key: cord-0979905-m16frw8i
authors: Gniazdowski, Victoria; Morris, C Paul; Wohl, Shirlee; Mehoke, Thomas; Ramakrishnan, Srividya; Thielen, Peter; Powell, Harrison; Smith, Brendan; Armstrong, Derek T; Herrera, Monica; Reifsnyder, Carolyn; Sevdali, Maria; Carroll, Karen C; Pekosz, Andrew; Mostafa, Heba H
title: Repeat COVID-19 Molecular Testing: Correlation of SARS-CoV-2 Culture with Molecular Assays and Cycle Thresholds
date: 2020-10-27
journal: Clin Infect Dis
DOI: 10.1093/cid/ciaa1616
sha: 01cf230a8660117dcea9a431ad1cf67f62181abf
doc_id: 979905
cord_uid: m16frw8i

BACKGROUND: Repeat COVID-19 molecular testing can lead to positive test results after negative tests and to multiple positive test results over time. The association between positive tests and infectious virus is important to quantify. METHODS: A two months cohort of retrospective data and consecutively collected specimens from COVID-19 patients or patients under investigation were used to understand the correlation between prolonged viral RNA positive test results, cycle threshold (Ct) values and growth of SARS-CoV-2 in cell culture. Whole genome sequencing was used to confirm virus genotype in patients with prolonged viral RNA detection. Droplet digital PCR (ddPCR) was used to assess the rate of false negative COVID-19 diagnostic tests. RESULTS: In two months, 29,686 specimens were tested and 2,194 patients received repeated testing. Virus recovery in cell culture was noted in specimens with SARS-CoV-2 target genes’ Ct value average of 18.8 ± 3.4. Prolonged viral RNA shedding was associated with positive virus growth in culture in specimens collected up to 20 days after the first positive result but mostly in individuals symptomatic at time of sample collection. Whole genome sequencing provided evidence the same virus was carried over time. Positive tests following negative tests had Ct values higher than 29.5 and were not associated with virus culture. ddPCR was positive in 5.6% of negative specimens collected from COVID-19 confirmed or clinically suspected patients. CONCLUSIONS: Low Ct values in SARS-CoV-2 diagnostic tests were associated with virus growth in cell culture. Symptomatic patients with prolonged viral RNA shedding can also be infectious.

Molecular methods for SARS-CoV-2 nucleic acid detection from nasopharyngeal swabs have been the gold standard for COVID-19 diagnosis. Although diagnostic approaches target different genes within the SARS-CoV-2 genome, they have shown comparable analytical sensitivity and high specificity (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) . Accuracy of the assay's result is associated with the shedding pattern of SARS-CoV-2 RNA, and can vary based on the source of respiratory specimen, sufficiency of specimen collection, and the course of illness (20) (21) (22) (23) (24) (25) .

Infection control personnel and physicians managing COVID-19 patients and patients under investigation (PUI) continue to face several diagnostic dilemmas related to a lack of understanding of the clinical sensitivities of SARS-CoV-2 molecular diagnostics and the correlation between viral RNA detection and shedding of infectious virus. Retesting of patients has become a common practice especially when there is a strong clinical suspicion or exposure history and there is an initial negative result (26) . A single positive molecular result should be sufficient for confirming COVID-19 diagnosis, however, repeated testing of hospitalized patients for determining isolation needs and infection control measures has become a part of managing this patient population. Two negative molecular assay results from two consecutively collected respiratory specimens more than 24 hours apart has been the strategy used in the USA initially for discontinuation of transmission precautions and returning to work (27) . Repeat testing on patients has revealed that SARS-CoV-2 RNA can be detectable for weeks after the onset of symptoms (28) . In general, molecular detection of SARS-CoV-2 RNA does not necessarily denote the presence of recoverable infectious virus.

A few studies, as well as data from the CDC, showed that higher viral loads are associated with recovery of infectious virus and that virus recovery is generally not reported after 9 days from symptom onset (22, 29, 30) . A case study, in which severe infection was associated with successful recovery of infectious SARS-CoV-2 from stool indicates that the duration of recovery of infectious virus particles might vary based on the severity of the disease or the duration of symptoms (31) . A careful interpretation of cell culture results is essential as A c c e p t e d M a n u s c r i p t 6 variables that include cell lines used for viral isolation, days cell cultures were held, among other technical factors might contribute to the success or failure of recovery of virus from clinical specimens.

