Multisite Mpox Infection and Viral Dynamics Among Persons With HIV in Metro Atlanta.

The 2022 mpox outbreak primarily involved sexual transmission among men who have sex with men and disproportionately affected persons with human immunodeficiency virus (HIV). We examined viral dynamics and clinical features in a cohort evaluated for mpox infection at a comprehensive HIV clinic in Atlanta, Georgia. Viral DNA was found in 8 oropharyngeal and 5 anorectal specimens among 10 mpox cases confirmed by lesion swab polymerase chain reaction. Within-participant anatomic site of lowest cycle threshold (Ct) value varied, and lower Ct values were found in oropharyngeal and anorectal swabs when corresponding symptoms were present. This provides insight into mpox infection across multiple anatomic sites among people with HIV.

Validation of short culture method for rapid bacterial identification of blood cultures via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

BACKGROUND: Development of rapid bacterial identification from blood cultures has been an area of intense study in diagnostic microbiology. Shortened turnaround time coupled with antimicrobial stewardship interventions have been shown to improve patient outcomes and decrease healthcare-associated costs. OBJECTIVES: We report the validation of a short incubation method for Gram-positive and Gram-negative bacterial identification utilizing MALDI-TOF MS without additional instrumentation, processing or cost compared with current practice. METHODS: Prospective, observational, single-centre study in a quaternary care academic hospital encompassing 376 blood cultures subjected to bacterial identification after short incubation periods of 3-4 and 6-8 h. RESULTS: There was 97.5% species-level identification agreement with tests undertaken after 3-4 h incubation with 83.6% isolates identified, and 99.7% species-level identification agreement after 6-8 h incubation with 96.7% isolates identified. CONCLUSIONS: The short incubation method provides a rapid MALDI-TOF MS bacterial identification method, reducing turnaround time by 10-18 h compared with standard practice without additional cost, processing or instrumentation.

Sensitivity of rapid antigen tests against SARS-CoV-2 Omicron and Delta variants.

Rapid antigen tests (RATs) have become an invaluable tool for combating the COVID-19 pandemic. However, concerns have been raised regarding the ability of existing RATs to effectively detect emerging SARS-CoV-2 variants. We compared the performance of 10 commercially available, emergency use authorized RATs against the Delta and Omicron SARS-CoV-2 variants using both individual patient and serially diluted pooled clinical samples. The RATs exhibited lower sensitivity for Omicron samples when using PCR cycle threshold (CT) value (a rough proxy for RNA concentration) as the comparator. Interestingly, however, they exhibited similar sensitivity for Omicron and Delta samples when using quantitative antigen concentration as the comparator. We further found that the Omicron samples had lower ratios of antigen to RNA, which offers a potential explanation for the apparent lower sensitivity of RATs for that variant when using C T value as a reference. Our findings underscore the complexity in assessing RAT performance against emerging variants and highlight the need for ongoing evaluation in the face of changing population immunity and virus evolution.

Efficacy of therapeutic plasma exchange in reducing the incidence of recurrent pancreatitis related to familial chylomicronemia.

BACKGROUND: Familial chylomicronemia syndrome (FCS) is caused by a genetic defect in triglyceride (TG) metabolism that leads to severe hypertriglyceridemia, which in turn is associated with multiple morbidities and may cause severe pancreatitis that is both recurrent and progressive. Management of hypertriglyceridemia in FCS is challenging, as both dietary and medical interventions are often ineffective. Therapeutic plasma exchange (TPE) has been shown to rapidly decrease circulating levels of chylomicrons and TGs in patients presenting with acute hypertriglyceridemic-associated pancreatitis. Conversely, limited evidence exists to suggest that prophylactic use of TPE is effective at preventing recurrence of acute pancreatitis. CASE REPORT: Herein, we report our experience with the use of chronic, prophylactic TPE to reduce the incidence of recurrent acute pancreatitis in a patient with FCS.

