Amplification of human β‐glucuronidase gene for appraising the accuracy of negative SARS‐CoV‐2 RT‐PCR results in upper respiratory tract specimens

Abstract

Real-time reverse transcription polymerase chain reaction (RT-PCR) is the mainstay of coronavirus disease 2019 (Covid-19) diagnosis.1 Up to 30% of the patients clinically suspected of Covid-19 may have initial or repeat RT-PCR negative results before positive test conversion, most notably when upper respiratory tract (URT) specimens are processed.2-7 False-negative RT-PCR results may hamper the clinical management of patients and hinder the adoption of epidemiological measures to control the pandemic. A number of pre-analytical and analytical factors may impact on the diagnostic efficiency of RT-PCR, including the type of and time to specimen processing, conservation before testing, quality of samples, the timing of sample collection after symptoms onset, or the intrinsic performance of the assay (ie, limit of detection [LOD]).2, 8 A large number of commercially available severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RT-PCR assays targeting one or more SARS-CoV-2 genes has been launched and have reached global widespread use.9 Most of these assays include a spike-in control for RT-PCR amplification, such as MS2 phage RNA genome, but provide no information on specimen cellularity, as no primers targeting human housekeeping genes (ie, RNase P) are usually included in the reaction. The current study was aimed at assessing whether amplification of β-glucuronidase (GUSB) gene would help estimate the accuracy of SARS-CoV-2 RT-PCR negative results in URT samples. As shown in Figure 1, out of 401 patients admitted to our center until 14th April with clinical suspicion of Covid-19, 202 were eventually diagnosed by microbiological means, either by RT-PCR (n = 191) or serological methods (n = 11). The median age of these patients was 65 years (range, 3-98 years); 115 were males and 87 females. A total of 199 patients received a final diagnosis of Covid-19 on clinical, laboratory, and imaging grounds and without microbiological documentation. Of the 202 patients, 34 (16.8%) tested negative by RT-PCR on first URT specimens, collected at a median of 5 days (range, 1-14 days) after the onset of symptoms. In these patients, as per protocol, URT swabs were collected every 24 to 72 hours until RT-PCR positive conversion. Twenty-three patients tested positive by RT-PCR in the second (n = 18) or third (n = 5) URT sample. Diagnosis of Covid-19 was achieved by serological methods in the remaining 11 patients. A total of 47 URT specimens from 21 patients testing positive by RT-PCR in the second or third specimen were subjected to GUSB gene RT-PCR analysis, which was performed in a parallel to viral RT-PCR testing. Twenty-six and 21 out of the 47 specimens yielded negative or positive SARS-CoV-2 RT-PCR results, respectively. To this end, we used the HEQC one-step kit (Seqplexing, Valencia, Spain), a one-step real-time RT-PCR. RNA was extracted from clinical samples using the DSP virus Pathogen Minikit on the QiaSymphony Robot instruments (Qiagen, Valencia, CA), reverse-transcribed to complementary DNA and subsequently amplified in the LightCycler 480 Real-Time PCR System Version II (Roche Diagnostics, Pleasanton). Cy5 fluorescent signal (618-660 nm) revealed amplification of the target gene. URT specimens that tested negative by SARS-CoV-2 RT-PCR displayed higher GUSB RT-PCR cycle thresholds (CT) (P = .070; the Mann-Whitney U test) than those testing positive (median, 30.7; range, 27.0-40.0, and median 29.7; range 25.5-36.8, respectively), thus reflecting poorer cellularity. Receiver operating characteristic (roc) curve analysis (not shown) indicated that a CT threshold of 31.2 discriminated best between positive and negative SARS-CoV-2 RT-PCRs (area under the curve, 0.66; 95% CI, 0.50-0.81; P = .08). This cut-off yielded a true negative ratio of 89% and an accuracy of 70% (Table 1). The current study has several limitations that should be acknowledged. First, a relatively scarce number of specimens were subjected to GUSB gene analysis