False negative molecular SARS-CoV-2 results occur and in some cases a single negative result is not sufficient for excluding COVID-19 diagnosis. False negative rates are estimated to range from 5 to 40%, yet a conclusive percentage is currently difficult to determine due to the lack of a diagnostic comparator gold standard (32, 33) . Initial false negative results in the setting of consistent respiratory symptoms have been reported, with some patients having subsequent positive results on serial testing (34) . The Infectious Diseases Society of America (IDSA) recommends repeated testing after initial negative RNA testing in cases with intermediate to high suspicion of COVID-19, but evidence that this practice positively affects outcomes is still lacking (35) . Clinical sensitivity has also been attributed to the specimen type collected and the time of collection in relation to the duration of symptoms (36) (37) (38) (39) (40) (41) (42) (43) (44) (45) (46) .

In this study, we analyzed the molecular diagnostics data from Johns Hopkins Hospital in the time frame March 11 th to May 11 th 2020. Our study aimed to dissect different diagnostic dilemmas by incorporating statistics of repeat testing, cycle threshold values, virus isolation in cell culture, whole genome sequencing, and ddPCR. We address questions that include: 1) How does a positive molecular test correlate with growth in cell culture? 2) Are patients with prolonged viral RNA shedding also shedding infectious virus? 3) Are there changes in viral sequences during prolonged shedding? 4) Does a positive test result following undetectable viral RNA correlate with virus recovery in cell culture? 5) Can false negative results due to an assay's analytical limitation (limit of detection) be detected by ddPCR?

A c c e p t e d M a n u s c r i p t 7

This study was performed in the Molecular Virology Laboratory, Johns Hopkins Hospital. Cell culture studies were conducted at the Johns Hopkins Bloomberg School of Public Health.

The study was approved by the Johns Hopkins University School of Medicine Institutional Review Board. Specimen handling for clinical diagnostic assays were performed in a BSL-3 level laboratory or a BSL-2 laboratory with BSL-3 level personal protective equipment including either a PAPR or N95 mask with face shield. Cell culture experiments were performed in a BSL-3 level laboratory using procedures approved by the Institutional Biosafety Committee.

Repeat testing was identified by pulling the data of all molecular COVID-19 testing that was conducted in the Johns Hopkins Hospital Microbiology laboratory from March 11 th to May 11 th 2019. Data were pulled using the laboratory information system (SOFT). Specimens used were remnant nasopharyngeal swab specimens collected in viral transport media (commercially purchased or custom made at Johns Hopkins University based on the CDC recipe (47) ) available at the completion of standard of care testing at the Johns Hopkins Laboratory. Standard of care testing was performed within less than 24 hours after specimens' receipt in the laboratory and they were refrigerated meanwhile. Left-over original specimens as well as nucleic acid extracts were frozen at -70°C. Cell culture and ddPCR were performed after a single freeze thaw cycle. During the time frame reported, several molecular diagnostic assays for SARS-CoV-2 were used including primarily The RealStar® comparable Ct values for the two genes (19) . Specimens were selected for further testing that include cell culture, sequencing, and ddPCR largely based on the availability of left-over clinical specimens or nucleic acid extracts. Clinical data were extracted by manual chart reviews. Positive serology results were extracted from patients charts and the methodology used for serology at Johns Hopkins Hospital was described in details previously (52) .

Nucleic acid extractions for the RealStar® SARS-CoV-2, the ddPCR assays, and Nanopore whole genome sequencing were performed as previously described in (3). The NucliSENS easyMag or eMAG instruments (bioMérieux, Marcy-l'Étoile, France) were used using software version 2.1.0.1. This extraction method was validated for our clinical diagnostic assays due to constraints of safety and throughput if compared to the manual approaches.

The input specimens' volumes were 500 µL and the final elution volume was 50 µL.

Specimens for automated systems were processed following each assay's FDA-EUA package insert.

