Results of the first German external quality assessment scheme for the detection of monkeypox virus DNA.

BACKGROUND: In May 2022, the monkeypox virus (MPXV) spread into non-endemic countries and the global community was quick to test the lessons learned from the SARS-CoV-2 pandemic. Due to its symptomatic resemblance to other diseases, like the non-pox virus varicella zoster (chickenpox), polymerase chain reaction methods play an important role in correctly diagnosing the rash-causing pathogen. INSTAND quickly established a new external quality assessment (EQA) scheme for MPXV and orthopoxvirus (OPXV) DNA detection to assess the current performance quality of the laboratory tests. METHODS: We analyzed quantitative and qualitative data of the first German EQA for MPXV and OPXV DNA detection. The survey included one negative and three MPXV-positive samples with different MPX viral loads. The threshold cycle (Ct) or other measures defining the quantification cycle (Cq) were analyzed in an assay-specific manner. A Passing Bablok fit was used to investigate the performance at laboratory level. RESULTS: 141 qualitative datasets were reported by 131 laboratories for MPXV detection and 68 qualitative datasets by 65 laboratories for OPXV detection. More than 96% of the results were correctly identified as negative and more than 97% correctly identified as positive. An analysis of the reported Ct/Cq values showed a large spread of these values of up to 12 Ct/Cq. Nevertheless, there is a good correlation of results for the different MPXV concentrations at laboratory level. Only a few quantitative results in copies/mL were reported (MPXV: N = 5; OPXV: N = 2), but the results correlated well with the concentration differences between the EQA samples, which were to a power of ten each. CONCLUSION: The EQA results show that laboratories performed well in detecting both MPXV and OPXV. However, Ct/Cq values should be interpreted with caution when conclusions are drawn about the viral load as long as metrological traceability is not granted.

RNA reference materials with defined viral RNA loads of SARS-CoV-2-A useful tool towards a better PCR assay harmonization.

SARS-CoV-2, the cause of COVID-19, requires reliable diagnostic methods to track the circulation of this virus. Following the development of RT-qPCR methods to meet this diagnostic need in January 2020, it became clear from interlaboratory studies that the reported Ct values obtained for the different laboratories showed high variability. Despite this the Ct values were explored as a quantitative cut off to aid clinical decisions based on viral load. Consequently, there was a need to introduce standards to support estimation of SARS-CoV-2 viral load in diagnostic specimens. In a collaborative study, INSTAND established two reference materials (RMs) containing heat-inactivated SARS-CoV-2 with SARS-CoV-2 RNA loads of ~107 copies/mL (RM 1) and ~106 copies/mL (RM 2), respectively. Quantification was performed by RT-qPCR using synthetic SARS-CoV-2 RNA standards and digital PCR. Between November 2020 and February 2021, German laboratories were invited to use the two RMs to anchor their Ct values measured in routine diagnostic specimens, with the Ct values of the two RMs. A total of 305 laboratories in Germany were supplied with RM 1 and RM 2. The laboratories were requested to report their measured Ct values together with details on the PCR method they used to INSTAND. This resultant 1,109 data sets were differentiated by test system and targeted gene region. Our findings demonstrate that an indispensable prerequisite for linking Ct values to SARS-CoV-2 viral loads is that they are treated as being unique to an individual laboratory. For this reason, clinical guidance based on viral loads should not cite Ct values. The RMs described were a suitable tool to determine the specific laboratory Ct for a given viral load. Furthermore, as Ct values can also vary between runs when using the same instrument, such RMs could be used as run controls to ensure reproducibility of the quantitative measurements.

Clinical evaluation of the automated Abbott RealTime SARS-CoV-2, Alinity m SARS-CoV-2, and Alinity m Resp-4-Plex assays.

BACKGROUND: Detection of SARS-CoV-2 infections relies on the use of sensitive, accurate and high throughput RT-PCR assays. OBJECTIVES: We assessed the analytical performance of the Abbott RealTime SARS-CoV-2 (RT-SARS), Alinity m SARS-CoV-2 (AlinSARS) assays and compared the clinical performance of the RT-SARS, AlinSARS, and Alinity m Resp-4-Plex (Alin4Plex) assays to the Seegene Allplex assay (Allplex) and an inhouse test (Inhouse). RESULTS: We found 100 % positive percent agreement (PPA) and 100 % negative percent agreement (NPA) comparing RT-SARS and Allplex. RT-SARS, AlinSARS and Inhouse showed 100 % NPA and 100 % PPA across all assays, except for the RdRp target of Inhouse (PPA = 84 %). Similarly, Alin4Plex and Allplex showed high agreement with specimens containing either SARS-CoV-2, influenza A, influenza B, or RSV. Detection rates of 100 % for SARS-CoV-2 at 50 copies/mL, high precision, and no cross-reactivity with non-SARS-CoV-2 respiratory pathogens were observed for RT-SARS and AlinSARS. AlinSARS detected SARS-CoV-2 in spiked throat washes and in specimens infected with SARS-CoV-2 Alpha or Beta variants. CONCLUSIONS: The newly developed RT-SARS, AlinSARS, and Alin4Plex assays proved to be useful for detecting SARS-CoV-2 RNA in clinical samples.

Preventing SARS-CoV-2 In-Hospital Infections in Cardiovascular Patients and Medical Staff: An Observational Study From the German Heart Center Berlin.

Objective: COVID-19 is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Preventing in-hospital infections is crucial to protect patients and hospital staff. Methods: At the very beginning of the COVID-19 pandemic, the German Heart Center initiated obligatory wearing of surgical face masks for patients and employees, SARS-CoV-2 screening for all patients, and symptom-based testing for employees. In addition, access restriction, closure of outpatient departments, and postponing non-urgent procedures were implemented with community-initiated regulations. Results: During the observation period (03/16/2020-04/27/2020), 1,128 SARS-CoV-2 tests were performed in 983 persons (1.1 tests/person; 589 in patients and 394 in hospital employees). Up to 60% of the clinical workforce was tested based on symptoms and risk (62.5% symptoms, 19.3% direct or indirect contact to known COVID-19, 4.5% returnee from risk area, 13.7% without specific reason). Patient testing for SARS-CoV-2 was obligatory (100% tested). The overall prevalence of positive tests during the observation period was 0.4% (n = 5 out of 1,128 tests performed). The incidence of new infections with SARS-CoV-2 was 0.5% (n = 5 out of 983 individuals; three healthcare workers, two patients). No nosocominal infections occurred, despite a mean number of 14.8 in-hospital contacts. Conclusion: Comprehensive SARS-CoV-2 testing and surgical face masks for patients and hospital staff, in addition to others measures, are key factors for the early detection of COVID-19 and to prevent spreading in the vulnerable hospital population.