key: cord-0794304-p6f436ot
authors: Adams, Nicholas M.; Leelawong, Mindy; Benton, Alison; Quinn, Criziel; Haselton, Frederick R.; Schmitz, Jonathan E.
title: COVID‐19 diagnostics for resource‐limited settings: Evaluation of “unextracted” qRT‐PCR
date: 2020-07-28
journal: J Med Virol
DOI: 10.1002/jmv.26328
sha: ec12056191b32c3676eeb2648b11e4e8fbebcf62
doc_id: 794304
cord_uid: p6f436ot

The coronavirus disease 2019 (COVID‐19) pandemic has created a precipitous increase in the need for molecular diagnostics. Unfortunately, access to RNA extraction reagents can represent a bottleneck for quantitative real‐time reverse transcriptase‐polymerase chain reaction (qRT‐PCR)‐based methodologies, stemming from both extraordinary supply‐chain stresses and the global reach of the virus into resource‐limited settings. To provide flexible diagnostic options for such environments, we report here an “unextracted modification” for qRT‐PCR using the Centers for Disease Control's (CDC's) widely utilized primers/probe sets for severe acute respiratory syndrome coronavirus 2 (N1/N2/N3 targeting viral nucleocapsid and RP‐control targeting human RNase P). This approach replaces RNA extraction/purification with a heat‐inactivation step of viral transport media (VTM), followed by direct inoculation—with or without VTM spin concentration—into PCR master mixes. Using derivatives of care from our clinical workflow, we compared traditional and unextracted CDC methodologies. Although some decrease in analytic sensitivity was evident (by higher C (t) values) without extraction, in particular for the N2 primer/probe‐set, we observed high categorical positive agreement between extracted and unextracted results for N1 (unconcentrated VTM‐38/40; concentrated VTM‐39/41), N3 (unconcentrated VTM‐38/40; concentrated VTM‐41/41), and RP (unconcentrated and concentrated VTM‐81/81). The negative categorical agreement for N1/N2/N3 was likewise high. Overall, these results suggest that laboratories could adapt and validate unextracted qRT‐PCR protocols as a contingency to overcome supply limitations, with minimal impact on categorical results.

individual components to formulate their own assays. Nationally and internationally, diagnostic demand has outstripped supply and contributed to variable availability of local testing. 4 A number of emergent COVID-19 diagnostics are based upon realtime reverse transcriptase-polymerase chain reaction (qRT-PCR). 5 While potentially quantitative, these assays have been deployed predominantly in a qualitative/categorical manner, with a cycle-threshold value (C t ) indicating the presence of the virus. 6 qRT-PCR allows laboratories to flexibly customize primers and probes with other reagents and instruments, validating their local combination for acceptable clinical performance and to meet regulatory requirements. 7 One common approach for SARS-CoV-2 has involved TaqMan-based assays, with nucleocapsidtargeting oligonucleotides developed by the Centers for Disease Control (CDC). 6 These primers/probes have been implemented with various reagents/instruments, including a number of combinations with FDA Emergency Use Authorization in the United States. 8 Initially promulgated with three locus-targets (N1, N2, N3), this "CDC-approach" has also been adapted by many labs to exclude N3, due to potential cross-reactivity with other betacoronaviruses. 3 To implement this (or any) qRT-PCR assay for SARS-CoV-2, viral RNA is first typically extracted from respiratory samples, often swabderived specimens in liquid viral transport media (VTM). 9 This extraction step serves two overarching purposes: (a) lysing viral particles to free genomic RNA and (b) removing potential PCR inhibitors. Various commercial products are available for this purpose, including automated and manual methods, although these reagents have experienced a massive surge in demand with widespread shortfalls in recent availability. 4 A potential solution to this bottleneck could direct inoculation of respiratory specimens into the qRT-PCR master mix, as has been recently proposed for COVID-19. 10 In theory, heat denaturation (before reverse transcription) could provide the necessary degree of virion denaturation and RNA access, while thoroughly inactivating the viable virus. 11 While this strategy does not attempt to remove inhibitors, similar methods have proven successful for other pathogens and specimen types. [12] [13] [14] In this context, we sought to evaluate this approach for the CDC primers/probes for SARS-CoV-2-both with and without additional viral preconcentration -to provide a contingency against supply-chain insecurities. We describe these results here, which may prove valuable to laboratories with resource limitations, either at baseline or due to the pandemic. To assess positive agreement for each experiment, we utilized all specimens from our clinical workflow with a "detected" result over a ∼24 hour period. At the time, COVID-19 testing at our institution covered exclusively symptomatic individuals from both outpatient and hospital/ emergency department settings (ie, no asymptomatic screens or tests-ofcure). To assess negative agreement, we employed a commensurate number of "not detected" specimens from clinical testing, randomly selected from the same times. Different specimen cohorts (positive and negative) were employed for experiments with unconcentrated and concentrated VTM. N1 (95%), N3 (95%), and RP (100%), although increased pairwise C t values were still observed, unextracted versus extracted (ΔC t = 5.2 ± 4.0, 5.2 ± 3.5, and 6.9 ± 3.3, respectively). C t discrepancies were even more pronounced for N2 (>15 ΔC t for 37/40 specimens), with a low resultant positive agreement (33%, 13/40). False positives in the unextracted/unconcentrated samples occurred at a low frequency (2/41 for N1, 0/41 for N2, and 1/41 for N3). Figure 1A summarizes, for each primer/probe, the range of C t values for extracted specimens that were also detected when unextracted, along with all discordant examples. Not surprisingly, discordant results for N1/N3 reflect speci- 

