratories are all located in the capital, Vientiane, and there are limited cold chain facilities to transport samples from rural areas. 15 In those areas, the use of immunochromatographic rapid diagnostic tests (RDTs) for dengue diagnosis has recently expanded. Therefore, as a potential solution, Vongsouvath and others showed that RDTs, which are transportable at room temperature, can be used as a source for DENV RNA for small-fragment amplification using real-time reverse transcription polymerase chain reaction (RT-qPCR).…”

“…Therefore, as a potential solution, Vongsouvath and others showed that RDTs, which are transportable at room temperature, can be used as a source for DENV RNA for small-fragment amplification using real-time reverse transcription polymerase chain reaction (RT-qPCR). 15–17…”

“…As previously described, 100 μL of the viral dilutions or patient samples was loaded on the NS1 side of the Standard Diagnostics BIOLINE Dengue Duo RDT (Standard Diagnostics, Gyeonggi-do, Republic of Korea). 15 Subsequently, the completely dry sample pad was cut out and submitted for RNA purification using QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to previously published procedure. 15 As reference, 140 μL of each sample was also directly extracted using QIAamp Viral RNA Mini Kit following the manufacturer’s instructions.…”

“…Other factors limiting the efficiency of the technique include a potential loss of RNA when extracting from RDTs versus direct extraction, and the storage duration of the samples used. 15 The samples were collected 7 years before this study and stored at −80°C; therefore, RNA degradation over time is likely to have affected amplification and sequencing results. Hence, testing the procedure with prospectively collected patient samples is required to determine if better results can be obtained.…”