VeroE6 cells (ATCC CRL-1586) were cultured at 37°C with 5% carbon dioxide in a humidified chamber using complete medium (CM) consisting of Dulbecco's modified Eagle Medium (Sigma Life Sciences #D5796) supplemented with 10% fetal bovine serum (Gibco, sterile filtered), 1mM glutamine (Invitrogen), 1mM sodium pyruvate (Invitrogen), 100µg/mL penicillin (Invitrogen) and 100 µg/mL streptomycin (Invitrogen). Cells were plated in 24 well A c c e p t e d M a n u s c r i p t 9 dishes and grown to 75% confluence. The use of a 24 well plates allowed for more convenient isolation of larger numbers of clinical samples. The CM was removed and replaced with 150 µL of infection media (IM) which is identical to CM but with the fetal bovine serum reduced to 2.5%. Fifty to one hundred µL of the clinical specimen was added to one well and the cells incubated at 37°C for one hour. The inoculum was aspirated and replaced with 0.5 ml IM and the cells cultured at 37°C for 4 days. Inoculum removal minimized nonspecific cytopathic effect associated with culture with VTM containing media. When cytopathic effect was visible in most of the cells, the IM was harvested and stored at -70°C.

Pilot experiments using 10 infectious units of SARS-CoV-2/USA-WA-1/2020 inoculated into one well of a 24 well plate routinely showed nearly complete cytopathic effect within 4 days of culture. The presence of SARS-CoV-2 was verified by one of two ways. SARS-CoV-2 viral RNA was extracted using the Qiagen Viral RNA extraction kit (Qiagen) and viral RNA detected using quantitative, reverse transcriptase PCR (qPCR) as described (53) .

Alternatively, SARS-CoV-2 viral antigen was detected by infecting VeroE6 cells grown on 4 chamber LabTek slides (Sigma Aldrich) with 50 µL of the VeroE6 virus isolate diluted in 150 µL of IM for 1 hour at 37°C. The inoculum was replaced with IM and the culture incubated at 37°C for 12-18 hours. The cultures were fixed with 4% paraformaldehyde for 20 minutes at room temperature and processed for indirect immunofluorescence microscopy as described (54) . The humanized monoclonal antibody D-006 (Sino Biological) was used as the primary antibody to detect Spike or S protein, followed by Alexa Fluor 488-conjugated goat antihuman IgG. The cells were mounted on Prolong antifade and imaged at 40X on a Zeiss Axio Imager M2 wide-field fluorescence microscope (55) .

Whole genome sequencing was conducted using the Oxford Nanopore platform following the ARTIC protocol for SARS-CoV-2 sequencing with the V3 primer set (56). Eleven indexed samples (and one negative control) were pooled for each sequencing run and 20 ng of the final pooled library was run on the Oxford Nanopore GridION instrument with R9.4.1 A c c e p t e d M a n u s c r i p t 10 flowcells. Basecalling and demultiplexing was performed with Guppy v3.5.2 and reads were assembled using a custom pipeline modified from the ARTIC network bioinformatics pipeline (https://artic.network/ncov-2019). As part of this custom pipeline, reads were mapped to a SARS-CoV-2 reference genome (GenBank MN908947.3) using minimap2 (57) . Coverage was normalized across the genome and variant calling was performed with Nanopolish v0.13.2 (58) . Sites with low coverage (based on the negative control coverage) were masked as 'N'. Variant calls were also independently validated with two other variant callersmedaka (https://nanoporetech.github.io/medaka/snp.html) and samtools( https://wikis.utexas.edu/display/bioiteam/Variant+calling+using+SAMtools)-and all sites with disagreements or allele frequency <75% were manually inspected in Integrated Genome Viewer (59) . Sites with minor allele frequency 25-75% were replaced with IUPAC ambiguity codes. Details about our SARS-CoV-2 sequencing protocols and analysis pipeline validation are available in (52) .

The ddPCR procedure followed the assay's EUA package insert (60) . Briefly, RNA isolated from NP specimens (5. Statistical analysis:

Paired t test was used to determine mean difference in Ct values between groups that showed successful virus growth on cell culture versus no growth. patients had an initial positive result that was followed by a negative test ( figure 1A and B).