In summary, the current study addresses a potential (and very practical) solution for a challenging workflow scenario-extraction reagent shortages-confronting many clinical laboratories during the COVID-19 pandemic. Although we observed a decrease in analytic sensitivity for all CDC primer/probe sets with unextracted VTM, indicated by higher pairwise C t values, categorical agreement between unextracted and extracted specimens was high for N1, N3, and RP, both with and without VTM concentration. These findings reflect the assay's qualitative nature-with any amplification categorized as "detected"-along with the high viral burden within most positive specimens (ie, far above the assay's limit of detection). As a result, the assay tolerated a loss of analytic sensitivity without a commensurate loss of diagnostic sensitivity. The N2 primer/probe-set represents a notable exception, as the prominent loss of sensitivity also compromised categorical results. In additional experiments (not shown), we determined that N2-inhibition was due to the T A B L E 1 Categorical and C t value agreement between extracted/unextracted specimens also includes the pairwise C t -value difference between extracted and unextracted specimens (ΔC t = C t-unextracted − C t-extracted , mean ± standard deviation). Abbreviations: CDC, Centers for Disease Control; CI, confidence interval; C t , cycle-threshold value; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; VTM, viral transport media. a A standard deviation could not be meaningfully calculated for N2-unextracted/unconcentrated, given the preponderance of specimens generated an undetected (>45) C t value. *Negative percent agreement for the RP primer/probe-set was not applicable (NA), as these SARS-CoV-2-negative specimens still appropriately demonstrate positivity for the RP internal control.

salt-solution base of the VTM itself. The addition of either HBSS (without Mg/Ca/phenol red) or saline alone to master mix drastically inhibited N2-amplification of defined positive specimens, but only marginally impacted N1/N3/RP. Accordingly, the reduced N2inhibition for the unextracted but concentrated VTM may be attributable (at least in part) to the aqueous dilution of VTM after spin concentration (see Section 2). It remains unclear why N2, among the CDC primer/probe sets, was particularly compromised by these salt conditions, although we hypothesize that it may due to its run of five consecutive cytosine residues. 4 Overall, this study suggests that laboratories might employ un- With either strategy, false-negative results are expected for occasional specimens with low viral burdens, near the limit-of-detection of the unmodified assay (the reason why our observed positive agreement was not uniformly 100%). The exact proportion of such low-abundance specimens within a laboratory's workflow could vary with the clinical circumstances of local testing-that is, inpatient or outpatient, symptomatic evaluation, or asymptomatic screening, test-of-diagnosis or test-of-cure. In implementing an "unextracted protocol," a laboratory would need to consider these factors as part of their preimplementation validation, as well as any other variations in VTM formulation, primers/probes (CDC-developed or otherwise), reagents, and instrumentation. Again, we must note that the specimens evaluated here were in the context of initial diagnostic testing of symptomatic individuals. We could see the approach being particularly unsuitable for test-of-cure scenarios where molecular positivity can linger with high C t values, although this practice (in general) is increasingly coming under scrutiny due to the questionable clinical F I G U R E 1 C t range of extracted VTM specimens with a positive unextracted agreement. These box-and-whisker plots summarize-for each primer/probe-set (N1/N2/N3)-the range of extracted C t values (vertical axis) for SARS-CoV-2-positive specimens that were also detected by qRT-PCR when unextracted. Pairwise data are shown for (a) extracted VTM versus unextracted/unconcentrated VTM and (b) extracted VTM versus unextracted/concentrated VTM. Also depicted (X's) are the individual extracted C t values for SARS-CoV-2positive specimens where the corresponding unextracted specimen was not detected. C t , cycle-threshold value; qRT-PCR, quantitative realtime reverse transcriptase-polymerase chain reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; VTM, viral transport media significance of such results vis-à-vis infectivity. 16 Overall, our data suggest that high categorical agreement can be achieved under realworld scenarios where management is impacted.

In conclusion, the myriad challenges of the COVID-19 pandemic include widespread limitations in laboratory supplies, including RNA extraction reagents and kits. Performing qRT-PCR using unextracted VTM, concentrated or unconcentrated, may serve as a contingency for resource-limited settings around the globe. Going forward, we could envision such places benefiting from unextracted PCR strategies (for COVID-19 or more broadly), even after supply-chain limitations return to baseline in resource-abundant locations.

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The authors would like to acknowledge the thoughtful input of 

The authors declare that there are no conflict of interests.

NMA, ML, and FRH made the initial observations that lead to the study. NMA, ML, AB, CQ, FRH, and JES designed the study. NMA, ML, and AB conducted the study. CQ and JES oversaw the study. JES performed statistical analysis. NMA, ML, and JES wrote the manuscript.

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

http://orcid.org/0000-0002-8341-5871