SARS-CoV-2 detection using a nanobody-functionalized voltammetric device.

Background: An ongoing need during the COVID-19 pandemic has been the requirement for accurate and efficient point-of-care testing platforms to distinguish infected from non-infected people, and to differentiate SARS-CoV-2 infections from other viruses. Electrochemical platforms can detect the virus via its envelope spike protein by recording changes in voltammetric signals between samples. However, this remains challenging due to the limited sensitivity of these sensing platforms. Methods: Here, we report on a nanobody-functionalized electrochemical platform for the rapid detection of whole SARS-CoV-2 viral particles in complex media such as saliva and nasopharyngeal swab samples. The sensor relies on the functionalization of gold electrode surface with highly-oriented Llama nanobodies specific to the spike protein receptor binding domain (RBD). The device provides results in 10 min of exposure to 200 µL of unprocessed samples with high specificity to SARS-CoV-2 viral particles in human saliva and nasopharyngeal swab samples. Results: The developed sensor could discriminate between different human coronavirus strains and other respiratory viruses, with 90% positive and 90% negative percentage agreement on 80 clinical samples, as compared to RT-qPCR. Conclusions: We believe this diagnostic concept, also validated for RBD mutants and successfully tested on Delta variant samples, to be a powerful tool to detect patients' infection status, easily extendable to other viruses and capable of overcoming sensing-related mutation effects.

A mask-based diagnostic platform for point-of-care screening of Covid-19.

Diagnostics of SARS-CoV-2 infection using real-time reverse-transcription polymerase chain reaction (RT-PCR) on nasopharyngeal swabs is now well-established, with saliva-based testing being lately more widely implemented for being more adapted for self-testing approaches. In this study, we introduce a different concept based on exhaled breath condensate (EBC), readily collected by a mask-based sampling device, and detection with an electrochemical biosensor with a modular architecture that enables fast and specific detection and quantification of COVID-19. The face mask forms an exhaled breath vapor containment volume to hold the exhaled breath vapor in proximity to the EBC collector to enable a condensate-forming surface, cooled by a thermal mass, to coalesce the exhaled breath into a 200-500 µL fluid sample in 2 min. EBC RT-PCR for SARS-CoV-2 genes (E, ORF1ab) on samples collected from 7 SARS-CoV-2 positive and 7 SARS-CoV-2 negative patients were performed. The presence of SARS-CoV-2 could be detected in 5 out of 7 SARS-CoV-2 positive patients. Furthermore, the EBC samples were screened on an electrochemical aptamer biosensor, which detects SARS-CoV-2 viral particles down to 10 pfu mL-1 in cultured SARS-CoV-2 suspensions. Using a "turn off" assay via ferrocenemethanol redox mediator, results about the infectivity state of the patient are obtained in 10 min.

Preanalytical Issues and Cycle Threshold Values in SARS-CoV-2 Real-Time RT-PCR Testing: Should Test Results Include These?

Since the emergence of SARS-CoV-2 pandemic, clinical laboratories worldwide are overwhelmed with SARS-CoV-2 testing using the current gold standard: real-time reverse-transcription polymerase chain reaction (RT-PCR) assays. The large numbers of suspected cases led to shortages in numerous reagents such as specimen transport and RNA extraction buffers. We try to provide some answers on how strongly preanalytical issues affect RT-PCR results by reviewing the utility of different transport buffer media and virus inactivation procedures and comparing the literature data with our own recent findings. We show that various viral inactivation procedures and transport buffers are available and are less of a bottleneck for PCR-based methods. However, efficient alternative lysis buffers remain more difficult to find, and several fast RT-PCR assays are not compatible with guanidine-containing media, making this aspect more of a challenge in the current crisis. Furthermore, the availability of different SARS-CoV-2-specific RT-PCR kits with different sensitivities makes the definition of a general cutoff level for the cycle threshold (Ct) value challenging. Only a few studies have considered how Ct values relate to viral infectivity and how preanalytical issues might affect viral infectivity and RNA detection. We review the current data on the correlation between Ct values and viral infectivity. The presence of the SARS-CoV-2 viral genome in its own is not sufficient proof of infectivity and caution is needed in evaluation of the infectivity of samples. The correlation between Ct values and viral infectivity revealed an RT-PCR cutoff value of 34 cycles for SARS-CoV-2 infectivity using a laboratory-developed RT-PCR assay targeting the RdRp gene. While ideally each clinical laboratory should perform its own correlation, we believe this perspective article could be a reference point for others, in particular medical doctors and researchers interested in COVID-19 diagnostics, and a first step toward harmonization.

