Search

Humans differ in their susceptibility to infectious disease, partly owing to variation in the immune response after infection. We used single-cell RNA sequencing to quantify variation in the response to influenza infection in peripheral blood mononuclear cells from European- and African-ancestry males. Genetic ancestry effects are common but highly cell type specific. Higher levels of European ancestry are associated with increased type I interferon pathway activity in early infection, which predicts reduced viral titers at later time points. Substantial population-associated variation is explained by cis-expression quantitative trait loci that are differentiated by genetic ancestry. Furthermore, genetic ancestryÐassociated genes are enriched among genes correlated with COVID-19 disease severity, suggesting that the early immune response contributes to ancestry-associated differences for multiple viral infection outcomes. [ FROM AUTHOR] Copyright of Science is the property of American Association for the Advancement of Science and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Background Penicillin allergy is reported in approximately 10% of the US population and is one of the most frequent medication allergies in children. Penicillin allergy labels are associated with more expensive care and adverse outcomes, such as MRSA and C. Diff infection. Studies suggest up to 90% of these patients could safely tolerate this class of drugs. Our team at M Health Fairview University of Minnesota Masonic Children’s Hospital (UMMCH) implemented an allergy assessment tool for inpatient use in January of 2020 to create an effective and accessible path for delabeling pediatric patients with penicillin and cephalosporin allergies. Methods Our pharmacists conducted the allergy assessment on inpatient pediatric patients with penicillin/cephalosporin allergies. Those deemed low to moderate risk for having a true IgE-mediated allergy by our assessment were recommended for outpatient allergy testing. Those deemed no risk were recommended for immediate delabeling. We performed manual chart reviews to describe pediatric patient risk stratification, allergy label status after assessment, outpatient allergy follow-up, and status and results of allergy testing. Results From January, 2020 to present, 140 patients received a pharmacist allergy assessment. During the 2020 calendar year, 51 allergy assessments were performed on 49 patients. 58 allergies were recorded, with penicillins as the most common (62%), followed by cephalosporins (24%), and penicillin combination drugs (14%). 1.9% of allergies were high risk, 68.8% moderate, 15.7% low, 9.8% no risk, and 3.8% difficult to determine. Of 42 patients deemed low to moderate risk, 8 (17%) were seen by an outpatient allergist. Of these, 5 proceeded with allergy testing and were found to be not allergic. Out of 58 allergy labels, 9 (15.5%) were removed, 5 after allergy testing, 2 immediately due to no risk, and 2 after further history review. Conclusion Our preliminary data show allergy assessments can inform pediatric allergy risk prior to outpatient allergy testing and assist with imminent delabeling. Next steps include analysis of our 2021 assessments as well as patient caregiver surveys to assess perception of the allergy assessment process and to elucidate on barriers to testing/delabeling. Disclosures Beth K. Thielen, MD, PhD, Horizon: Advisor/Consultant|Horizon: Honoraria|Merck: Grant/Research Support.

Background Plasma metagenomic next-generation sequencing (mNGS) is an emerging diagnostic tool. As the clinical use of mNGS increases, efforts to better understand the role of mNGS in diagnosis and management of infectious diseases are essential. Current literature is limited to small retrospective reviewers and a prospective study therefore subject to institutional variability. Here, we aim to describe the use, sensitivity, time to diagnosis, clinical impact, and cost effectiveness of mNGS in the largest patient cohort to date. Methods We included pediatric and adult patients who had plasma mNGS testing as part of care from December 2017 through December 2021 at University of Minnesota (UMN). Patients were identified using the Karius database. Electronic medical records were reviewed for every mNGS test, recording patient demographics, underlying conditions, indications, and results within 30 days of mNGS. All cases were assessed by 2 reviewers including one board-certified infectious diseases doctor. Sensitivity of mNGS was determined relative to conventional tests. The primary end point was change in clinical management. Secondary end points included accuracy, time to diagnosis, and infectious disease consultation. UMN IRB approved this study as non human subjects research. Results 584 mNGS tests were ordered in the study period, with 203 reviews completed to date. Solid organ transplantation (SOT) (32%) was the most common underlying condition, followed by hemopoietic stem cell transplantation (HSCT) (28%). Fever (45.8%), then pulmonary findings (26%) were the most common indications for ordering mNGS. Conventional and mNGS tests identified an infectious cause of the clinical syndrome in 49% of cases, while a contaminant was found in 52% of cases. The sensitivity of mNGS in identification of infectious cause was 47%, approximately 7% more sensitive than conventional testing. Despite this increased sensitivity, mNGS changed clinical management in 20% of cases. Conclusion mNGS demonstrated superiority in accurate detection of infectious causes of clinical syndromes, however, changed clinical management in a minority of cases, suggesting timing of the mNGS test is critical. This study identified SOT and HSCT as high yield patient groups for implementation of a prospective study. Disclosures Beth K. Thielen, MD, PhD, Horizon: Advisor/Consultant|Horizon: Honoraria|Merck: Grant/Research Support.

