Diagnostic Microbiology and Infectious Disease

The SARS-CoV-2 virus has emerged as a global pandemic, severely impacting everyday life. Significant resources have been dedicated towards profiling the viral genome in the adult population. We present an analysis of viral genomes acquired from pediatric patients presenting to Childrens National Hospital in Washington D.C, including 24 with primary SARS CoV2 infection and 3 with Multisystem Inflammatory Syndrome in Children (MIS-C) undergoing treatment at our facility. Viral genome analysis using next generation sequencing indicated that approximately 81% of the analyzed strains were of the GH clade, 7% of the cases belonged to the GR clade, and 12% of the cases belonged to S, V, or G clades. One sample, acquired from a neonatal patient, presented with the highest viral RNA load of all patients evaluated at our center. Viral sequencing of this sample identified a SARS-CoV-2 spike variant, S:N679S. Analysis of data deposited in the GISAID global database of viral sequences shows the S:N679S variant is present in eight other sequenced samples within the US mid-Atlantic region. The similarity of the regional sequences suggests transmission and persistence of the SARS-CoV-2 variant within the Capitol region, raising the importance of increasing the frequency of SARS-CoV-2 genomic surveillance. IMPORTANCEA variant in the SARS-CoV-2 spike protein was identified in a febrile neonate who was hospitalized with COVID-19. This patient exhibited the highest viral RNA load of any COVID-19 patient tested at our center. Viral sequencing identified a spike protein variant, S:N679S, which is proximal to the cleavage site at residue 681. The SARS-CoV-2 surface spike is a protein trimer (three subunits) which serves as the key target for antibody therapies and vaccine development. Study of viral sequences from the GISAID database revealed eight related sequences from the US mid-Atlantic region. The identification of this variant in a very young patient, its critical location in the spike polyprotein, and the evidence that it has been detected in other patients in our region underscores the need for increased viral sequencing to monitor variant prevalence and emergence, which may have a direct impact on recommended public health measures and vaccination strategies.

BackgroundThe increasing number of cases and hospital admissions due to COVID-19 created an urgent need for rapid, reliable testing procedures for SARS-CoV-2 in Emergency Departments (ED) in order to effectively manage hospital resources, allocate beds and prevent nosocomial spread of infection. The ID NOW COVID-19 assay is a simple, user-friendly, rapid molecular test run on an instrument with a small footprint enabling point-of-care diagnostics. MethodsIn the first wave, outsourced RT-PCR testing regularly required 36-48 hours before results were available. This prospective study was conducted in the second wave (October 2020-April 2021) and evaluated the impact the implementation of the ID NOW COVID-19 test in the ED had on clinical care processes and patient pathways. 710 patients were recruited upon arrival at the ED which included those presenting clinical symptoms, asymptomatic individuals or persons fulfilling epidemiological criteria. The first anterior nasal swab was taken by trained nurses in the ambulance or a separate consultation room. The ID NOW COVID-19 test was performed in the ED in strict compliance with the manufacturers instructions and positive or suspected cases were additionally tested with RT_PCR (cobas SARS-COV-2 RT-PCR, Roche) following collection of a second nasopharyngeal NP specimen. ResultsSwabs directly tested with the ID NOW COVID-19 test showed a diagnostic concordance of 98 % (sensitivity 99.59 %, specificity 94.55 %, PPV 97.6 %, NPV 99.05 %) compared to RT-PCR as reference. The 488 patients that tested positive with the ID NOW COVID-19 had a Ct range in RT-PCR results between 7.94 to 37.42 (in 23.2 % > 30). Two false negative results (0.28%) were recorded from patients with Ct values > 30. 14 (1.69%) discordant results were reviewed case-by-case and usually associated with either very early or very advanced stages of infection. Furthermore, patients initially negative with the ID NOW COVID-19 test and admitted to the hospital were tested again on days 5 and 12: no patient became positive. DiscussionThe ID NOW COVID-19 test for detection of SARS-CoV-2 demonstrated excellent diagnostic agreement with RT-PCR under the above-mentioned patients pathways implemented during the second wave. The main advantage of the system was the provision of reliable results within a few minutes. This not only allowed immediate initiative of appropriate therapy and care for COVID-19 (patient benefit) but provided essential information on isolation and thus available beds. This drastically helped the overall finances of the department and additionally allowed more patients to be admitted including those requiring immediate attention; this was not possible during the first wave since beds were blocked waiting for diagnostic confirmation. Our findings also show that when interpreting the results, the clinical condition and epidemiological history of the patient must be taken into account, as with any test procedure. Overall, the ID NOW COVID-19 test for SARS-CoV-2 provided a rapid and reliable alternative to laboratory-based RT-PCR in the real clinical setting which became an acceptable part of the daily routine within the ED and demonstrated that early patient management can mitigate the impact of the pandemic on the hospital.

