SARS-CoV-2 coinfections with variant genomic lineages identified by multiplex fragment analysis.

Immunocompromised patients can experience prolonged SARS-CoV-2 infections in the setting of a lack of protectivity immunity despite vaccination. As circulating SARS-CoV-2 strains become more heterogeneous, concomitant infection with multiple SARS-CoV-2 variants has become an increasing concern. Immunocompromised patient populations represent potential reservoirs for the emergence of novel SARS-CoV-2 variants through mutagenic change or coinfection followed by recombinatory events. Identification of SARS-CoV-2 coinfections is challenging using traditional next generation sequencing pipelines; however, targeted genotyping approaches can facilitate detection. Here we describe five COVID-19 cases caused by coinfection with different SARS-CoV-2 variants (Delta/Omicron BA.1 and Omicron BA.1/BA.2) as identified by multiplex fragment analysis.

SARS-CoV-2 coinfections with variant genomic lineages identified by multiplex fragment analysis

Immunocompromised patients can experience prolonged SARS-CoV-2 infections in the setting of a lack of protectivity immunity despite vaccination. As circulating SARS-CoV-2 strains become more heterogeneous, concomitant infection with multiple SARS-CoV-2 variants has become an increasing concern. Immunocompromised patient populations represent potential reservoirs for the emergence of novel SARS-CoV-2 variants through mutagenic change or coinfection followed by recombinatory events. Identification of SARS-CoV-2 coinfections is challenging using traditional next generation sequencing pipelines;however, targeted genotyping approaches can facilitate detection. Here we describe five COVID-19 cases caused by coinfection with different SARS-CoV-2 variants (Delta/Omicron BA.1 and Omicron BA.1/BA.2) as identified by multiplex fragment analysis.

COVID-19 Vaccination is Associated With Favorable Outcomes Among Lung Transplant Patients With Breakthrough Infections.

BACKGROUND: There are limited data regarding the clinical efficacy of COVID-19 vaccines among lung transplant (LT) patients. METHODS: We included all LT patients diagnosed with COVID-19 between March 1, 2020, and December 10, 2021 (n = 84; median age 55, range, 20-73 years; males 65.5%). The study group was divided into 3 groups based on the vaccination status (patients who did not complete the primary series for any of the vaccines: n = 58; those with 2 doses of messenger RNA (mRNA) or 1 dose of the adenoviral vector vaccine, vaccinated group: n = 16; those with at least 1 additional dose beyond the primary series, boosted group: n = 10). RESULTS: Pulmonary parenchymal involvement on chest computed tomography scan was less common among the boosted group (P = .009). The proportion of patients with new or worsening respiratory failure was significantly lower among the vaccinated and boosted groups and these patients were significantly more likely to achieve the composite endpoint of oxygen-dependence free survival (P = .02). On multivariate logistic regression analysis, higher body mass index, restrictive lung disease as the transplant indication, and preinfection chronic lung allograft dysfunction were independently associated with acute or acute on chronic respiratory failure while being on therapeutic dose anticoagulation and having received the booster dose had a protective effect. CONCLUSION: COVID-19 vaccines appear to have several favorable effects among LT patients with breakthrough infections including lower likelihood of allograft involvement on imaging (among boosted patients), need of hospitalization, and complications such as new or worsening respiratory failure.

Multiplex Fragment Analysis for Flexible Detection of All SARS-CoV-2 Variants of Concern.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge, and effective tracking requires rapid return of results. Surveillance of variants is typically performed by whole genome sequencing (WGS), which can be financially prohibitive and requires specialized equipment and bioinformatic expertise. Genotyping approaches are rapid methods for monitoring SARS-CoV-2 variants but require continuous adaptation. Fragment analysis may represent an approach for improved SARS-CoV-2 variant detection. METHODS: A multiplex fragment analysis approach (CoVarScan) was validated using PCR targeting variants by size and fluorescent color. Eight SARS-CoV-2 mutational hot spots in variants of concern (VOCs) were targeted. Three primer pairs (recurrently deleted region [RDR] 1, RDR2, and RDR3-4) flank RDRs in the S-gene. Three allele-specific primers target recurrent spike receptor binding domain mutants. Lastly, 2 primer pairs target recurrent deletions or insertions in ORF1A and ORF8. Fragments were resolved and analyzed by capillary electrophoresis (ABI 3730XL), and mutational signatures were compared to WGS results. RESULTS: We validated CoVarScan using 3544 clinical respiratory specimens. The assay exhibited 96% sensitivity and 99% specificity compared to WGS. The limit of detection for the core targets (RDR1, RDR2, and ORF1A) was 5 copies/reaction. Variants were identified in 95% of samples with cycle threshold (CT) <30 and 75% of samples with a CT 34 to 35. Assay design was frozen April 2021, but all subsequent VOCs have been detected including Delta (n = 2820), Mu, (n = 6), Lambda (n = 6), and Omicron (n = 309). Genotyping results are available in as little as 4 h. CONCLUSIONS: Multiplex fragment analysis is adaptable and rapid and has similar accuracy to WGS to classify SARS-CoV-2 variants.

