Intrathecal inflammatory responses in the absence of SARS-CoV-2 nucleic acid in the CSF of COVID-19 hospitalized patients

Objective: Evaluate SARS-CoV-2 RNA and inflammatory cytokines and chemokines in the CSF of patients with acute COVID-19 and neurologic symptoms, and to compare these to controls and patients with known neurotropic pathogens. Background: Neurologic symptoms have been described in 30-60% of hospitalized patients with Coronavirus Disease 2019 (COVID-19). However, little is known about CSF profiles in these patients. Design/Methods: CSF from twenty-seven consecutive patients with COVID-19 and neurological symptoms was assayed for SARS-CoV-2 RNA using quantitative reverse transcription PCR (RT-qPCR) and unbiased metagenomic sequencing. Assays for blood brain barrier (BBB) breakdown (CSF:serum albumin ratio (Q-Alb)), and proinflammatory cytokines and chemokines (IL-6, IL-8, IL-15, IL-16, monocyte chemoattractant protein -1 (MCP-1) and monocyte inhibitory protein - 1β (MIP-1β)) were performed in 23 patients and compared to CSF from patients with HIV-1 (16 virally suppressed, 5 unsuppressed), West Nile virus (WNV) (n=4) and 16 healthy controls (HC). Results: Median CSF cell count for COVID-19 patients was 1 white blood cell/μL;two patients were infected with a second pathogen (Neisseria, Cryptococcus neoformans). No CSF samples had detectable SARS-CoV-2 RNA by either detection method. In patients with COVID-19 only, CSF IL-6, IL-8, IL-15, and MIP-1β levels were higher than HC and suppressed HIV (corrected-p < 0.05). MCP-1 and MIP-1β levels were higher, while IL-6, IL-8, IL-15 were similar in COVID-19 compared to WNV patients. Q-Alb correlated with all proinflammatory markers, with IL-6, IL-8, and MIP-1β (r≥0.6, p<0.01) demonstrating the strongest associations. Conclusions: Lack of SARS-CoV-2 RNA in CSF is consistent with pre-existing literature. Evidence of intrathecal proinflammatory markers in a subset of COVID-19 patients with BBB breakdown despite minimal CSF pleocytosis is atypical for neurotropic pathogens.

Single-amplicon, Multiplex Real-time RT-PCR with Tiled Probes to Detect SARS-CoV-2 spike Mutations Associated with Variants of Concern

Background. Detection and surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is of great public health importance. Broadly accessible and inexpensive assays are needed to enhance variant surveillance and detection globally. We developed and validated a single-reaction multiplex real-time RT-PCR (the Spike SNP assay) to detect specific mutations associated with variants of concern (VOC). Methods. A single primer pair was designed to amplify a 348 bp region of spike. Probes were initially designed with locked nucleic acids (LNAs) to increase probe melting temperature, shorten probe length, and specifically detect 417K, E484K, and N501Y (Figure). The assay was optimized and evaluated using characterized variant sample pools. Clinical evaluation was performed on a convenience set of residual nasopharyngeal swabs, and variant calls were confirmed by SARS-CoV-2 genomic sequencing in a subset of samples. Following the initial evaluation, unmodified probes (without LNAs) were designed to detect L452R, L452Q, and E484Q. Figure. Spike SNP distinguishes mutations occurring in different lineages (A-C). Representative results of variant detection a single Spike SNP run are shown for mutations in the codons for 4177K (A) and mutations that encode 484K (B) and 501Y (C). Curves show dilutions of the following variants: blue, BEI 52286 (wild type);pink B.1.1.7;purple, B1.525;and green, P.1. Variant pools were used for B.1.17, B.1.525, and P.1 strains. Curves are displayed for a given dilution in each channel and result interpretation is shown (D). Results. The lower limit of 95% detection was 2.46 to 2.48 log10 GE/mL for the three targets (~1-2 GE/reaction). Among 253 nasopharyngeal swabs with detectable SARS-CoV-2 RNA, the Spike SNP assay was positive in 238 (94.1%), including all samples with Ct values < 30 (220/220) for the N2 target and 18/33 samples with N2 Ct values ≥ 30. Results were confirmed by SARS-CoV-2 genomic sequencing in 50/50 samples (100%). Subsequent addition of the 452R probe did not affect performance for the original targets, and probes for 452Q and 484Q performed similarly to LNAmodified probes. Conclusion. The Spike SNP assay provides fast, inexpensive and sensitive detection of specific mutations associated with SARS-CoV-2 VOCs, and the assay can be quickly modified to detect new mutations in the receptor binding domain. Similar analytical performance of LNA-modified and unmodified probes presents options for future assay customization that balance the shorter probe length (LNAs) and increased accessibility (unmodified). The Spike SNP assay, if implemented across laboratories offering SARS-CoV-2 testing, could greatly increase capacity for variant detection and surveillance globally.