Our data indicate that of all the patients that had repeat testing, 81.5% continued to have negative results, 5.7% had an initial negative followed by a repeat positive test, and 6.8% had a final negative test result after an initial positive ( figure 1B) . Figure 1C Table 2 ). Of note, two different isolates collected from patient #14 in the same day were included in this analysis for validating our sequencing reproducibility. 

The molecular detection of SARS-CoV-2 genome has been valuable not only in diagnosis, but also in making infection control related decisions . Several outcomes were observed with repeat molecular testing including: i) prolonged viral RNA shedding, ii) alternating negative and positive results, and iii) false negative results. Our data show that prolonged positivity could be associated with growth of virus in cell culture especially when symptoms persist.

Our data also show that RNA positive specimens after a negative result are not associated with viral culture growth. In general, we believe that our data support the current CDC guidelines updated to discourage the use of testing-based methods for return to work. In addition, our data indicate that although Ct values might be used to indicate if a patient is likely infectious, caution is warranted as higher Ct values were occasionally associated with viral growth on cell culture.

The ddPCR assay detected a few positives at very low viral loads that were missed by our standard of care testing in the subset of patients who were highly suspected of infection.

Overall, our data confirm that SARS-CoV-2 RNA is detectable for a prolonged time and

show that the standard of care molecular diagnostics' analytical sensitivities are largely influenced by variables other than the assay's performance.

The use of a diagnostic test's Ct values as an indicator of the presence of infectious virus has been proposed. One report suggested that a Ct value above 33-34 is not associated with cell culture viral recovery (61) and another report concluded that cell culture infectivity is observed when the Ct values were below 24 and within 8 days from symptoms onset (29) .

Our data show that the average Ct value that was associated with cell culture growth is 18.8. due to the lack of sufficient clinical outcome studies. This is especially important due to variabilities with specimen collection, assays used for diagnosis, the lack of a standardized quantification assays, and inconsistencies in cell culture protocols between different laboratories.

A significant number of our cultured specimens that yielded no infectious virus had low Ct values (28.6% Ct < 23, figure 1) indicating that variables other than the viral genome copies play a role in isolating infectious virus on cell culture. The integrity of the viral genome and variables related to sampling and storage of specimens have been proposed to impact virus recovery in cell culture (63) . Virus particles may be bound to neutralizing antibodies and therefore unable to initiate infection (64) . Generally, prolonged shedding of viral RNA was previously noted for many other viruses, including SARS-CoV, MERS-CoV, influenza, and measles viruses (65) (66) (67) (68) (69) . Subgenomic SARS-CoV-2 RNA was assessed as compared to cell culture as a surrogate of infectiousness with good agreement (22, 70) . Validating this approach will be valuable as it might overcome variabilities recovering the virus in cell culture.

Positive molecular results after negative tests were noticed in patients with COVID-19 and it is not certain if that indicates a relapsed infection or reinfection. Our data showed that RNA detection after RNA negative tests were not associated with positive viral growth in cell culture. It is likely that detectable viral RNA in convalescence is associated with prolonged viral RNA shedding especially since the viral loads are usually lower than that detectable during the early stages of infection. In addition, positive test results after negative molecular RNA tests that are associated with new symptoms are more perplexing, and reinfection has not been ruled out. Our ddPCR data also show that some of these negatives are associated with viral loads below the assays analytical sensitivities. Comprehensive studies that A c c e p t e d M a n u s c r i p t 16 combine understanding the development of protective immunity and compare isolated viral genomes will help understanding the enigma of reinfection by SARS-CoV-2. We previously showed that different viral clades circulate in the Baltimore/ DC metropolitan area (52) .

Dissecting the differences between viral clades in the efficiency for growth in cell culture, development of immune responses, and prolonged shedding or reinfection is under investigation by our group.