Ad interim recommendations for diagnosing SARS-CoV-2 infection by the IFCC SARS-CoV-2 variants working group.

This document, endorsed by the IFCC Working Group on SARS-CoV-2 Variants, aims to update previous indications for diagnosing acute SARS-CoV-2 infection, taking into consideration the evidence that has emerged after the origin and spread of new lineages and sub-lineages of the virus characterized by mutated genetics and altered biochemical, biological and clinical characteristics. These indications encompass the use of different diagnostic strategies in specific clinical settings, such as high risk of SARS-CoV-2 infection (symptomatic patients), low risk of SARS-CoV-2 infection (asymptomatic subjects) at hospital admission/contact tracing, testing in asymptomatic subjects, in epidemiologic surveys and/or population screening, along with tentative indications for identification of new lineages and/or sub-lineages of SARS-CoV-2.

Clinical Evaluation of the Abbott Alinity SARS-CoV-2 Spike-Specific Quantitative IgG and IgM Assays among Infected, Recovered, and Vaccinated Groups.

The coronavirus disease 19 (COVID-19) pandemic continues to impose a significant burden on global health infrastructure. While identification and containment of new cases remain important, laboratories must now pivot and consider an assessment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunity in the setting of the recent availability of multiple COVID-19 vaccines. Here, we have utilized the latest Abbott Alinity semiquantitative IgM and quantitative IgG spike protein (SP) serology assays (IgMSP and IgGSP) in combination with Abbott Alinity IgG nucleocapsid (NC) antibody test (IgGNC) to assess antibody responses in a cohort of 1,236 unique participants comprised of naive, SARS-CoV-2-infected, and vaccinated (including both naive and recovered) individuals. The IgMSP and IgGSP assays were highly specific (100%) with no cross-reactivity to archived samples collected prior to the emergence of SARS-CoV-2, including those from individuals with seasonal coronavirus infections. Clinical sensitivity was 96% after 15 days for both IgMSP and IgGSP assays individually. When considered together, the sensitivity was 100%. A combination of NC- and SP-specific serologic assays clearly differentiated naive, SARS-CoV-2-infected, and vaccine-related immune responses. Vaccination resulted in a significant increase in IgGSP and IgMSP values, with a major rise in IgGSP following the booster (second) dose in the naive group. In contrast, SARS-CoV-2-recovered individuals had several-fold higher IgGSP responses than naive following the primary dose, with a comparatively dampened response following the booster. This work illustrates the strong clinical performance of these new serological assays and their utility in evaluating and distinguishing serological responses to infection and vaccination.

The effect of the Covid-19 shutdown on glycemic testing and control.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic caused a halt to in-person ambulatory care. We evaluated how the reduction in access to care affected HbA1c testing and patient HbA1c levels. METHODS: HbA1c data from 11 institutions were extracted to compare testing volume and the percentage of abnormal results between a pre-pandemic period (January-June 2019, period 1) and a portion of the COVID-19 pandemic period (Jan-June 2020, period 2). HbA1c results greater than 6.4% were categorized as abnormal. RESULTS: HbA1C testing volumes decreased in March, April and May by 23, 61 and 40% relative to the corresponding months in 2019. The percentage of abnormal results increased in April, May and June (25, 23, 9%). On average, we found that the frequency of abnormal results increased by 0.31% for every 1% decrease in testing volume (p < 0.0005). CONCLUSION: HbA1c testing volume for outpatients decreased by up to 70% during the early months of the pandemic. The decrease in testing was associated with an increase in abnormal HbA1c results.

Digital Droplet PCR for SARS-CoV-2 Resolves Borderline Cases.

OBJECTIVES: The Bio-Rad SARS-CoV-2 ddPCR Kit (Bio-Rad Laboratories) was the first droplet digital polymerase chain reaction (ddPCR) assay to receive Food and Drug Administration (FDA) Emergency Use Authorization approval, but it has not been evaluated clinically. We describe the performance of ddPCR-in particular, its ability to confirm weak-positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) results. METHODS: We clinically validated the Bio-Rad Triplex Probe ddPCR Assay. The limit of detection was determined by using serial dilutions of SARS-CoV-2 RNA in an artificial viral envelope. The ddPCR assay was performed according to the manufacturer's specifications on specimens confirmed to be positive (n = 48) or negative (n = 30) by an FDA-validated reverse transcription-polymerase chain reaction assay on the m2000 RealTime system (Abbott). Ten borderline positive cases were also evaluated. RESULTS: The limit of detection was 50 copies/mL (19 of 20 positive). Forty-seven specimens spanning a range of quantification cycles (2.9-25.9 cycle numbers) were positive by this assay (47 of 48; 97.9% positive precent agreement), and 30 negative samples were confirmed as negative (30 of 30; 100% negative percent agreement). Nine of 10 borderline cases were positive when tested in triplicate. CONCLUSIONS: The ddPCR of SARS-CoV-2 is an accurate method, with superior sensitivity for viral RNA detection. It could provide definitive evaluation of borderline positive cases or suspected false-negative cases.