Prolonged partial thromboplastin time: To mix or not to mix - is that the question?

Routine mixing studies are frequently used to evaluate patients presenting with prolonged partial thromboplastin times (PTT) and/or prothrombin times (PT). Unfortunately, mixing studies have a number of inherent limitations including lack of standardization in terms of what defines normal pooled plasma (NPP), the processing of a patient's plasma for platelet removal (platelet poor plasma versus platelet-free plasma), performance of appropriate controls, conducting an incubation step to evaluate for a time and temperature dependent inhibitor, and finally interpretation of test results. Moreover, misinterpretation of study results can lead to a delayed or incorrect diagnosis or worse, inappropriate treatment. Within this manuscript, we present four cases illustrating the shortcomings associated with inappropriate utilization and interpretation of routine mixing studies; and present practical steps for managing abnormal PT or PTT results.

Cross-talk among flesh-eating Aeromonas hydrophila strains in mixed infection leading to necrotizing fasciitis.

Necrotizing fasciitis (NF) caused by flesh-eating bacteria is associated with high case fatality. In an earlier study, we reported infection of an immunocompetent individual with multiple strains of Aeromonas hydrophila (NF1-NF4), the latter three constituted a clonal group whereas NF1 was phylogenetically distinct. To understand the complex interactions of these strains in NF pathophysiology, a mouse model was used, whereby either single or mixed A. hydrophila strains were injected intramuscularly. NF2, which harbors exotoxin A (exoA) gene, was highly virulent when injected alone, but its virulence was attenuated in the presence of NF1 (exoA-minus). NF1 alone, although not lethal to animals, became highly virulent when combined with NF2, its virulence augmented by cis-exoA expression when injected alone in mice. Based on metagenomics and microbiological analyses, it was found that, in mixed infection, NF1 selectively disseminated to mouse peripheral organs, whereas the other strains (NF2, NF3, and NF4) were confined to the injection site and eventually cleared. In vitro studies showed NF2 to be more effectively phagocytized and killed by macrophages than NF1. NF1 inhibited growth of NF2 on solid media, but ExoA of NF2 augmented virulence of NF1 and the presence of NF1 facilitated clearance of NF2 from animals either by enhanced priming of host immune system or direct killing via a contact-dependent mechanism.

Combating Multidrug-Resistant Pathogens with Host-Directed Nonantibiotic Therapeutics.

Earlier, we reported that three Food and Drug Administration-approved drugs, trifluoperazine (TFP; an antipsychotic), amoxapine (AXPN; an antidepressant), and doxapram (DXP; a breathing stimulant), identified from an in vitro murine macrophage cytotoxicity screen, provided mice with 40 to 60% protection against pneumonic plague when administered at the time of infection for 1 to 3 days. In the present study, the therapeutic potential of these drugs against pneumonic plague in mice was further evaluated when they were administered at up to 48 h postinfection. While the efficacy of TFP was somewhat diminished as treatment was delayed to 24 h, the protection of mice with AXPN and DXP increased as treatment was progressively delayed to 24 h. At 48 h postinfection, these drugs provided the animals with significant protection (up to 100%) against challenge with the agent of pneumonic or bubonic plague when they were administered in combination with levofloxacin. Likewise, when they were used in combination with vancomycin, all three drugs provided mice with 80 to 100% protection from fatal oral Clostridium difficile infection when they were administered at 24 h postinfection. Furthermore, AXPN provided 40 to 60% protection against respiratory infection with Klebsiella pneumoniae when it was administered at the time of infection or at 24 h postinfection. Using the same in vitro cytotoxicity assay, we identified an additional 76/780 nonantibiotic drugs effective against K. pneumoniae For Acinetobacter baumannii, 121 nonantibiotic drugs were identified to inhibit bacterium-induced cytotoxicity in murine macrophages. Of these 121 drugs, 13 inhibited the macrophage cytotoxicity induced by two additional multiple-antibiotic-resistant strains. Six of these drugs decreased the intracellular survival of all three A. baumannii strains in macrophages. These results provided further evidence of the broad applicability and utilization of drug repurposing screening to identify new therapeutics to combat multidrug-resistant pathogens of public health concern.

