Performance of the RT-LAMP-based eazyplex® SARS-CoV-2 as a novel rapid diagnostic test.

BACKGROUND: Diagnostic assays for severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) that are easy to perform and produce fast results are essential for timely decision making regarding the isolation of contagious individuals. OBJECTIVE: We evaluated the CE-approved eazyplex® SARS-CoV-2, a ready-to-use real time RT-LAMP assay for identification of the SARS-CoV-2 N and ORF8 genes from swabs in less than 30 min without RNA extraction. STUDY DESIGN: Oropharyngeal and nasal swabs from 100 positive and 50 negative patients were inoculated into 0.9 % saline and tested by NeuMoDx™ RT-PCR. An aliquot was diluted fivefold in Copan sputum liquefying (SL) solution and directly analyzed by eazyplex® SARS-CoV-2. In addition, 130 patient swabs were prospectively tested with both methods in parallel. Analytical sensitivity of the assay was determined using virus stock dilutions. RESULTS: Positive percent agreement (PPA) between the eazyplex® SARS-CoV-2 and RT-PCR was 74 % for samples with Ct values < 35. When using a Ct cut-off ≤ 28 the PPA increased to 97.4 %. In the prospective part of the study overall PPA of the eazyplex® kit was 66.7 % but increased to 100 % when only Ct values ≤ 28 were considered. There were no false positive results. The median time to positivity was 12.5 min for the N gene and 16.75 min for ORF8. Analytical sensitivity was 3.75 TCID50/mL. 105 virus copies/mL were reproducibly detected. CONCLUSION: The eazyplex® SARS-CoV-2 is a rapid assay that accurately identifies samples with high viral loads. It may be useful for near-patient testing outside of a molecular diagnostic laboratory.

Use of the variplex™ SARS-CoV-2 RT-LAMP as a rapid molecular assay to complement RT-PCR for COVID-19 diagnosis.

BACKGROUND: Molecular assays based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) may be useful for rapid diagnosis of the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) because of the easy performance and the option to bypass RNA extraction. OBJECTIVES: This study was designed to evaluate the clinical performance of the CE-labeled variplexTM real time SARS-CoV-2 RT-LAMP assay in comparison to commercial RT-PCRs. STUDY DESIGN: RNA extracted from pharyngeal swabs was tested by variplex™ RT-LAMP and Corman's LightMix™ E gene RT-PCR as reference. Samples of respiratory secretions from Coronavirus infection disease (COVID-19) and negative control patients were analyzed by variplex™ without RNA extraction and tested in parallel with the Allplex™ and VIASURE BD MAX RT-PCRs. RESULTS: Using isolated RNA variplex™ RT-LAMP showed a sensitivity of 75 % compared to LightMix E gene RT-PCR but contrary to the latter it produced no false-positive results. For the evaluation of samples from respiratory secretions concordance analysis showed only a moderate agreement between the variplex™ RT-LAMP conducted on unprocessed samples and Allplex™ and VIASURE RT-PCRs (Cohen's κ ranging from 0.52-0.56). Using the approach to define a sample as true-positive when at least two assays gave a positive result the clinical sensitivities were as follows: 76.3 % for variplex™, 84.2 % for Allplex™ and 68.4 % for VIASURE. However, when results of RT-PCR and RT-LAMP were combined diagnostic sensitivity was increased to 92-100 %. CONCLUSION: The variplex RT-LAMP may serve as a rapid test to be combined with a RT-PCR assay to increase the diagnostic accuracy in patients with suspected COVID-19 infection.

Viruses and atypical bacteria in the respiratory tract of immunocompromised and immunocompetent patients with airway infection.

Respiratory tract infections (RTI) can take a serious course under immunosuppression. Data on the impact of the underlying pathogens are still controversial. Samples from the upper (n = 322) and lower RT (n = 169) were collected from 136 children and 355 adults; 225 among them have been immunocompromised patients. Exclusion criteria were presence of relevant cultivable microorganisms, C-reactive protein > 20 mg/dl, or procalcitonin > 2.0 ng/ml. Samples were tested by PCR for the presence of herpesviruses (HSV-1/-2; VZV; CMV; HHV6; EBV), adenoviruses, bocaviruses, entero-/rhinoviruses (HRV), parechoviruses, coronaviruses, influenza viruses (IV), parainfluenza viruses as well as for pneumoviruses (HMPV and RSV), and atypical bacteria (Mycoplasma pneumoniae, M.p.; Chlamydia pneumoniae, C.p.). Viral/bacterial genome equivalents were detected in more than two-thirds of specimens. Under immunosuppression, herpesviruses (EBV 30.9%/14.6%, p < 0.001; CMV 19.6%/7.9%, p < 0.001; HSV-1: 14.2%/7.1%, p = 0.012) were frequently observed, mainly through their reactivation in adults. Immunocompromised adults tended to present a higher RSV prevalence (6.4%/2.4%, p = 0.078). Immunocompetent patients were more frequently tested positive for IV (15.0%/5.8%, p = 0.001) and M.p. (6.4%/0.4%, p < 0.001), probably biased due to the influenza pandemic of 2009 and an M.p. epidemic in 2011. About 41.8% of samples were positive for a single pathogen, and among them EBV (19.9%) was most prevalent followed by HRV (18.2%) and IV (16.6%). HSV-2 and C.p. were not found. Marked seasonal effects were observed for HRV, IV, and RSV. Differences in pathogen prevalence were demonstrated between immunocompetent and immunocompromised patients. The exact contribution of some herpesviruses to the development of RTI remains unclear.