Immunocompromised Patients with Prolonged Viral Shedding of SARS-COV-2

Background. Most individuals diagnosed with mild to moderate COVID-19 are no longer infectious after day 10 of symptom onset and those with severe or critical illness from COVID are typically not infection after day 20 day of symptom onset. Recovered persons can continue to test positive for SARS-CoV-2 by PCR via detection of non-viable RNA in nasopharyngeal specimens for up to three months (or longer) after illness onset. It is also know known that severely immunocompromised patients may produce replication-competent virus greater than 20 days from symptom onset and may require, per CDC recommendations, "additional testing and consultation with infectious diseases specialists and infection control experts". We aim to discuss four case studies of severely immunocompromised patients who exhibited signs of persistent COVID-19 infection of COVID and how we managed transmission-based precautions in our hospital through sequencing and evaluation of cycle thresholds (CT) values and subgenomic RNA detection. Methods. Residual nasopharyngeal (NP) samples were collected on patients exhibiting persistent COVID like symptoms. These samples underwent N gene and N gene subgenomic RNA (sgRNA) real-time reverse transcription polymerase chain reaction (rRT-PCR) testing. Results. Analysis of longitudinal SARS-CoV-2 sequence data demonstrated within-patient virus evolution, including mutations in the receptor binding domain and deletions in the N-terminal domain of the spike protein, which have been implicated in antibody escape. See Figures 1 and 2. Figure 1. Timelines of Identified Patients 1 and 2 Patient 1: 46-year-old woman with recently diagnosed stage IV diffuse large B-cell lymphoma for which she was treated with 2 cycles of R-CHOP. Patient 2: 38-year-old woman with history of myelodysplastic syndrome, peripheral blood stem cell transplant with chronic graft versus host disease of the GI tract, skin, and eyes as well as CMV enteritis, and she was maintained on rituximab, mycophenolate mofetil, prednisone, and monthly IVIG without recent changes to her immunosuppression. Figure 2. Timeline of Identified Patients 3 and 4 Patient 3: 44 year-old man with prior history of thymoma s/p thymectomy Patient 4: 46 year-old man who was initially diagnosed with marginal zone lymphoma approximately 2.5 years ago. He was initially treated with bendamustine and rituximab and achieved remission. He was then continued on maintenance rituximab without significant complications for a planned two years. Conclusion. Differentiating between prolonged viral shedding of non-infectious RNA and persistent replicating viable virus can be difficult to determine without full evaluation of a patient's clinical picture and timeline. Consultation between laboratory, infectious diseases, and infection prevention experts to provide appropriate level of guidance for precautions and treatment may be warranted. Testing by PCR and analysis of CT values may provide key findings of viral replication in immunocompromised hosts, indicating the need for evaluation of additional treatment and maintaining isolation status in healthcare settings.

Investigation of patient and viral characteristics associated with SARS-CoV-2 vaccine breakthrough infections in Atlanta, GA

Purpose of Study Despite the tremendous success of SARSCoV- 2 vaccines, breakthrough infections occur and are being recognized with increasing frequency. It is unclear whether breakthrough infections are the result of host and/or viral factors. We examined clinical and viral genomic data from patients with SARS-CoV-2 infection after vaccination to elucidate factors contributing to breakthrough. Methods Used This study was conducted in the Emory Healthcare (EHC) System. Patients with vaccine breakthrough infection, defined as a positive PCR test ≥14 days after the final dose of an FDA approved vaccine, were identified by both routine surveillance and notification by treating clinicians. Vaccination status was obtained from the Georgia Registry of Immunization Transactions and Services records by the Georgia Emerging Infections Program. Clinical information was derived from electronic medical records and was compared to data from 2-3 matched controls per case. Residual SARS-CoV-2 positive nasopharyngeal (NP) samples were collected and underwent RNA extraction. SARSCoV- 2 genome sequencing was performed using random-primer cDNA synthesis, Nextera XT library preparation, and Illumina sequencing. Summary of Results Forty vaccine breakthrough cases were identified between March 22 and July 16, 2021. The median time from final vaccine dose to positive COVID-19 test was 91 days (range 15-163). Compared to 94 controls, vaccine breakthrough cases were significantly older (median 57.5 years vs 42.0 years, p<.0001). Individuals over 60 accounted for half of all breakthrough cases, and individuals over 40 accounted for 80%. Immunosuppressed individuals represented 37.5% of breakthrough cases compared to 25% of unvaccinated controls. Rates of symptomatic infection and severe disease leading to hospitalization were similar between cases and controls. There was no difference in SARS-CoV-2 RT-PCR cycle threshold (Ct) between cases (n=32, median Ct=20.7, interquartile range (IQR)- 10.3) and controls (n=94, median Ct=24.0, IQR= 7.0;p=0.34). SARS-CoV-2 genome sequences from 24 cases were compared to 116 baseline surveillance sequences from unvaccinated EHC patients. There was no distinct phylogenetic clustering of vaccine breakthrough cases, and their sequences belonged to the predominant lineage of the time. From March 22-June 19, B.1.1.7 (alpha) accounted for 78% of breakthrough infections and 77% of surveillance sequences. From June 20-July 16, B.1.617.2 (delta) accounted for 86% of breakthrough infections and 72% of surveillance sequences. No spike mutations or deletions were associated with vaccine breakthrough infections. Conclusions Overall, our findings suggest that host factors, such as older age and immunosuppression, play a more important role than viral factors in SARS-CoV-2 vaccine breakthrough infections. Further studies are needed to understand the potential impacts of waning immunity or poor immunogenicity in individuals who experience vaccine breakthrough infections.