Compared to the standard diagnostic molecular techniques, ddPCR offers an absolute quantification of targets after partitioning the specimen into thousands of droplets which increases the accuracy of detection (71) (72) (73) . DdPCR showed a slightly higher sensitivity in detecting SARS-CoV-2 RNA in a subset of specimens from patients with high suspicion of COVID-19 and negative RT-PCR. Our data is consistent with published reports that compared ddPCR with RT-PCR (37, 74) It is important to note that the analytical sensitivity of the ddPCR assay as reported by the EUA package insert (645 copies/ mL) is comparable to standard of care RT-PCR methods used for diagnosis including the CDC panel assay among others (3) . All the positives detected by the ddPCR assay in this study were below the ddPCR assay's analytical limit of detection which explains a few conflicting results when a few specimens were repeated ( Figure 5 ). The Bio-Rad ddPCR assay uses primers and probes that are same as reported by the CDC assay including the human RNase P gene as an internal control. Including this control is very valuable to exclude insufficient sampling as a cause of false negative results (75) . Only a few samples that tested negative by the standard PCR methodologies were later positive by ddPCR (5.6%), even in a cohort with a high suspicion of COVID-19. Overall, this suggests that false negative results in some cases are secondary to low viral loads likely associated with temporal aspects of viral shedding.

Our study indicates that prolonged viral RNA shedding is associated with growth of the virus in cell culture in a subset of patients and seems to correlate with persistence of symptoms.

Higher Ct values and positive RNA tests detected after viral RNA clearance were not A c c e p t e d M a n u s c r i p t 30 Figure 5 

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Correlation of Chest CT and RT-PCR Testing in Coronavirus Disease 2019 (COVID-19) in China: A Report of 1014 Cases

Comparison of Unsupervised Home Self-collected Midnasal Swabs With Clinician-Collected Nasopharyngeal Swabs for Detection of SARS-CoV-2 Infection

Swabs Collected by Patients or Health Care Workers for SARS-CoV-2 Testing

Utility of retesting for diagnosis of SARS-CoV-2/COVID-19 in hospitalized patients: Impact of the interval between tests

Discontinuation of Transmission-Based Precautions and Disposition of Patients with COVID-19 in Healthcare Settings (Interim Guidance)

Temporal dynamics in viral shedding and transmissibility of COVID-19

Predicting infectious SARS-CoV-2 from diagnostic samples

Prolonged virus shedding even after seroconversion in a patient with COVID-19

Infectious SARS-CoV-2 in Feces of Patient with Severe COVID-19

COVID-19 diagnostics in context

Occurrence and Timing of Subsequent SARS-CoV-2 RT-PCR Positivity Among Initially Negative Patients

Sensitivity of Chest CT for COVID-19: Comparison to RT-PCR

Guidelines on the Diagnosis of COVID-19

Detection of SARS-CoV-2 in Different Types of Clinical Specimens

Quantitative Detection and Viral Load Analysis of SARS-CoV-2 in Infected Patients

Clinical and virologic characteristics of the first 12 patients with coronavirus disease 2019 (COVID-19) in the United States

Swabs Collected by Patients or Health Care Workers for SARS-CoV-2 Testing

Posterior oropharyngeal saliva for the detection of SARS-CoV-2

Sensitivity of nasopharyngeal swabs and saliva for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

Saliva sample as a non-invasive specimen for the diagnosis of coronavirus disease 2019: a cross-sectional study

Saliva as a non-invasive specimen for detection of SARS-CoV-2

Variation in False-Negative Rate of Reverse Transcriptase Polymerase Chain Reaction-Based SARS-CoV-2 Tests by Time Since Exposure

Profiling Early Humoral Response to Diagnose Novel Coronavirus Disease (COVID-19)

Evidence Supporting Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 While Presymptomatic or Asymptomatic

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FDA. BD SARS-CoV-2 Reagents for BD MAX™ System

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Genomic Diversity of SARS-CoV-2 During Early Introduction into the United States National Capital Region. medRxiv

Triplex Real-Time RT-PCR for Severe Acute Respiratory Syndrome Coronavirus 2

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Integrative genomics viewer

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Persistent SARS-CoV-2 replication in severe COVID-19

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SARS-CoV-2 Virus Culture and Subgenomic RNA for Respiratory Specimens from Patients with Mild Coronavirus Disease

Highthroughput droplet digital PCR system for absolute quantitation of DNA copy number

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