Side-Effects of COVID-19 on Patient Care: An INR Story.

BACKGROUND: Numerous studies have documented reduced access to patient care due to the COVID-19 pandemic, including access to diagnostic or screening tests, prescription medications, and treatment for an ongoing condition. In the context of clinical management for venous thromboembolism, this could result in suboptimal therapy with warfarin. We aimed to determine the impact of the pandemic on utilization of International Normalized Ratio (INR) testing and the percentage of high and low results. METHODS: INR data from 11 institutions were extracted to compare testing volume and the percentage of INR results ≥3.5 and ≤1.5 between a pre-pandemic period (January-June 2019, period 1) and a portion of the COVID-19 pandemic period (January-June 2020, period 2). The analysis was performed for inpatient and outpatient cohorts. RESULTS: Testing volumes showed relatively little change in January and February, followed by a significant decrease in March, April, and May, and then returned to baseline in June. Outpatient testing showed a larger percentage decrease in testing volume compared to inpatient testing. At 10 of the 11 study sites, we observed an increase in the percentage of abnormal high INR results as test volumes decreased, primarily among outpatients. CONCLUSION: The COVID-19 pandemic impacted INR testing among outpatients which may be attributable to several factors. Increased supratherapeutic INR results during the pandemic period when there was reduced laboratory utilization and access to care is concerning because of the risk of adverse bleeding events in this group of patients. This could be mitigated in the future by offering drive-through testing and/or widespread implementation of home INR monitoring.

SARS-CoV-2 Antibody Responses Do Not Predict COVID-19 Disease Severity.

OBJECTIVES: Initial reports indicate adequate performance of some serology-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) assays. However, additional studies are required to facilitate interpretation of results, including how antibody levels impact immunity and disease course. METHODS: A total of 967 subjects were tested for IgG antibodies reactive to SARS-CoV-2, including 172 suspected cases of SARS-CoV-2, 656 plasma samples from healthy donors, 49 sera from patients with rheumatic disease, and 90 specimens from individuals positive for polymerase chain reaction (PCR)-based respiratory viral panel. A subgroup of SARS-CoV-2 PCR-positive cases was tested for IgM antibodies by proteome array method. RESULTS: All specificity and cross-reactivity specimens were negative for SARS-CoV-2 IgG antibodies (0/795, 0%). Positive agreement of IgG with PCR was 83% of samples confirmed to be more than 14 days from symptom onset, with less than 100% sensitivity attributable to a case with severe immunosuppression. Virus-specific IgM was positive in a higher proportion of cases less than 3 days from symptom onset. No association was observed between mild and severe disease course with respect to IgG and IgM levels. CONCLUSIONS: The studied SARS-CoV-2 IgG assay had 100% specificity and no adverse cross-reactivity. Measures of IgG and IgM antibodies did not predict disease severity in our patient population.

Clinical Validation of a SARS-CoV-2 Real-Time Reverse Transcription PCR Assay Targeting the Nucleocapsid Gene.

BACKGROUND: Detection of SARS-CoV-2 viral RNA is important for the diagnosis and management of COVID-19. METHODS: We present a clinical validation of a reverse transcription PCR (RT-PCR) assay for the SARS-CoV-2 nucleocapsid (N1) gene. Off-board lysis on an automated nucleic acid extraction system was optimized with endemic coronaviruses (OC43 and NL63). Genomic RNA and SARS-CoV-2 RNA in a recombinant viral protein coat were used as control materials and compared for recovery from nucleic acid extraction. RESULTS: Nucleic acid extraction showed decreased recovery of endemic Coronavirus in vitro transcribed RNA (NL63) compared with attenuated virus (OC43). SARS-CoV-2 RNA had more reliable recovery from extraction through amplification than genomic RNA. Recovery of genomic RNA was improved by combining lysis buffer with clinical matrix before adding RNA. The RT-PCR assay demonstrated 100% in silico sensitivity and specificity. The accuracy across samples was 100% (75 of 75). Precision studies showed 100% intra-run, inter-run, and inter-technologist concordance. The limit of detection was 264 copies per milliliter (estimated 5 copies per reaction; 35.56 mean threshold cycle value). CONCLUSIONS: This SARS-CoV-2 assay demonstrates appropriate characteristics for use under an Emergency Use Authorization. Endemic coronavirus controls were useful in optimizing the extraction procedure. In the absence of live or attenuated virus, recombinant virus in a protein coat is an appropriate control specimen type for assay validation during a pandemic.