Outbreak of SARS-CoV-2 in hospitalized hemodialysis patients: An epidemiologic and genomic investigation

Purpose of Study Healthcare-associated transmission of SARSCoV- 2 is relatively rare and may be difficult to quantify. We performed an epidemiological investigation and SARSCoV- 2 genome sequencing to define the source and scope of a SARS-CoV-2 outbreak in a cluster of hospitalized patients. Methods Used We conducted an outbreak investigation after identifying hospital-onset COVID-19 in patients receiving hemodialysis in January 2021. Electronic medical record review, staff interviews, review of employee schedule logs, and contact tracing were used to determine the outbreak timeline and identify potentially exposed healthcare workers (HCW). SARS-CoV-2 genomes were sequenced from residual nasopharyngeal swab samples from 6 individuals in the outbreak investigation and compared to sequences from 14 patients in the same facility, 54 patients in nearby facilities, and 375 publicly available sequences from individuals in the state of Georgia. Summary of Results Eight patients with hospital-onset COVID- 19 were identified (Cases 1-8);all were receiving hemodialysis and 5 were bedded in a single inpatient nursing unit. Among 53 potentially exposed HCW, 29 underwent testing and 5 were positive (Cases 9-13). The suspected index patient (Case 1) was found to have been coughing and inconsistently wearing a mask during a hemodialysis session on the same day that 5 of the 7 other patients and one HCW (Case 10) were in close proximity in the hemodialysis unit (figure 1A). Further investigation revealed lack of use of curtain barriers in the hemodialysis bays, inconsistent use of personal protective equipment by HCW, and overcrowding of staff breakrooms. Among the only 6 samples available for phylogenetic analysis, SARS-CoV-2 sequences from 5 (4 patients and 1 HCW, Case 9) were identical and at least 4 SNPs removed from the next closest sequence in this study, supporting a transmission cluster (figure 1B ). The sequence from the sixth sample (HCW Case 10 ) was phylogenetically distinct, indicating an independent source of infection. Conclusions Lack of appropriate respiratory hygiene led to SARS-CoV-2 transmission during a single hemodialysis session, based on clinical and molecular epidemiology. Use of appropriate PPE for both patients and HCW and other infection prevention measures are critical to prevent SARS-CoV-2 transmission.

Metagenomic sequencing to identify alternative infections and co-infections in persons under investigation for COVID-19

Background: Broad testing for respiratory viruses among persons under investigation (PUI) for SARS-CoV-2 is performed inconsistently, limiting our understanding of alternative infections and co-infections in these patients. Here, we used unbiased metagenomic next-generation sequencing (mNGS) to assess the frequencies of 1) alternative viral infections in SARS-CoV-2 RT-PCR negative PUIs and 2) viral co-infections in SARS-CoV-2 RT-PCR positive PUIs. Methods: A convenience sample set was selected from PUIs who were tested for SARS-CoV-2 in the Emory Healthcare system during the first 2 months of the pandemic from 02/26-04/23/20. Laboratory results were extracted by chart review;Flu/RSV and multiplex respiratory pathogen PCRs had been performed at the discretion of treating physicians. Excess nasopharyngeal swab samples were retrieved within 72 hours of collection and underwent RNA extraction and SARS-CoV-2 testing by triplex RT-PCR. mNGS was performed by DNAse treatment, random primer cDNA synthesis, Nextera XT tagmentation, and high-depth Illumina sequencing. Reads underwent taxonomic classification by KrakenUniq, as implemented in viral-ngs. Results: 53 PUIs were included, 30 negative and 23 positive for SARS-CoV-2 by RT-PCR. Among SARS-CoV-2 negative PUIs, 28 (93%) underwent clinical testing for alternative infections, and 8 (29%) tested positive for another respiratory virus. In all cases, mNGS identified the same virus (Table 1). In another 3 PUIs, mNGS identified two viruses that were not tested for and one that was missed by routine testing. No SARS-CoV-2 was detected by mNGS among RT-PCR negative PUIs. Among SARS-CoV-2 RT-PCR positive PUIs, 18 (69%) underwent clinical testing for co-infections, and none were detected. mNGS did not identify any viral co-infections but did detect SARS-CoV-2 in all 23 PUIs. Conclusion: Unbiased mNGS offers the powerful opportunity to streamline testing for PUIs by assessing for SARS-CoV-2 and alternative infections simultaneously;this technique can also be used to identify co-infections, but none were observed in our study population. Interestingly, many PUIs had no infection identified on routine testing or mNGS, which may reflect inadequate sampling, rapid virus clearance, or a non-viral process. (Table Presented).