BACKGROUNDCirculating cell-free DNA (cfDNA) has become a valuable analyte for molecular testing, but requires specialized collection tubes or immediate processing. We investigated the feasibility of using residual plasma from heparin separators, which are routinely used in clinical chemistry, as an accessible and underutilized source for cfDNA biobanking and testing. METHODSWe analyzed matched plasma samples from healthy volunteers in two experiments: an immediate-processing tube comparison across EDTA, Streck, and heparin separators (n = 5) and a clinical-handling simulation that paired EDTA and heparin separator tubes and delayed processing at room temperature versus 4{degrees}C (n = 6). We also analyzed matched EDTA and heparin separator plasma samples from viral PCR-positive patients (Hospital Cohort; n =38). Whole-genome sequencing and genome-wide enriched methylation sequencing were performed to evaluate concordance across multiple benchmarks, including metagenomics, chromosomal copy number, methylome, and fragmentomics. RESULTSUnder immediate processing, heparin separator plasma showed high concordance with EDTA and Streck plasma for methylation patterns (Pearsons r = 0.92-0.93, Spearmans {rho}=0.65-0.70) and fragmentation features (n = 5). In the clinical-handling simulation, cfDNA integrity in heparin separators was comparable to that in EDTA at 4{degrees}C (n=6). In the Hospital Cohort, heparin separators showed a strong concordance with matched EDTA tubes for viral detection (n=38, Pearsons r=0.96), copy number alteration profiling (n=6, Pearsons r=0.96-1.00), and methylation patterns (n=12, r=0.83-0.93). CONCLUSIONHospital residual plasma from routine clinical chemistry tests that are processed within a short pre-centrifugation window and refrigerated can provide a vast, untapped resource for cfDNA biobanking and potential testing.

Concomitant infection of multiple SARS-CoV-2 variants has become an increasing concern, as this scenario increases the likelihood of recombinant variants. Detecting co-infection of SARS-CoV-2 variants is difficult to detect by whole genome sequencing approaches, but genotyping methods facilitate detection. We describe 2 cases of Delta/Omicron and 2 cases of Omicron sublineage BA.1/ BA.2 co-infection as detected by a multiplex genotyping fragment analysis method. Findings were confirmed by whole genome sequencing. Review of the patient characteristics revealed co-morbidities and conditions which weaken the immune system and may make them more susceptible to harboring SARS-CoV-2 variant co-infections.