Chronic granulomatous disease (CGD) is a rare but serious primary immunodeficiency with varying prevalence and rates of X-linked and autosomal recessive disease worldwide. Functional defects in the phagocyte nicotinamide adenine dinucleotide phosphate oxidase complex predispose patients to a relatively narrow spectrum of bacterial and fungal infections that are sometimes fastidious and often difficult to identify. When evaluating and treating patients with CGD, it is important to consider their native country of birth, climate, and living situation, which may predispose them to types of infections that are atypical to your routine practice. In addition to recurrent and often severe infections, patients with CGD and X-linked female carriers are also susceptible to developing many non-infectious complications including tissue granuloma formation and autoimmunity. The DHR-123 oxidation assay is the gold standard for making the diagnosis and it along with genetic testing can help predict the severity and prognosis in patients with CGD. Disease management focuses on prophylaxis with antibacterial, antifungal, and immunomodulatory medications, prompt identification and treatment of acute infections, and prevention of secondary granulomatous complications. While hematopoietic stem-cell transplantation is the only widely available curative treatment for patients with CGD, recent advances in gene therapy may provide a safer, more direct alternative.

Background During the Covid-19 pandemic, clinical trials necessitated rapid testing to be performed remotely. Dried blood spot (DBS) techniques have enabled remote HIV virologic testing globally and more recently, antibody testing as well. We evaluated DBS testing for SARS-CoV-2 antibody testing in outpatients to assess seropositivity. Methods In 2020, we conducted three internet-based randomized clinical trials and offered serologic testing via self-collected DBS as a voluntary sub-study. COVID-19 diagnosis was based on CDC case definition with epidemiological link to cases. A minority reported PCR testing at an outside facility. We tested for anti-SARS-CoV-2 immunoglobulin via Antibody Detection by Agglutination-PCR (ADAP) and compared with ELISA. Results Of 2727 participants in the primary studies, 60% (1648/2727) consented for serology testing; 56% (931/1648) returned a usable DBS sample. Of those asymptomatic, 5% (33/707) had positive ADAP serology. Of participants with a positive PCR, 67% (36/54) had positive SARS-CoV-2 antibodies. None of those who were PCR-positive and asymptomatic were seropositive (0/7). Of 77 specimens tested for concordance via ELISA, 83% (64/77) were concordant. Challenges of completing a remote testing program during a pandemic included sourcing and assembling collection kits, delivery and return of the kits, and troubleshooting testing. Self-collection was successful for >95% of participants. Delays in US mail with possible sample degradation and timing of DBS collection complicated the analysis. Conclusion We found remote antibody testing during a global pandemic feasible although challenging. We identified an association between symptomatic COVID-19 and positive antibody results at similar prevalence to other outpatient cohorts.

To evaluate if cost and availability of antimalarials are barriers to malaria chemoprophylaxis and treatment, we surveyed retail pharmacies in Minneapolis/Saint Paul and New York City on the price and stocking of antimalarials. We demonstrated extreme price variability among pharmacies and limited availability for some recommended antimalarials.

Humans vary in their susceptibility to infectious disease, partly due to variation in the immune response following infection. Here, we used single-cell RNA-sequencing to quantify genetic contributions to this variation in peripheral blood mononuclear cells, focusing specifically on the transcriptional response to influenza infection. We find that monocytes are the most responsive to influenza infection, but that all cell types mount a conserved interferon response, which is stronger in individuals with increased European ancestry. By comparing European American and African American individuals, we show that genetic ancestry effects on expression are common, influencing 29% of genes, but highly cell type-specific. Further, we demonstrate that much of this population-associated expression variation is explained by cis expression quantitative trait loci, which are enriched for signatures of recent positive selection. Our findings establish common cis-regulatory variants—including those that are differentiated by genetic ancestry—as important determinants of the antiviral immune response.

