Detection of SARS-COV N2 Gene: Very low amounts of viral RNA or false positive?

Falasca, Francesca; Sciandra, Ilaria; Carlo, Daniele Di; Gentile, Massimo; Deales, Alberto; Antonelli, Guido; Turriziani, Ombretta

doi:10.1016/j.jcv.2020.104660

Cited by 31 publications

(35 citation statements)

References 9 publications

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“…Decreasing viral load might also occur in true negative cases, i.e., non-infected people, of whom the first sample was a false-positive, possibly due to contamination or high test sensitivity. This can be more often the case in people with no history of clinical signs relevant to COVID-19, as shown in the current study, findings that are in line with those of Falasca et al [18].…”

Section: Discussionsupporting

confidence: 93%

“…In the same study, a second NPS collected 24 h after the first, indicated that 12 of 17 samples were negative (70.5%). Laboratory data correlated well with clinical data further supported these laboratory findings, as only 2 among these 17 persons developed clinical signs relevant to COVID-19 [18]. Katz et al [19] reported false-positive rRT-PCR reactions in a setting for urgent head and neck surgery and otolaryngologic emergencies, leading to delay of urgent surgical procedures and transfer of people to COVID-19-designated units [19].…”

Section: Discussionmentioning

confidence: 69%

“…To avoid false positive or negative results, each clinical setting must define its Ct cutoff value, taking under consideration the limit of detection of the assay and the distribution of Cts in the population being tested [17]. In a recent study by Falasca et al [18], 1% of samples tested (17 of 1639) were found to contain a very low viral RNA load, since positivity was detected at high Ct. In the same study, a second NPS collected 24 h after the first, indicated that 12 of 17 samples were negative (70.5%).…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Combination of rRT-PCR and Anti-Nucleocapsid/Anti-Spike Antibodies to Characterize Specimens with Very Low Viral SARs-CoV-2 Load: A Real-Life Experience

et al. 2021

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The objective of the present study was to evaluate the true positivity among people, whose results of initial testing of nasopharyngeal swabs (NPS) showed a very low viral load of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Seventy-seven people detected with low viral loads of SARs-CoV-2 in nasopharyngeal samples (Ct ≥ 35) were enrolled in the study. For this purpose, a second NPS was collected for rRT-PCR (real-time reverse transcription polymerase chain reaction) combined with a pair of serum samples for detection of anti-nucleocapsid (anti-N) and anti-spike (anti-S) antibodies. In 8 people, subsequent examinations indicated an increase in viral loads, thereafter, followed by an increase of anti-N and anti-S antibodies, findings compatible with an early stage of COVID-19 infection. In 9 people, who already had increased anti-N antibodies, subsequent examination showed a decrease or absence of viral load and an increase in antibodies, indicative of a late stage of COVID-19 infection. In 60 people, subsequent examination showed absence of infection (as indicated by absence of viral load and antibodies). We propose that the combination of a second NPS and one serum-specimen, both taken three days after the first NPS, helps significantly to avoid false-positive results.

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Section: Discussionsupporting

confidence: 93%

Section: Discussionmentioning

confidence: 69%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Combination of rRT-PCR and Anti-Nucleocapsid/Anti-Spike Antibodies to Characterize Specimens with Very Low Viral SARs-CoV-2 Load: A Real-Life Experience

et al. 2021

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“…35 , 36 False-positive real-time RT-PCR results in clinical material with low viral loads have been observed for PCR assays. 37 By comparing three RT-RPA assays and validating them with clinical samples, we were able to pick a highly accurate RPA assay, i.e., RdRP RT-RPA.…”