The emergence of new SARS-CoV-2 variants necessitates the reevaluation of current COVID-19 tests to ensure continued accuracy and reliability. The new SARS-CoV-2 variant, Omicron, is heavily mutated, with over 50 mutations within its RNA genome. Any of these mutations could adversely affect the ability of diagnostic assays to detect the virus in patient samples, potentially leading to inconclusive or false negative results. In fact, the U.S. Food and Drug Administration (FDA) has identified over two dozen diagnostic tests that contain a gene target that is expected to have "significantly reduced sensitivity due to a mutation in the SAS-CoV-2 Omicron variant"1. Additionally, one of the U.S. Centers for Disease Control and Prevention (CDC) Emergency Use Authorization (EUA) targets for COVID-19 tests, 2019-nCoV_N1, overlaps an Omicron mutation within the sequence targeted by the fluorescent probe. This target from the CDC has been used in many other EUA assays. Using in vitro transcribed (IVT) N gene RNA representing the wild-type (GenBank/GISAID ID MN908947.3) and Omicron variant (BA.1, GISAID ID EPI_ISL_6752027), we evaluated the performance of two different amplification protocols, both of which include the CDC 2019-nCoV_N1 primer-probe set. Both assays were able to detect the mutant N1 sequence as efficiently as the wild-type sequence. Consequently, these data suggest that diagnostic assays that use the 2019-nCoV-N1 primer-probe set are unlikely to be impacted by currently circulating Omicron lineage viruses.

BackgroundIn recent studies, up to half of immunocompromised (IC) subject populations fail to develop antibodies after COVID-19 vaccination. Purpose and MethodsHere, we explore whether T-cells which respond to the spike (S) antigenic sequence and its less conserved S1, and the conserved S2 component are present in serial samples before and after each dose of mRNA1273 or BNT162b2 vaccines in 20 healthy immunocompetent subjects. Single samples from 7 vaccinated IC subjects were also tested. Simultaneously, we measured IgG antibodies to the receptor binding domain (RBD) of S1, and anti-S IgG, and frequencies of monocytic CD14+HLA-DR-(M-MDSC) and polymorphonuclear CD14-CD15+CD11b+ (PMN-MDSC) myeloid-derived suppressor cells. ResultsIn healthy subjects, S1-, S2-, and S-reactive CD4 and CD8 T-cell frequencies showed a numeric but not statistically significant decrease after the first vaccine dose and were accompanied by increased MDSC frequencies (p<0.05). After the second dose, S2-and S-reactive CD4 and CD8 cells and MDSC approached pre-vaccination levels. In healthy subjects, a) S1-reactive CD8 frequencies were significantly higher after the second dose compared with pre-vaccination levels (p=0.015), b) anti-RBD and anti-S IgG were present in all after the second dose. Among seven IC subjects, anti-RBD and anti-S IgG were absent in 4 and 3 subjects, respectively. S1-reactive CD8 cells were identified in 2 of 4 anti-RBD negative subjects. S-reactive CD4 or CD8 cells were identified in all three anti-S negative subjects. ConclusionsIn healthy immunocompetent subjects, mRNA vaccines induce antibodies to the spike antigenic sequences and augment CD8 cells reactive to the S1 spike sequence, which is more specific for the SARS-CoV-2 virus. In this exploratory cohort of vaccinated immunocompromised subjects, S1-reactive CD8 cells can be detected in some who are negative for RBD antibody, and S-reactive T-cells are present in all who are negative for spike antibody.