Chronic granulomatous disease (CGD) is a rare but serious primary immunodeficiency with varying prevalence and rates of X-linked and autosomal recessive disease worldwide. Functional defects in the phagocyte nicotinamide adenine dinucleotide phosphate oxidase complex predispose patients to a relatively narrow spectrum of bacterial and fungal infections that are sometimes fastidious and often difficult to identify. When evaluating and treating patients with CGD, it is important to consider their native country of birth, climate, and living situation, which may predispose them to types of infections that are atypical to your routine practice. In addition to recurrent and often severe infections, patients with CGD and X-linked female carriers are also susceptible to developing many non-infectious complications including tissue granuloma formation and autoimmunity. The DHR-123 oxidation assay is the gold standard for making the diagnosis and it along with genetic testing can help predict the severity and prognosis in patients with CGD. Disease management focuses on prophylaxis with antibacterial, antifungal, and immunomodulatory medications, prompt identification and treatment of acute infections, and prevention of secondary granulomatous complications. While hematopoietic stem-cell transplantation is the only widely available curative treatment for patients with CGD, recent advances in gene therapy may provide a safer, more direct alternative.

Background Respiratory syncytial virus (RSV) typically causes winter outbreaks in temperate climates. During summer 2017, the Minnesota Department of Health received a report of increased cases of severe RSV-B infection. Methods We compared characteristics of summer 2017 cases with those of 2014–2018 summers. To understand the genetic relatedness among viruses, we performed high-throughput sequencing of RSV from patients with a spectrum of illness from sites in Minnesota and Wisconsin. Results From May to September 2017, 58 RSV cases (43 RSV-B) were reported compared to 20–29 cases (3–7 RSV-B) during these months in other years. Median age and frequency of comorbidities were similar, but 55% (24/43) were admitted to the ICU in 2017 compared to 12% in preceding 3 years (odds ratio, 4.84, P Conclusions We identified an outbreak of severe RSV-B disease associated with a new genetic lineage among urban Minnesota children during a time of expected low RSV circulation.

The Mucorales fungi—formerly classified as the zygomycetes—are environmentally ubiquitous fungi, but generally rare causes of clinical infections. In the immunocompromised host, however, they can cause invasive, rapidly spreading infections that confer a high risk of morbidity and mortality, often despite surgical and antifungal therapy. Patients with extensive burn injuries are particularly susceptible to skin and soft-tissue infections with these organisms. Here, we present a case of Lichtheimia infection in a patient with extensive full-thickness burns that required significant and repeated surgical debridement successfully treated with isavuconazole and adjunctive topical amphotericin B washes. We also review the available literature on contemporary antifungal treatment for Lichtheimia species and related Muco- rales fungi.

Background Determining the incidence, disease-associated serotypes and antimicrobial susceptibility of invasive pneumococcal disease (IPD) among children in Africa is essential in order to monitor the impact of these infections prior to widespread introduction of the pneumococcal conjugate vaccine (PCV). Methods To provide updated estimates of the incidence, serotype distribution, and antimicrobial susceptibility profile of Streptococcus pneumoniae causing disease in Africa, we performed a systematic review of articles published from 2000 to 2015 using Ovid Medline and Embase. We included prospective and surveillance studies that applied predefined diagnostic criteria. Meta-analysis for all pooled analyses was based on random-effects models. Results We included 38 studies consisting of 386,880 participants in 21 countries over a total of 350,613 person-years. The pooled incidence of IPD was 62.6 (95% CI 16.9, 226.5) per 100,000 person-years, including meningitis which had a pooled incidence of 24.7 (95% CI 11.9, 51.6) per 100,000 person-years. The pooled prevalence of penicillin susceptibility was 78.1% (95% CI 61.9, 89.2). Cumulatively, PCV10 and PCV13 included 66.9% (95% CI 55.9, 76.7) and 80.6% (95% CI 66.3, 90.5) of IPD serotypes, respectively. Conclusions Our study provides an integrated and robust summary of incidence data, serotype distribution and antimicrobial susceptibility for S. pneumoniae in children ≤5 years of age in Africa prior to widespread introduction of PCV on the continent. The heterogeneity of studies and wide range of incidence rates across the continent indicate that surveillance efforts should be intensified in all regions of Africa to improve the integrity of epidemiologic data, vaccine impact and cost benefit. Although the incidence of IPD in young children in Africa is substantial, currently available conjugate vaccines are estimated to cover the majority of invasive disease-causing pneumococcal serotypes. These data provide a reliable baseline from which to monitor the impact of the broad introduction of PCV.