Relapsing Pityriasis Rosea With HHV-7 Reactivation in an 11-Year-Old Girl.

Pityriasis rosea (PR) usually presents as acute exanthema with oval erythematous-squamous lesions localized on the trunk, arms, and legs with spontaneous remission. We present an unusual case of PR with frequent relapses during a period of 7 years. An 11-year-old white female patient presented with many pruritic erythematous oval lesions on her trunk. A second episode followed 2 years later with several pruritic erythematous lesions on her lower limbs. During the following 5 years, the patient had several relapses per year, with 1 to 3 lesions on changing localizations. PR was diagnosed on the basis of the clinical presentation and detection of human herpesvirus 7 DNA. Spontaneous remission occurred without treatment in each episode. Relapsing PR is a rare form of PR characterized by a lower number of lesions and smaller sized lesions compared with the classic form of PR. Pediatricians should consider the diagnosis of relapsing PR even if only a single or few erythematous lesions are present.

Comparison of two commercial quantitative PCR assays and correlation with the first WHO International Standard for human CMV.

Comparability between CMV assays could be facilitated by the first WHO International Standard for human CMV (standard). Standard dilutions were submitted to nucleic acid extraction with Versant kPCR Molecular systems SP or MagNA Pure LC System followed by the kPCR PLX™ CMV DNA (kPCR) or the CMV R-gene™ assay (R-gene), respectively; 139 clinical specimens were tested. Both assays correlated well with the standard (R2 > 0.96) and a matrix effect was observed. Quantitative results correlated reasonably between both assays for whole blood (R2 = 0.79) and well for other specimen types (R2 = 0.93). Quantification differences were within one log10 of the averaged log10 results for 25/27 blood specimens and for 32/33 other specimens. Calibration to the standard did not increase this percentage. In conclusion, results of both assays showed reasonable correlation with each other and good correlation with the standard. Calibration to the standard did not improve comparability of quantitative results.

Comparison of two commercial quantitative PCR assays for EBV DNA detection and their correlation with the first WHO International Standard for EBV.

PURPOSE: There are few data on the performance of automated Epstein-Barr virus (EBV) PCR assays. This study compared EBV quantification for the kPCR PLX EBV DNA (kPCR; Siemens, France) and the EBV R-gene (R-gene; Argene, Biomerieux, France) assays and their correlation with the World Health Organization (WHO) standard. METHODOLOGY: WHO International Standard for EBV (WHO standard) dilution panels in different matrices were submitted to nucleic acid extraction with Versant kPCR Molecular Systems SP followed by the kPCR assay, or to nucleic acid extraction with the MagNA Pure LC System or NucliSENS easyMag followed by the R-gene assay. Seventy-four clinical specimens were tested in both assays. Bland-Altman analysis and linear regression analysis were performed. RESULTS: The correlation between the WHO standard diluted in different matrices and the R-gene and kPCR assays was good (R2 >0.96 and R2 >0.92, respectively). A matrix effect was observed. The correlation of quantitative results between both assays yielded a coefficient of determination R2 higher than 0.74. The quantification differences were within one log10 of the averaged results for 34 of the 38 specimens (89 %). Calibration to the WHO standard did not increase the comparability of quantitative results. CONCLUSIONS: The quantitative results of both assays showed reasonable correlation with each other and a good correlation with the WHO standard.