Phospholipase A2-activating protein is associated with a novel form of leukoencephalopathy.

Leukoencephalopathies are a group of white matter disorders related to abnormal formation, maintenance, and turnover of myelin in the central nervous system. These disorders of the brain are categorized according to neuroradiological and pathophysiological criteria. Herein, we have identified a unique form of leukoencephalopathy in seven patients presenting at ages 2 to 4 months with progressive microcephaly, spastic quadriparesis, and global developmental delay. Clinical, metabolic, and imaging characterization of seven patients followed by homozygosity mapping and linkage analysis were performed. Next generation sequencing, bioinformatics, and segregation analyses followed, to determine a loss of function sequence variation in the phospholipase A2-activating protein encoding gene (PLAA). Expression and functional studies of the encoded protein were performed and included measurement of prostaglandin E2 and cytosolic phospholipase A2 activity in membrane fractions of fibroblasts derived from patients and healthy controls. Plaa-null mice were generated and prostaglandin E2 levels were measured in different tissues. The novel phenotype of our patients segregated with a homozygous loss-of-function sequence variant, causing the substitution of leucine at position 752 to phenylalanine, in PLAA, which causes disruption of the protein's ability to induce prostaglandin E2 and cytosolic phospholipase A2 synthesis in patients' fibroblasts. Plaa-null mice were perinatal lethal with reduced brain levels of prostaglandin E2 The non-functional phospholipase A2-activating protein and the associated neurological phenotype, reported herein for the first time, join other complex phospholipid defects that cause leukoencephalopathies in humans, emphasizing the importance of this axis in white matter development and maintenance.

New Role for FDA-Approved Drugs in Combating Antibiotic-Resistant Bacteria.

Antibiotic resistance in medically relevant bacterial pathogens, coupled with a paucity of novel antimicrobial discoveries, represents a pressing global crisis. Traditional drug discovery is an inefficient and costly process; however, systematic screening of Food and Drug Administration (FDA)-approved therapeutics for other indications in humans offers a rapid alternative approach. In this study, we screened a library of 780 FDA-approved drugs to identify molecules that rendered RAW 264.7 murine macrophages resistant to cytotoxicity induced by the highly virulent Yersinia pestis CO92 strain. Of these compounds, we identified 94 not classified as antibiotics as being effective at preventing Y. pestis-induced cytotoxicity. A total of 17 prioritized drugs, based on efficacy in in vitro screens, were chosen for further evaluation in a murine model of pneumonic plague to delineate if in vitro efficacy could be translated in vivo Three drugs, doxapram (DXP), amoxapine (AXPN), and trifluoperazine (TFP), increased animal survivability despite not exhibiting any direct bacteriostatic or bactericidal effect on Y. pestis and having no modulating effect on crucial Y. pestis virulence factors. These findings suggested that DXP, AXPN, and TFP may modulate host cell pathways necessary for disease pathogenesis. Finally, to further assess the broad applicability of drugs identified from in vitro screens, the therapeutic potential of TFP, the most efficacious drug in vivo, was evaluated in murine models of Salmonella enterica serovar Typhimurium and Clostridium difficile infections. In both models, TFP treatment resulted in increased survivability of infected animals. Taken together, these results demonstrate the broad applicability and potential use of nonantibiotic FDA-approved drugs to combat respiratory and gastrointestinal bacterial pathogens.

High-throughput, signature-tagged mutagenic approach to identify novel virulence factors of Yersinia pestis CO92 in a mouse model of infection.

