Dengue diagnostics: serious inaccuracies are likely to occur if pre-analytical conditions are not strictly followed

Abstract

BACKGROUNDThe heat-labile nature of Dengue virus (DENV) in serum samples must be considered when applying routine diagnostic tests to avoid issues that could impact the accuracy of test results with direct implications for case management and disease reporting.OBJECTIVES To check if pre-analytical variables, such as storage time and temperature, have an impact on the accuracy of the main routine diagnostic tests for dengue.METHODS Virus isolation, reverse transcription real-time polymerase chain reaction (RT-PCR) and NS1 enzyme-linked immunosorbent assay (ELISA) were evaluated using 84 samples submitted to different pre-analytical conditions. FINDINGS Sensitivity and negative predictive value were directly affected by sample storage conditions. RT-PCR and virus isolation showed greater dependence on well-conserved samples for an accurate diagnosis. Interestingly, even storage at -30ºC for a relatively short time (15 days) was not adequate for accurate results using virus isolation and RT-PCR tests. On the other hand, NS1 ELISA showed no significant reduction in positivity for aliquots tested under the same conditions as in the previous tests. MAIN CONCLUSIONSOur results support the stability of the NS1 marker in ELISA diagnosis and indicate that the accuracy of routine tests such as virus isolation and RT-PCR is significantly affected by inadequate transport and storage conditions of serum samples.

Epidemiology, biology, pathogenesis, clinical manifestations, and diagnosis of dengue virus infection, and its trend in Ethiopia: a comprehensive literature review

“…In the early stages of the disease (seven or less than 7 days after onset of illness), DENV infection can be diagnosed from serum, plasma, circulating blood cells, or from other tissues by detecting viral RNA with nucleic acids amplification tests, NS1 protein using some commercial tests and viral isolation in mammalian or mosquito cell culture to further genotyping and lineage for virus characterization [122]. DENV is thermally labile; RNA detection and isolation of the virus are highly dependent on well-preserved specimens for accurate diagnosis results [127]. The samples awaiting shipment to the laboratory should be stored in a refrigerator or freezer.…”

Experience in the validation of a rapid test for NS1 and IgM for early diagnosis during a dengue epidemic in Colombia

“…Tests such as RT-PCR and viral isolation showed a greater dependence on well-preserved samples to make an accurate diagnosis. Their results showed that storage of samples for 15 days at -30°C yielded inaccurate results in RT-PCR and viral isolation tests, while in the same samples the NS1 ELISA tests did not show a significant reduction in positivity (46). In favor of the hypothesis of sample quality deterioration due to transportation, this study shows that the samples that remained at the local level for viral culture processing gave positive results for virus isolation and subsequent genomic sequencing.…”

Dengue: A review of laboratory diagnostics in the vaccine age

“…As an RNA virus, DENV is labile and sensitive to deterioration, which is advantageous in reducing the risk of contamination in laboratory assays, but can reduce the sensitivity of molecular assays if pre-analytical handling is inappropriate [16]. A 37% reduction in specimens testing RT-PCR positive after 48 h storage refrigerated at 2–8 °C was observed, and and 20 % reduction after 15 days storage at −30 °C [39]. Whichever specimen is chosen, appropriate handling is required to optimize yield.…”

“…Specimens are sensitive to pre-analytical handling, which impacts sensitivity. A significant decrease in culture detection of virus from serum was noted with 24 h of refrigerator storage or 15 days of freezing at −30 °C, when compared to immediate processing after retrieval from liquid nitrogen storage [39]. Because of this, many studies now use molecular assays as a gold standard instead.…”