BACKGROUND: Existing literature suggests that influenza C typically causes mild respiratory tract disease. However, clinical and epidemiological data are limited. METHODS: Four outpatient clinics and three hospitals submitted clinical data and respiratory specimens through a surveillance network for acute respiratory infection (ARI) during May 2013 through December 2016. Specimens were tested using multi-target nucleic acid amplification tests (NAAT) for 19–22 respiratory pathogens, including influenza C. RESULTS: Influenza C virus was detected among 59 of 10,202 (0.58%) hospitalized SARI cases and 11 of 2,282 (0.48%) outpatients. Most detections occurred from December to March, with 73% during the 2014–2015 season. Influenza C detections occurred among patients of all ages, with similar rates between inpatients and outpatients. The highest rate of detection occurred among children aged 6 to 24 months (1.2%). Among hospitalized cases, seven required intensive care. Medical co-morbidities were reported in 58% of hospitalized cases and all who required intensive care. At least one other respiratory pathogen was detected in 40 (66%) cases, most commonly rhinovirus/enterovirus (25%) and respiratory syncytial virus (RSV) (20%). The hemagglutinin-esterase-fusion (HEF) gene was sequenced in 37 specimens, and both C/Kanagawa and C/Sao Paulo lineages were detected in inpatients and outpatients. CONCLUSIONS: We found seasonal circulation of influenza C with year-to-year variability. Detection was most frequent among young children, but occurred in all ages. Some cases positive for influenza C, particularly those with co-morbid conditions, had severe disease, suggesting a need for further study of the role of influenza C virus in the pathogenesis of respiratory disease.

Background Respiratory syncytial virus (RSV) causes seasonal outbreaks peaking from October to April in the United States. While symptoms are typically mild and limited to the upper respiratory tract in older children and adults, RSV can cause severe lower respiratory disease, hospitalization and death, particularly among younger children, the immunocompromised and the elderly. During summer 2017, the Minnesota Department of Health (MDH) received a report of a cluster of severe respiratory illness in children with RSV-B infection admitted at an urban county hospital. Methods MDH conducts surveillance for RSV in Minneapolis-St. Paul with collection of respiratory specimens and clinical and demographic data. We compared characteristics of cases reported in summer 2017 with the previous 4 summer seasons. To understand the genetic relatedness among viruses, we performed complete genome sequencing from primary specimens using an Illumina MiSeq platform employing both a sequence-independent sequencing method and an RSV-specific, overlapping PCR-based approach. Results From May to September 2017, 58 cases of RSV (48 RSV-B) were reported to MDH compared with 18 (7 RSV-B) during the same period in 2016, 27 in 2015, and 29 in 2014. The median age and frequency of co-morbidities were similar across years; however, 50% required ICU admission in 2017 compared with 12% in preceding 3 years. The RSV-B genome was sequenced from 10 specimens from March to May 2016, 20 specimens from May to September 2017 and 30 specimens from October 2017-January 2018. Phylogenic analysis revealed that 15 cases from May to September. 2017 formed a unique clade distinct from the lineage encompassing 2016 cases from Minnesota and 35 representative complete RSV genomes in GenBank isolated from 6 continents over 13 years. From October 2017 to January 2018, we detected co-circulation of viruses from both lineages among older adults and children. Conclusion We identified an outbreak of RSV-B associated with severe disease among urban Minnesota children during a time of expected low RSV circulation. Complete genome sequencing data suggested emergence of a new lineage distinct from viruses circulating in Minnesota during the previous season. Genomic characterization can provide useful insights into epidemiologic variations. Disclosures All authors: No reported disclosures.

Background Seroprevalence and surveillance studies indicate that influenza C virus (ICV) infection is common among humans, and initial exposure occurs early in life. ICV often causes milder disease than influenza A and B viruses, but the mechanisms underlying differences in pathogenicity remain poorly understood. Methods To compare early events of infection in natural target sites, we cultured primary human tracheal/bronchial epithelial cells under air-liquid interface conditions to allow differentiation. We-infected these cells with human strains of influenza A, B or C virus. Cells were infected at low MOI (0.1) to ensure populations of directly infected cells and uninfected neighboring cells. To compare the early immune response and cell tropism among these viruses, we performed single-cell RNA sequencing of mock- and influenza-infected cells. In parallel, we infected cells pretreated with interferon to mimic later rounds of infection after an early immune response is initiated. Results Infection of primary cells by all three viruses was confirmed by RT-qPCR of bulk cell lysates. As expected, prior exposure to interferon β results resulted in reduced levels of viral transcripts. At the single-cell level, we identified expression of genes associated with specific cell types, including basal, ciliated and secretory cells. We also identified expression of interferon stimulated genes, but these genes were not homogeneously expressed among all cell subpopulations and varied among cultures infected with different influenza viruses. We also found different patterns in gene expression in cells previously exposed to interferon, suggesting that host environment varies over subsequent rounds of infection. Conclusion Single-cell sequencing is an important tool for studying the host response to influenza infection in complex cellular environments such as the respiratory tract, in which cells vary in their susceptibility to infection and antiviral response. Further analysis will characterize differences among directly infected vs. neighboring cells and correlate responses with pathogenicity. Disclosures All authors: No reported disclosures.