The identification of new virulence factors in Yersinia pestis and understanding their molecular mechanisms during an infection process are necessary in designing a better vaccine or to formulate an appropriate therapeutic intervention. By using a high-throughput, signature-tagged mutagenic approach, we created 5,088 mutants of Y. pestis strain CO92 and screened them in a mouse model of pneumonic plague at a dose equivalent to 5 50% lethal doses (LD50) of wild-type (WT) CO92. From this screen, we obtained 118 clones showing impairment in disseminating to the spleen, based on hybridization of input versus output DNA from mutant pools with 53 unique signature tags. In the subsequent screen, 20/118 mutants exhibited attenuation at 8 LD50 when tested in a mouse model of bubonic plague, with infection by 10/20 of the aforementioned mutants resulting in 40% or higher survival rates at an infectious dose of 40 LD50. Upon sequencing, six of the attenuated mutants were found to carry interruptions in genes encoding hypothetical proteins or proteins with putative functions. Mutants with in-frame deletion mutations of two of the genes identified from the screen, namely, rbsA, which codes for a putative sugar transport system ATP-binding protein, and vasK, a component of the type VI secretion system, were also found to exhibit some attenuation at 11 or 12 LD50 in a mouse model of pneumonic plague. Likewise, among the remaining 18 signature-tagged mutants, 9 were also attenuated (40 to 100%) at 12 LD50 in a pneumonic plague mouse model. Previously, we found that deleting genes encoding Braun lipoprotein (Lpp) and acyltransferase (MsbB), the latter of which modifies lipopolysaccharide function, reduced the virulence of Y. pestis CO92 in mouse models of bubonic and pneumonic plague. Deletion of rbsA and vasK genes from either the Δlpp single or the Δlpp ΔmsbB double mutant augmented the attenuation to provide 90 to 100% survivability to mice in a pneumonic plague model at 20 to 50 LD50. The mice infected with the Δlpp ΔmsbB ΔrbsA triple mutant at 50 LD50 were 90% protected upon subsequent challenge with 12 LD50 of WT CO92, suggesting that this mutant or others carrying combinational deletions of genes identified through our screen could potentially be further tested and developed into a live attenuated plague vaccine(s).

Further characterization of a highly attenuated Yersinia pestis CO92 mutant deleted for the genes encoding Braun lipoprotein and plasminogen activator protease in murine alveolar and primary human macrophages.

We recently characterized the Δlpp Δpla double in-frame deletion mutant of Yersinia pestis CO92 molecularly, biologically, and immunologically. While Braun lipoprotein (Lpp) activates toll-like receptor-2 to initiate an inflammatory cascade, plasminogen activator (Pla) protease facilitates bacterial dissemination in the host. The Δlpp Δpla double mutant was highly attenuated in evoking bubonic and pneumonic plague, was rapidly cleared from mouse organs, and generated humoral and cell-mediated immune responses to provide subsequent protection to mice against a lethal challenge dose of wild-type (WT) CO92. Here, we further characterized the Δlpp Δpla double mutant in two murine macrophage cell lines as well as in primary human monocyte-derived macrophages to gauge its potential as a live-attenuated vaccine candidate. We first demonstrated that the Δpla single and the Δlpp Δpla double mutant were unable to survive efficiently in murine and human macrophages, unlike WT CO92. We observed that the levels of Pla and its associated protease activity were not affected in the Δlpp single mutant, and, likewise, deletion of the pla gene from WT CO92 did not alter Lpp levels. Further, our study revealed that both Lpp and Pla contributed to the intracellular survival of WT CO92 via different mechanisms. Importantly, the ability of the Δlpp Δpla double mutant to be phagocytized by macrophages, to stimulate production of tumor necrosis factor-α and interleukin-6, and to activate the nitric oxide killing pathways of the host cells remained unaltered when compared to the WT CO92-infected macrophages. Finally, macrophages infected with either the WT CO92 or the Δlpp Δpla double mutant were equally efficient in their uptake of zymosan particles as determined by flow cytometric analysis. Overall, our data indicated that although the Δlpp Δpla double mutant of Y. pestis CO92 was highly attenuated, it retained the ability to elicit innate and subsequent acquired immune responses in the host similar to that of WT CO92, which are highly desirable in a live-attenuated vaccine candidate.