Section: Discussionmentioning

confidence: 99%

Suitcase Lab for Rapid Detection of SARS-CoV-2 Based on Recombinase Polymerase Amplification Assay

et al. 2021

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In March 2020, the SARS-CoV-2 virus outbreak was declared as a world pandemic by the World Health Organization (WHO). The only measures for controlling the outbreak are testing and isolation of infected cases. Molecular real-time polymerase chain reaction (PCR) assays are very sensitive but require highly equipped laboratories and well-trained personnel. In this study, a rapid point-of-need detection method was developed to detect the RNA-dependent RNA polymerase (RdRP), envelope protein (E), and nucleocapsid protein (N) genes of SARS-CoV-2 based on the reverse transcription recombinase polymerase amplification (RT-RPA) assay. RdRP, E, and N RT-RPA assays required approximately 15 min to amplify 2, 15, and 15 RNA molecules of molecular standard/reaction, respectively. RdRP and E RT-RPA assays detected SARS-CoV-1 and 2 genomic RNA, whereas the N RT-RPA assay identified only SARS-CoV-2 RNA. All established assays did not cross-react with nucleic acids of other respiratory pathogens. The RT-RPA assay’s clinical sensitivity and specificity in comparison to real-time RT-PCR (n = 36) were 94 and 100% for RdRP; 65 and 77% for E; and 83 and 94% for the N RT-RPA assay. The assays were deployed to the field, where the RdRP RT-RPA assays confirmed to produce the most accurate results in three different laboratories in Africa (n = 89). The RPA assays were run in a mobile suitcase laboratory to facilitate the deployment at point of need. The assays can contribute to speed up the control measures as well as assist in the detection of COVID-19 cases in low-resource settings.

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“…False negative results may be due to sample inhibitors, poor amplification efficiency, and reduced precision in low concentration samples. False positives could arise from contaminants or poor test specificity [41]. In any case, the sensitivity and specificity of the RT-PCR methods used to detect SARS-CoV-2 in seminal fluid have not been evaluated [42].…”

Section: Discussionmentioning

confidence: 99%

Molecular diagnosis of SARS-CoV-2 in seminal fluid

Paoli

Pallotti

Nigro

et al. 2021

J Endocrinol Invest

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Purpose Due to relevant repercussions on reproductive medicine, we aimed to evaluate feasibility of RT-PCR as a detection method of SARS-CoV-2 RNA in seminal fluid. Methods A qualitative determination of the RT-PCR assays in semen was performed through different approaches: (1) efficiency of RNA extraction from sperm and seminal plasma was determined using PRM1 and PRM2 mRNA and a heterologous system as control; (2) samples obtained by diluting viral preparation from a SARS-CoV-2 panel (virus cultured in Vero E6 cell lines) were tested; (3) viral presence in different fractions of seminal fluid (whole sample, seminal plasma and post-centrifugation pellet) was evaluated. Semen samples from mild and recovered COVID-19 subjects were collected by patients referring to the Infectious Disease Department of the Policlinico Umberto I Hospital - “Sapienza” University of Rome. Control subjects were recruited at the Laboratory of Seminology-Sperm Bank “Loredana Gandini'' of the same hospital. Results The control panel using viral preparations diluted in saline and seminal fluid showed the capability to detect viral RNA presence with Ct values depending on the initial viral concentration. All tested semen samples were negative for SARS-CoV-2, regardless of the nasopharyngeal swab result or seminal fluid fraction. Conclusion These preliminary data show that RT-PCR for SARS-CoV-2 RNA testing appears to be a feasible method for the molecular diagnosis of SARS-CoV-2 in seminal fluid, supported by results of the control panel. The ability to detect SARS-CoV-2 in semen is extremely important for reproductive medicine, especially in assisted reproductive technology and sperm cryopreservation.

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Detection of SARS-COV N2 Gene: Very low amounts of viral RNA or false positive?

Cited by 31 publications

References 9 publications

Combination of rRT-PCR and Anti-Nucleocapsid/Anti-Spike Antibodies to Characterize Specimens with Very Low Viral SARs-CoV-2 Load: A Real-Life Experience

Combination of rRT-PCR and Anti-Nucleocapsid/Anti-Spike Antibodies to Characterize Specimens with Very Low Viral SARs-CoV-2 Load: A Real-Life Experience

Suitcase Lab for Rapid Detection of SARS-CoV-2 Based on Recombinase Polymerase Amplification Assay

Molecular diagnosis of SARS-CoV-2 in seminal fluid

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