The perplexing problem of persistently PCR-positive personnel

We report the results of a review of the evidence from studies comparing SARS-CoV-2 culture with reverse transcriptase polymerase chain reaction (rt-PCR), as viral culture represents the best indicator of current infection and infectiousness of the isolate. We identified fourteen studies succeeding in culturing or observing tissue invasion by SARS-CoV in sputum, naso or oropharyngeal, urine, stool and environmental samples from patients diagnosed with Covid-19. The data are suggestive of a relation between the time from collection of a specimen to test, copy threshold, and symptom severity, but the quality of the studies was moderate with lack of standardised reporting and lack of testing of PCR against viral culture or infectivity in animals. This limits our current ability to quantify the relationship between viral load, cycle threshold and viable virus detection and ultimately the usefulness of PCR use for assessing infectiousness of patients. Prospective routine testing of reference and culture specimens are necessary for each country involved in the pandemic to establish the usefulness and reliability of PCR for Covid-19 and its relation to patients factors such as date of onset of symptoms and copy threshold, in order to help predict infectivity.

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“…Calibration of assays could then be done to estimate virus viability from the cycle threshold with some certainty. 45…”

Section: Discussionmentioning

confidence: 99%

Viral cultures for COVID-19 infectivity assessment – a systematic review (Update 4)

Jefferson

Spencer

Brassey

et al. 2020

Preprint

101

115

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“…The gradual decrease in PPA in samples after 7 days of symptom onset is consistent with seroconversion 13 and clearance of antigen from the body. It has been suggested that infectivity of individuals with high Ct values and symptom onset to testing > 8 days is low 14 , and it was hypothesized that the sensitive molecular methods detect viral genomic fragments, rather than transmissible viruses 4,15,16 . Our data in Table 1 and Figure 4A are consistent with this hypothesis.…”

Section: Discussionmentioning

confidence: 99%

Femtomolar SARS-CoV-2 Antigen Detection Using the Microbubbling Digital Assay with Smartphone Readout Enables Antigen Burden Quantitation and Dynamics Tracking

Chen

Zhao

Feng

et al. 2021

Preprint

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Background: Little is known about the dynamics of SARS-CoV-2 antigen burden in respiratory samples in different patient populations at different stages of infection. Current rapid antigen tests cannot quantitate and track antigen dynamics with high sensitivity and specificity in respiratory samples. Methods: We developed and validated an ultra-sensitive SARS-CoV-2 antigen assay with smartphone readout using the Microbubbling Digital Assay previously developed by our group, which is a platform that enables highly sensitive detection and quantitation of protein biomarkers. A computer vision-based algorithm was developed for microbubble smartphone image recognition and quantitation. A machine learning-based classifier was developed to classify the smartphone images based on detected microbubbles. Using this assay, we tracked antigen dynamics in serial swab samples from COVID patients hospitalized in ICU and immunocompromised COVID patients. Results: The limit of detection (LOD) of the Microbubbling SARS-CoV-2 Antigen Assay was 0.5 pg/mL (10.6 fM) recombinant nucleocapsid (N) antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs, comparable to many rRT-PCR methods. The assay had high analytical specificity towards SARS-CoV-2. Compared to EUA-approved rRT-PCR methods, the Microbubbling Antigen Assay demonstrated a positive percent agreement (PPA) of 97% (95% confidence interval (CI), 92-99%) in symptomatic individuals within 7 days of symptom onset and positive SARS-CoV-2 nucleic acid results, and a negative percent agreement (NPA) of 97% (95% CI, 94-100%) in symptomatic and asymptomatic individuals with negative nucleic acid results. Antigen positivity rate in NP swabs gradually decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity of the same samples. The computer vision and machine learning-based automatic microbubble image classifier could accurately identify positives and negatives, based on microbubble counts and sizes. Total microbubble volume, a potential marker of antigen burden, correlated inversely with Ct values and days-after-symptom-onset. Antigen was detected for longer periods of time in immunocompromised patients with hematologic malignancies, compared to immunocompetent individuals. Simultaneous detectable antigens and nucleic acids may indicate the presence of replicating viruses in patients with persistent infections. Conclusions: The Microbubbling SARS-CoV-2 Antigen Assay enables sensitive and specific detection of acute infections, and quantitation and tracking of antigen dynamics in different patient populations at various stages of infection. With smartphone compatibility and automated image processing, the assay is well-positioned to be adapted for point-of-care diagnosis and to explore the clinical implications of antigen dynamics in future studies.

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“…This is a problem in all the occupational environments, 15 but it is especially regrettable in a healthcare system already stressed by the pandemic. 16 …”

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confidence: 99%

“…The proposal to consider safe a PCR cycle threshold (Ct) instead of the positive-negative dichotomy 18 does not consider that the intra- and extra-test variability of these assays is still too high. 16 Probably the best strategy is the one that considers the PCR Ct together with the severity of symptoms, immunocompromised status and, if available, the antibody serological level. 16 , 17 Whereas there is not a “zero risk” approach to this issue, certainly an excessive search for negative PCR tests leads also to waste resources and to a further stress on the healthcare system.…”

mentioning

confidence: 99%

“… 16 Probably the best strategy is the one that considers the PCR Ct together with the severity of symptoms, immunocompromised status and, if available, the antibody serological level. 16 , 17 Whereas there is not a “zero risk” approach to this issue, certainly an excessive search for negative PCR tests leads also to waste resources and to a further stress on the healthcare system.…”

mentioning

confidence: 99%

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