Functional genomic characterization of virulence factors from necrotizing fasciitis-causing strains of Aeromonas hydrophila.

The genomes of 10 Aeromonas isolates identified and designated Aeromonas hydrophila WI, Riv3, and NF1 to NF4; A. dhakensis SSU; A. jandaei Riv2; and A. caviae NM22 and NM33 were sequenced and annotated. Isolates NF1 to NF4 were from a patient with necrotizing fasciitis (NF). Two environmental isolates (Riv2 and -3) were from the river water from which the NF patient acquired the infection. While isolates NF2 to NF4 were clonal, NF1 was genetically distinct. Outside the conserved core genomes of these 10 isolates, several unique genomic features were identified. The most virulent strains possessed one of the following four virulence factors or a combination of them: cytotoxic enterotoxin, exotoxin A, and type 3 and 6 secretion system effectors AexU and Hcp. In a septicemic-mouse model, SSU, NF1, and Riv2 were the most virulent, while NF2 was moderately virulent. These data correlated with high motility and biofilm formation by the former three isolates. Conversely, in a mouse model of intramuscular infection, NF2 was much more virulent than NF1. Isolates NF2, SSU, and Riv2 disseminated in high numbers from the muscular tissue to the visceral organs of mice, while NF1 reached the liver and spleen in relatively lower numbers on the basis of colony counting and tracking of bioluminescent strains in real time by in vivo imaging. Histopathologically, degeneration of myofibers with significant infiltration of polymorphonuclear cells due to the highly virulent strains was noted. Functional genomic analysis provided data that allowed us to correlate the highly infectious nature of Aeromonas pathotypes belonging to several different species with virulence signatures and their potential ability to cause NF.

Performance of the Xpert™ Mpox PCR assay with oropharyngeal, anorectal, and cutaneous lesion swab specimens.

Anorectal and oropharyngeal exposures are implicated in sexual transmission of mpox, but authorized assays in the United States are only validated with cutaneous lesion swabs. Diagnostic assays for anorectal and oropharyngeal swabs are needed to address potential future outbreaks. The Cepheid Xpert® Mpox is the first point-of-care assay to receive FDA emergency use authorization in the United States and would be a valuable tool for evaluating these sample types. Our exploratory study demonstrates 100 % positive agreement with our in-house PCR assay for natural positive anorectal and oropharyngeal specimens and 92 % sensitivity with low-positive spiked specimens. The Xpert® assay detected viral DNA in specimens not detected by our reference PCR assay from four participants with mpox DNA at other sites, suggesting it may be more sensitive at low viral loads. In conclusion, the validation of the Xpert® for oropharyngeal and anorectal sample types can be rapidly achieved if clinical need returns and prospective samples become available.

Deletion of the Braun lipoprotein-encoding gene and altering the function of lipopolysaccharide attenuate the plague bacterium.