BackgroundThe COVID-19 pandemic has been characterized by ongoing evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with concomitant variation in viral transmissibility and morbidity. Within specific timeframes and geographic areas, multiple SARS-CoV-2 variants have coexisted in the human population, each characterized by distinct biologic and clinical features, such as varying susceptibility to neutralizing monoclonal antibodies (nMAbs), a major frontline treatment. As part of an observational real-world data study of the effectiveness of nMAbs for treatment of COVID-19, SARS-CoV-2 viral samples were obtained from patients under treatment, generating paired clinical and genomics data. This paper describes the processing pipeline and findings from the genomics portion of this combined data set. MethodsSARS-CoV-2 sequences were generated from 14,796 diagnostic samples from four large U.S. health systems between July 2020 and March 2022. Among nMAbs-treated patients, samples were collected on the same day as, or prior to, treatment with nMAbs. Thus, these samples represent a snapshot of SARS-CoV-2 variants circulating in the respective patient groups, as opposed to variants that arose in response to specific treatments. Health systems collected viral samples and performed library creation and sequencing according to local protocols, using tiled ARTIC amplicon primers. FASTQ files were submitted to a study data platform and processed through a common pipeline. This pipeline enabled a unified approach to quality control, assembly, and production of genomics features for downstream analysis. ResultsAlpha and pre-Alpha SARS-CoV-2 lineages were predominant in the data set prior to June 2021. From June 2021 through November 2021, Delta was the dominant variant. Beginning in December 2021, Omicron was dominant. A variety of mutations associated with decreased nMAbs binding to the spike protein in vitro were detected, including lineage-defining mutations and non-lineage-defining mutations such as E340A, G446V, and S494P. Distinct patterns of sequence gaps and ambiguous base calls were associated with distinct variants. ConclusionsThe distribution of SARS-CoV-2 variants, per WHO nomenclature, across epochs in this data set matched concurrent CDC genomic surveillance results across the U.S. Detection of putative nMAbs escape mutations within clinical samples was consistent with FDA decisions to amend EUAs as variants emerged. This genomics data set provides an opportunity to examine associations between SARS-CoV-2 genomic variation and clinical outcomes in the associated EHR data set. The expansion of real-world data sets such as this to study the relationship between viral sequence and treatment outcomes could provide the foundation for future efforts to achieve near-real-time understanding of clinical outcomes related to genomic variation over time, and evidence to update treatment decisions more rapidly and to greater effect during ongoing and future pandemics.

Rapid molecular diagnostic tests have been critical in the response to the COVID-19 pandemic. It is important to evaluate the ability of these assays to identify variants of concern, particularly across varying collection and storage conditions. Nasal swabs positive for Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.167.2), Gamma (P.1), or Omicron (B.1.1.529) variants of concern (VOCs) were stored in TE buffer and viral transport media (VTM). We evaluated the sensitivity of the Cepheid Xpert(R) Xpress SARS-CoV-2 assay in detecting VOC samples and validated TE buffer for use with the assay. Testing of known VOC positives revealed no substantial reduction of PCR sensitivity. Comparison of TE and VTM samples also revealed no reduction in performance when using TE buffer, validating the use of TE buffer to store SARS-CoV-2 samples. SARS-CoV-2 VOCs collected and stored across various conditions can be detected by the Cepheid Xpert(R) Xpress SARS-CoV-2 assay.

Diagnostic testing and evaluation of patient immunity against the novel severe acute respiratory syndrome (SARS) corona virus that emerged last year (SARS-CoV-2) are essential for health and economic crisis recovery of the world. It is suggested that potential acquired immunity against SARS-CoV-2 from prior exposure may be determined by detecting the presence of circulating IgG antibodies against viral antigens, such as the spike glycoprotein and its receptor binding domain (RBD). Testing our asymptomatic population for evidence of COVID-19 immunity would also offer valuable epidemiologic data to aid health care policies and health care management. Currently, there are over 100 antibody tests that are being used around the world without approval from the FDA or similar regulatory bodies, and they are mostly for rapid and qualitative assessment, with different degrees of error rates. ELISA-based testing for sensitive and rigorous quantitative assessment of SARS-CoV-2 antibodies can potentially offer mechanistic insights into the COVID-19 disease and aid communities uniquely challenged by limited financial resources and access to commercial testing products. Employing recombinant SARS-CoV-2 RBD and spike protein generated in the laboratory, we devised a quantitative ELISA for the detection of circulating serum antibodies. Serum from twenty SARS-CoV-2 RT-PCR confirmed COVID-19 hospitalized patients were used to detect circulating IgG titers against SARS-CoV-2 spike protein and RBD. Quantitative detection of IgG antibodies to the spike glycoprotein or the RBD in patient samples was not always associated with faster recovery, compared to patients with borderline antibody response to the RBD. One patient who did not develop antibodies to the RBD completely recovered from COVID-19. In surveying 99 healthy donor samples (procured between 2017-February 2020), we detected RBD antibodies in one donor from February 2020 collection with three others exhibiting antibodies to the spike protein but not the RBD. Collectively, our study suggests that more rigorous and quantitative analysis, employing large scale samples sets, is required to determine whether antibodies to SARS-CoV-2 spike protein or RBD is associated with protection from COVID-19 disease. It is also conceivable that humoral response to SARS-CoV-2 spike protein or RBD works in association with adaptive T cell response to determine clinical sequela and severity of COVID-19 disease.

