Performance and workflow assessment of six nucleic acid extraction technologies for use in resource limited settings

Abstract

Infectious disease nucleic acid amplification technologies (NAAT) have superior sensitivity, specificity, and rapid time to result compared to traditional microbiological methods. Recovery of concentrated, high quality pathogen nucleic acid (NA) from complex specimen matrices is required for optimal performance of several NA amplification/detection technologies such as polymerase chain reaction (PCR). Fully integrated NAAT platforms that enable rapid sample-to-result workflows with minimal user input are generally restricted to larger reference lab settings, and their complexity and cost are prohibitive to widespread implementation in resource limited settings (RLS). Identification of component technologies for incorporation of reliable and affordable sample preparation with pathogen NA amplification/detection into an integrated platform suitable for RLS, is a necessary first step toward achieving the overarching goal of reducing infectious disease-associated morbidity and mortality globally. In the current study, we evaluate the performance of six novel NA extraction technologies from different developers using blinded panels of stool, sputum and blood spiked with variable amounts of quality-controlled DNA- and/or RNA-based microbes. The extraction efficiencies were semi-quantitatively assessed using validated real-time reverse transcription (RT)-PCR assays specific for each microbe and comparing target-specific RT-PCR results to those obtained with reference NA extraction methods. The technologies were ranked based on overall diagnostic accuracy (analytical sensitivity and specificity). Sample input and output volumes, total processing time, user-required manual steps and cost estimates were also examined for suitability in RLS. Together with the performance analysis, these metrics were used to select the more suitable candidate technologies for further optimization of integrated NA amplification and detection technologies for RLS.

Fig 1. A comparison of sample processing times and manual steps to extract NAs from sputum, blood and stool specimens.

Comparison or turnaround time (min) and number of manual steps for extraction methods for developers (A-F) for each specimen type (sputum, blood, and stool). The area surrounded by the dashed line indicates where the more optimal technologies are in terms of processing time and the number of user steps. Note, to prevent obfuscation with clusters of multiple symbols located on a single point, in such instances each symbol is slightly diffused (where applicable) to provide clarity to the reader.

Fig 2

The comparison of each technology (developer A–F) via radar plots using ASSUR. The ASSUR criteria, as defined in the text, was applied for each target microbe used in the three specimen panels of sputum, blood and stool. Each component for ASSUR was normalized from 0.0 to 1.0 where 1.0 represents the most optimal value within each component in the set of data points for each component. The uppermost set of three images are examples to indicate the polar curves generated to indicate analysis via ASSUR as Good (1.0), Intermediate (0.5) and Poor (0.0) respectively. The developers are listed as A–F and with panels for sputum (SP), Blood (BL) and sputum (SP) and plots for each target nucleic acid associated within each panel. When the radar plot is read clockwise the letters read ASSUR; A, affordable; S, sensitivity; S, specificity; U, Number of user steps; R, rapid turnaround time; INF, influenza A; MS2, male specific bacteriophage; MTB, M. tuberculosis; SPN, S. pneumoniae; STM, S. Typhimurium.