Braun (murein) lipoprotein (Lpp) and lipopolysaccharide (LPS) are major components of the outer membranes of Enterobacteriaceae family members that are capable of triggering inflammatory immune responses by activating Toll-like receptors 2 and 4, respectively. Expanding on earlier studies that demonstrated a role played by Lpp in Yersinia pestis virulence in mouse models of bubonic and pneumonic plague, we characterized an msbB in-frame deletion mutant incapable of producing an acyltransferase that is responsible for the addition of lauric acid to the lipid A moiety of LPS, as well as a Δlpp ΔmsbB double mutant of the highly virulent Y. pestis CO92 strain. Although the ΔmsbB single mutant was minimally attenuated, the Δlpp single mutant and the Δlpp ΔmsbB double mutant were significantly more attenuated than the isogenic wild-type (WT) bacterium in bubonic and pneumonic animal models (mouse and rat) of plague. These data correlated with greatly reduced survivability of the aforementioned mutants in murine macrophages. Furthermore, the Δlpp ΔmsbB double mutant was grossly compromised in its ability to disseminate to distal organs in mice and in evoking cytokines/chemokines in infected animal tissues. Importantly, mice that survived challenge with the Δlpp ΔmsbB double mutant, but not the Δlpp or ΔmsbB single mutant, in a pneumonic plague model were significantly protected against a subsequent lethal WT CO92 rechallenge. These data were substantiated by the fact that the Δlpp ΔmsbB double mutant maintained an immunogenicity comparable to that of the WT strain and induced long-lasting T-cell responses against heat-killed WT CO92 antigens. Taken together, the data indicate that deletion of the msbB gene augmented the attenuation of the Δlpp mutant by crippling the spread of the double mutant to the peripheral organs of animals and by inducing cytokine/chemokine responses. Thus, the Δlpp ΔmsbB double mutant could provide a new live-attenuated background vaccine candidate strain, and this should be explored in the future.

A non-invasive in vivo imaging system to study dissemination of bioluminescent Yersinia pestis CO92 in a mouse model of pneumonic plague.

The gold standard in microbiology for monitoring bacterial dissemination in infected animals has always been viable plate counts. This method, despite being quantitative, requires sacrificing the infected animals. Recently, however, an alternative method of in vivo imaging of bioluminescent bacteria (IVIBB) for monitoring microbial dissemination within the host has been employed. Yersinia pestis is a Gram-negative bacterium capable of causing bubonic, septicemic, and pneumonic plague. In this study, we compared the conventional counting of bacterial colony forming units (cfu) in the various infected tissues to IVIBB in monitoring Y. pestis dissemination in a mouse model of pneumonic plague. By using a transposon mutagenesis system harboring the luciferase (luc) gene, we screened approximately 4000 clones and obtained a fully virulent, luc-positive Y. pestis CO92 (Y. pestis-luc2) reporter strain in which transposition occurred within the largest pMT1 plasmid which possesses murine toxin and capsular antigen encoding genes. The aforementioned reporter strain and the wild-type CO92 exhibited similar growth curves, formed capsule based on immunofluorescence microscopy and flow cytometry, and had a similar LD(50). Intranasal infection of mice with 15 LD(50) of CO92-luc2 resulted in animal mortality by 72 h, and an increasing number of bioluminescent bacteria were observed in various mouse organs over a 24-72 h period when whole animals were imaged. However, following levofloxacin treatment (10 mg/kg/day) for 6 days 24 h post infection, no luminescence was observed after 72 h of infection, indicating that the tested antimicrobial killed bacteria preventing their detection in host peripheral tissues. Overall, we demonstrated that IVIBB is an effective and non-invasive way of monitoring bacterial dissemination in animals following pneumonic plague having strong correlation with cfu, and our reporter CO92-luc2 strain can be employed as a useful tool to monitor the efficacy of antimicrobial countermeasures in real time.

Investigation of Blood Plasma Viral Nucleocapsid Antigen as a Marker of Active Severe Acute Respiratory Syndrome Coronavirus 2 Omicron Variant Infection.