Rapid antigen detection tests (RADTs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are now in widespread use in the United States. RADTs play an important role in maintaining an open society but require periodic reassessment to ensure test performance remains intact as the virus evolves. The nucleocapsid (N) protein is the target for the majority of RADTs and the SARS-CoV-2 Omicron variant has several N protein mutations that are previously uncharacterized. We sought to assess the impact of these mutations by testing 30 Omicron variant samples across a wide range of viral loads on three widely used RADTs: the iHealth COVID-19 Antigen Rapid Test, the ACON Laboratories FlowFlex COVID-19 Antigen Home Test, and the Abbott BinaxNOW COVID-19 Antigen Card, using 30 Delta variant samples as a comparator. We found no change in the analytic sensitivity of all three RADTs for detection of Omicron versus Delta, but noted differences in performance between assays. No RADT was able to detect samples with a cycle threshold (Ct) value of [&ge;]27.5 for the envelope gene target on the Roche cobas RT-PCR assay. Epidemiologic studies are necessary to correlate these findings with their real-world performance.

BackgroundRapid identification of emergency department (ED) patients with a possible COVID-19 infection is needed. PCR-testing all ED patients is neither feasible nor effective in most centers, therefore a rapid, objective, low-cost screening tool to triage ED patients is necessary. MethodsResults from all routine lab tests from ED patients at the Catharina Hospital were collected from July 2019 to July 2020 and used in a statistical model to obtain the CoLab-score. The score was validated temporally and externally in three independent centers. ResultsThe CoLab-score consists of 10 routine lab results and can be used to safely rule-out a COVID-19 infection in more than one third of ED presentations with a negative predictive value of 0.997 (95% CI: 0.994 - 0.999). ConclusionsThe CoLab-score is a valuable tool to rule out COVID-19, guide PCR testing and is available to any center with access to routine laboratory tests.

ImportanceHereditary hematopoietic malignancies (HHMs) are hereditary cancer syndromes that constitute at least 14% of all myeloid malignancies, but genetic assays used to diagnose HHMs have historically been of variable quality. Here, we demonstrate that HHM assays continue to have persistent shortcomings. These diagnostic gaps place patients with HHMs at high risk for missed diagnoses, missed opportunities for cancer screening, and donor-derived leukemias following stem cell transplant. ObjectiveTo determine if the quality of HHM diagnostic assays has improved since 2020, when our group first demonstrated that most HHM diagnostic tests were insufficient for the accurate diagnosis of these syndromes. We hypothesized that the number of genes tested on each HHM assay increased from 2020 to 2022, in keeping with a more comprehensive sequencing approach. Design, Setting, and ParticipantsWe analyzed assays from eight commercial laboratories to determine which HHM-related genes were sequenced by these assays. We compared these assays to panels from 2020 to determine trends in sequencing quality. ResultsThe majority of HHM diagnostic assays did not change over time and are insensitive for the detection of the full spectrum of HHM-related mutations. The majority (75%) of HHM assays do not sequence CHEK2, the gene most frequently mutated in HHMs, and 25% of HHM assays do not sequence DDX41, the second most frequently mutated HHM gene. ConclusionsThe quality of HHM diagnostic assays has stagnated despite the discovery of novel HHM-related genes as well as prior work demonstrating heterogeneity in quality of HHM testing. The majority of commercially available HHM tests remain insufficient for the diagnosis of the full spectrum of HHM-related germline mutations. Key PointsO_ST_ABSQuestionC_ST_ABSHow have diagnostic assays for hereditary hematopoietic malignancies (HHMs) changed since 2020, when most HHM diagnostic assays were inadequate for the accurate diagnosis of HHMs? FindingsMost HHM assays have significant deficiencies in quality and do not sequence the most relevant HHM-related genes. No meaningful improvements in the quality of HHM diagnostic testing have occurred since 2020. MeaningThe quality of HHM diagnostic testing must be improved to universally include the most common HHM-related germline mutations. This will reduce the risk for false negatives, donor derived leukemias, improve genetic counseling, and improve screening for other HHM-related malignancies.

Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection.1-4 It has been used by the Centers for Disease Control and Prevention (CDC) and several other companies in their Emergency Use Authorization (EUA) assays. With many PCR-based molecular assays, an extraction step is routinely used as part of the protocol. This step can take up a significant amount of time and labor, especially if the extraction is performed manually. Long assay time, partly caused by slow sample preparation steps, has created a large backlog when testing patient samples suspected of COVID-19. Using flu and RSV clinical specimens, we have collected evidence that the RT-qPCR assay can be performed directly on patient sample material from a nasal swab immersed in virus transport medium (VTM) without an RNA extraction step. We have also used this approach to test for the direct detection of SARS-CoV-2 reference materials spiked in VTM. Our data, while preliminary, suggest that using a few microliters of these untreated samples still can lead to sensitive test results. If RNA extraction steps can be omitted without significantly affecting clinical sensitivity, the turn-around time of COVID-19 tests and the backlog we currently experience can be reduced drastically. Next, we will confirm our findings using patient samples.

IntroductionConcerns have been raised regarding the accuracy of diagnostic antigen testing for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant. We compared the performance of the LumiraDx SARS-CoV-2 Antigen Test between symptomatic participants recruited prospectively during the Delta to Omicron variant transition in the USA. MethodsTwo paired anterior nasal swabs were collected from each participant (adults and children) within 12 days of symptom onset between November 24th, 2021 and February 1st, 2022, during which time Omicron replaced Delta as the dominant variant in the sample population. Swabs were tested by the LumiraDx SARS-CoV-2 Antigen Test and compared using real-time polymerase chain reaction (RT-PCR) reference testing. Reference samples identified as positive were sequenced to identify the SARS-CoV-2 variant. Positive percent agreement (PPA) was calculated, with results stratified by RT-PCR cycle threshold (Ct). ResultsOf the 38 participants for whom LumiraDx SARS-CoV-2 Antigen Test results were available, 36 were confirmed positive by RT-PCR. Overall, PPA of the LumiraDx SARS-CoV-2 Antigen Test was 94.7% (95% confidence interval: 82.3%, 99.4%) and PPA was 100% for samples with a Ct <33. Sufficient viral load for sequencing was present in nine samples (six Delta, three Omicron), all of which returned a positive result using the LumiraDx SARS-CoV-2 Antigen Test. There were no performance differences observed between participants with the Delta and Omicron variants. ConclusionsSARS-CoV-2 differences between Delta and Omicron variant mutations did not affect the performance of the LumiraDx SARS-CoV-2 Antigen Test which detects the nucleocapsid protein antigen. The LumiraDx SARS-CoV-2 Antigen Test can be a useful antigen test to diagnose emerging variants of coronavirus disease 2019.

IntroductionAntibodies to the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can increase as soon as 10-13 days after infection. We describe our evaluation of the Abbott SARS-CoV-2 IgG assay on the Architect immunoassay analyser. MethodsWe assessed the precision, sensitivity, and specificity of the Abbott SARS-CoV-2 IgG assay in samples from polymerase chain reaction (PCR) positive patients and healthy healthcare workers. The manufacturer cut-off index (COI) of 1.4 was adopted to identify positive results. We examined the assay cross-reactivity with other viral antibodies (influenza/dengue/hepatitis C/hepatitis B) and rheumatoid factor (RF). The sample throughput of the Abbott assay was also assessed. ResultsThe Abbott assay showed excellent precision, with a CV of 3.4% for the negative control (COI = 0.06) and 1.6% for a high positive serum sample (COI = 8.6). Residual serum was available from 57 inpatients not initially suspected of having COVID-19, 29 of whom tested positive for SARS-CoV-2 IgG. The Abbott assay has a sensitivity of 90.9-100% when tested in 54 subjects [&ge;]14 days post PCR positive, and a specificity of 100% (N = 358). There was no cross-reactivity with other viral antibodies (influenza/dengue/hepatitis C/hepatitis B) and RF. The Architect Abbott assay has a throughput of 100 samples in 70 minutes. ConclusionThe Abbott SARS-CoV-2 IgG assay shows excellent performance that is well within FDA and CDC guidelines when testing patients [&ge;]14 days POS with little cross-reactivity from other viral antibodies. There is some evidence that SARS-CoV-2 IgG develops early in the disease process. IMPACT STATEMENTWith the current SARS-CoV-2 pandemic still ongoing, laboratories are hard pressed to introduce SARS-CoV-2 antibody testing to help as an indirect marker for infection to identify patients with prior infection/exposure. The Abbott SARS-CoV-2 IgG assay has excellent performance with good precision, specificity, and sensitivity [&ge;]14 days after a positive SARS-CoV-2 PCR test, with a competitive throughput of 100 samples in 70 minutes. It shows no cross-reactivity with other viral antibodies (influenza/dengue/hepatitis B/hepatitis C) and rheumatoid factor. We have also found evidence of early antibody development in some patients before they tested positive on the SARS-CoV-2 PCR test.

Cytomegalovirus (CMV) infection monitoring is a key element in the management of immunocompromised patients. CMV DNA quantification in plasma or whole blood is the best indicator for clinicians to adjust immunosuppressive or antiviral therapies. Despite the availability of internationally standardized material, the commutability of CMV quantification results across laboratories remains inadequate. To assess inter-laboratory variability in CMV DNA quantification, we conducted a blinded study in seven independent laboratories. Each participant received a panel of 92 specimens for CMV quantification using their routinely used standard platform. While quantifications were highly correlated and reproducible, large discrepancies were observed with differences up to 1.45 log10 IU/mL between techniques for identical specimens. However, quantification scattering was lower for the WHO international standard or a commercially tested control (IQR=0.129) than for clinical specimens (0.469; p=0.0142). Blind quantification of the WHO or the commercial standard indicated that all techniques, except for fully integrated platforms, did not align well with the expected values and most platforms tended to quantify specimens and standards differently. Recalibration of all platforms against the same standard improved the spread of results, but differences of up to 1.19 log10 IU/mL remained for the same specimens. Achieving commutability in CMV quantification remains an elusive goal. Efforts should focus on improving both the assay calibrators and the run controls, which currently do not appear to simulate the unique characteristics of circulating CMV in patients. Until this is resolved, each transplanted patient should be consistently monitored by the same laboratory on the same platform.

Rapid classification and detection of SARS-CoV-2 variants have been critical in comprehending the viruss transmission dynamics. Clinical manifestation of the infection is influenced by comorbidities such as age, immune status, diabetes, and the infecting variant. Thus, clinical management may differ for new variants. For example, some monoclonal antibody treatments are variant-specific. Yet, an FDA-approved test for detecting the SARS-CoV-2 variant is unavailable. A laboratory-developed test (LDT) remains a viable option for reporting the infecting variant for clinical intervention or epidemiological purposes. Accordingly, we have validated the Illumina COVID-Seq assay as an LDT according to the guidelines prescribed by the College of American Pathologists (CAP) and Clinical Laboratory Improvement Amendments (CLIA). The limit of detection (LOD) of this test is Ct<30 ([~]15 viral copies) and >200X genomic coverage, and the test is 100% specific in the detection of existing variants. The test demonstrated 100% precision in inter-day, intra-day, and intra-laboratory reproducibility studies. It is also 100% accurate, defined by reference strain testing and split sample testing with other CLIA laboratories. Advanta Genetics LDT COVID Seq has been reviewed by CAP inspectors and is under review by FDA for Emergency Use Authorization.