Background: Nasopharyngeal qualitative reverse-transcription polymerase chain reaction (RT-PCR) is the gold standard for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but it is not practical or sufficient in every clinical scenario due to its inability to distinguish active from resolved infection. Alternative or adjunct testing may be needed to guide isolation precautions and treatment in patients admitted to the hospital. Methods: We performed a single-center, retrospective analysis of residual clinical specimens and medical record data to examine blood plasma nucleocapsid antigen as a candidate biomarker of active SARS-CoV-2. Adult patients admitted to the hospital or presenting to the emergency department with SARS-CoV-2 ribonucleic acid (RNA) detected by RT-PCR from a nasopharyngeal swab specimen were included. Both nasopharyngeal swab and a paired whole blood sample were required to be available for analysis. Results: Fifty-four patients were included. Eight patients had positive nasopharyngeal swab virus cultures, 7 of whom (87.5%) had concurrent antigenemia. Nineteen (79.2%) of 24 patients with detectable subgenomic RNA and 20 (80.0%) of 25 patients with N2 RT-PCR cycle threshold ≤ 33 had antigenemia. Conclusions: Most individuals with active SARS-CoV-2 infection are likely to have concurrent antigenemia, but there may be some individuals with active infection in whom antigenemia is not detectable. The potential for high sensitivity and convenience of a blood test prompts interest in further investigation as a screening tool to reduce reliance on nasopharyngeal swab sampling and as an adjunct diagnostic test to aid in clinical decision making during the period after acute coronavirus disease 2019.

Sensitivity of Rapid Antigen Tests Against SARS-CoV-2 Omicron and Delta Variants.

Rapid Antigen Tests (RAT) have become an invaluable tool for combating the COVID-19 pandemic. However, concerns have been raised regarding the ability of existing RATs to effectively detect emerging SARS-CoV-2 variants. We compared the performance of eight commercially available, emergency use authorized RATs against the Delta and Omicron SARS-CoV-2 variants using individual patient and serially diluted pooled clinical samples. The RATs exhibited lower sensitivity for Omicron samples when using PCR Cycle threshold (C T ) value (a proxy for RNA concentration) as the comparator. Interestingly, however, they exhibited similar sensitivity for Omicron and Delta samples when using quantitative antigen concentration as the comparator. We further found that the Omicron samples had lower ratios of antigen to RNA, which offers a potential explanation for the apparent lower sensitivity of RATs for that variant when using C T value as a reference. Our findings underscore the complexity in assessing RAT performance against emerging variants and highlight the need for ongoing evaluation in the face of changing population immunity and virus evolution.

Viral and host factors associated with SARS-CoV-2 disease severity in Georgia, USA.

While SARS-CoV-2 vaccines have shown strong efficacy, their suboptimal uptake combined with the continued emergence of new viral variants raises concerns about the ongoing and future public health impact of COVID-19. We investigated viral and host factors, including vaccination status, that were associated with SARS-CoV-2 disease severity in a setting with low vaccination rates. We analyzed clinical and demographic data from 1,957 individuals in the state of Georgia, USA, coupled with viral genome sequencing from 1,185 samples. We found no difference in disease severity between individuals infected with Delta and Omicron variants among the participants in this study, after controlling for other factors, and we found no specific mutations associated with disease severity. Compared to those who were unvaccinated, vaccinated individuals experienced less severe SARS-CoV-2 disease, and the effect was similar for both variants. Vaccination within 270 days before infection was associated with decreased odds of moderate and severe outcomes, with the strongest association observed at 91-270 days post-vaccination. Older age and underlying health conditions, especially immunosuppression and renal disease, were associated with increased disease severity. Overall, this study provides insights into the impact of vaccination status, variants/mutations, and clinical factors on disease severity in SARS-CoV-2 infection when vaccination rates are low. Understanding these associations will help refine and reinforce messaging around the crucial importance of vaccination in mitigating the severity of SARS-CoV-2 disease.

Nucleocapsid Antigenemia in Patients Receiving Anti-CD20 Therapy With Protracted COVID-19.

Immunocompromised patients with prolonged coronavirus disease 2019 symptoms present diagnostic and therapeutic challenges. We measured viral nucleocapsid antigenemia in 3 patients treated with anti-CD20 immunotherapy who acquired severe acute respiratory syndrome coronavirus 2 infection and experienced protracted symptoms. Our results support nucleocapsid antigenemia as a marker of persistent infection and therapeutic response.