SARS-CoV-2 infection has been playing havoc with emerging omicron variants of concern (VoC). Here, we report sequencing of the omicron variant in 13 patients from India using Oxford Nanopore Technology (ONT) Minion, wherein a rapid amplicon based sequence analysis was performed to assess and compare with existing 34 mutations in spike glycoprotein. We highlight and discuss the nature of these mutations that are unique and common to other populations. This is perhaps the first report on omicron variants from India using a long read sequencing chemistry.

IntroductionCoronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is diagnosed by molecular-based detection of SARS-CoV-2 RNA. Serologic testing detects antibodies specific to SARS-CoV-2 and IgM specifically may serve as an adjunct test to PCR early in disease. We evaluated the Abbott anti-SARS-CoV-2 IgM and IgG assays along with DiaSorin anti-SARS-CoV-2 IgG and Roche anti-SARS-CoV-2 Total. MethodsSpecimens from 175 PCR-positive patients and 107 control specimens were analyzed using Abbott IgM and IgG, DiaSorin IgG, and Roche Total (IgA, IgG, IgM) assays. Sensitivity, specificity, cross-reactivity, concordance between assays, trends over time, positive predictive value (PPV), and negative predictive value (NPV) were determined. ResultsAbbott IgM sensitivity was 63.6% at 0 days post-PCR positivity, 76.5% at 1-5d, 76.3% at 6-14d, 85.2% at 15-30d, and 63.6% at >30d. All assays exhibited highest sensitivity 15-30d post-PCR positivity (83.3-85.2%). Combining Abbott IgM and IgG improved sensitivity by 22.7% compared to IgG alone when tested 0d post-PCR positivity. All assays had a specificity of 100% and only Abbott IgG exhibited cross-reactivity (anti-dsDNA). Cohens kappa varied between 0.86-0.93. Time to seroconversion from PCR positivity was lowest for Abbott IgM and highest for Abbott IgG. NPV was highest for Abbott IgM <14 days post-PCR positivity and Abbott IgG [&ge;]14 days. ConclusionThe Abbott IgM assay exhibited the earliest response and greatest signal in most patients evaluated for serial sampling and had the highest NPV <14 days post-PCR positivity, suggesting its potential utility as an adjunct test to PCR early in disease course.

BackgroundDetermining the severity of COVID-19 remains an unmet medical need. Our objective was to develop a blood-based host-gene-expression classifier for the severity of viral infections and validate it in independent data, including COVID-19. MethodsWe developed the classifier for the severity of viral infections and validated it in multiple viral infection settings including COVID-19. We used training data (N=705) from 21 retrospective transcriptomic clinical studies of influenza and other viral illnesses looking at a preselected panel of host immune response messenger RNAs. ResultsWe selected 6 host RNAs and trained logistic regression classifier with a cross-validation area under curve of 0.90 for predicting 30-day mortality in viral illnesses. Next, in 1,417 samples across 21 independent retrospective cohorts the locked 6-RNA classifier had an area under curve of 0.91 for discriminating patients with severe vs. non-severe infection. Next, in independent cohorts of prospectively (N=97) and retrospectively (N=100) enrolled patients with confirmed COVID-19, the classifier had an area under curve of 0.89 and 0.87, respectively, for identifying patients with severe respiratory failure or 30-day mortality. Finally, we developed a loop-mediated isothermal gene expression assay for the 6-messenger-RNA panel to facilitate implementation as a rapid assay. ConclusionsWith further study, the classifier could assist in the risk assessment of COVID-19 and other acute viral infections patients to determine severity and level of care, thereby improving patient management and reducing healthcare burden.