In HIV-1-infected individuals, G-to-A hypermutation is found in HIV-1 DNA isolated from peripheral blood mononuclear cells (PBMCs). These mutations are thought to result from editing by one or more host enzymes in the APOBEC3 (A3) family of cytidine deaminases, which act on CC (APOBEC3G) and TC (other A3 proteins) dinucleotide motifs in DNA (edited cytidine underlined). Although many A3 proteins display high levels of deaminase activity in model systems, only low levels of A3 deaminase activity have been found in primary cells examined to date. In contrast, here we report high levels of deaminase activity at TC motifs when whole PBMCs or isolated primary monocyte-derived cells were treated with interferon-alpha (IFNalpha) or IFNalpha-inducing toll-like receptor ligands. Induction of TC-specific deaminase activity required new transcription and translation and correlated with the appearance of two APOBEC3A (A3A) isoforms. Knockdown of A3A in monocytes with siRNA abolished TC-specific deaminase activity, confirming that A3A isoforms are responsible for all TC-specific deaminase activity observed. Both A3A isoforms appear to be enzymatically active; moreover, our mutational studies raise the possibility that the smaller isoform results from internal translational initiation. In contrast to the high levels of TC-specific activity observed in IFNalpha-treated monocytes, CC-specific activity remained low in PBMCs, suggesting that A3G deaminase activity is relatively inhibited, unlike that of A3A. Together, these findings suggest that deaminase activity of A3A isoforms in monocytes and macrophages may play an important role in host defense against viruses.

The deoxycytidine deaminase APOBEC3G (A3G) is expressed in human T cells and inhibits HIV-1 replication. When transfected into A3G-deficient epithelial cell lines, A3G induces catastrophic hypermutation by deaminating the HIV-1 genome. Interestingly, studies suggest that endogenous A3G in T cells induces less hypermutation than would be expected. However, to date, the specific deaminase activity of endogenous A3G in human CD4+ T cells has not been examined directly. Here, we compared deaminase activity of endogenous and exogenous A3G in various human cell lines using a standard assay and a novel, quantitative, high-throughput assay. Exogenous A3G in epithelial cell lysates displayed deaminase activity only following RNase treatment, as expected given that A3G is known to form an enzymatically inactive RNA-containing complex. Surprisingly, comparable amounts of endogenous A3G from T cell lines or from resting or activated primary CD4+ T cells exhibited minimal deaminase activity, despite RNase treatment. Specific deaminase activity of endogenous A3G in H9, CEM, and other T cell lines was up to 36-fold lower than specific activity of exogenous A3G in epithelial-derived cell lines. Furthermore, RNase-treated T cell lysates conferred a dose-dependent inhibition to epithelial cell lysates expressing enzymatically active A3G. These studies suggest that T cells, unlike epithelial-derived cell lines, express an unidentified RNase-resistant factor that inhibits A3G deaminase activity. This factor could be responsible for reduced levels of hypermutation in T cells, and its identification and blockade could offer a means for increasing antiretroviral intrinsic immunity of T cells., Author Summary APOBEC3G (A3G) is an antiviral enzyme that is expressed in human T cells and macrophages, which are the cell types infected by HIV. Early in the HIV life cycle, the HIV RNA genome is reverse transcribed into DNA. A3G can modify this DNA enzymatically, leading to high rates of mutation such that the virus can no longer replicate. To date, most studies of A3G's enzymatic activity have utilized cell lines (293T and HeLa) that can be transfected to express A3G but do not express it endogenously. A report of unexpectedly low levels of mutation in viral DNA from HIV-infected human T cells led us to investigate regulation of A3G enzymatic activity in T cells. We developed a high-throughput assay to compare the enzymatic activity of endogenous A3G in T cells versus transfected (exogenous) A3G. Surprisingly, enzymatic activity of A3G from human T cell lines and primary T cells was very low relative to A3G from transfected cells, even when corrected for A3G protein amount. Moreover, T cell lysates inhibited enzymatic activity of exogenously expressed A3G. These data suggest that enzymatic activity of endogenous A3G in human T cells is inhibited by an uncharacterized mechanism that may protect the host from this DNA mutator and could have important implications for A3G antiviral activity in vivo.

For many years it has been known that viral capsid proteins are capable of self-assembly, but increasing evidence over the past decade indicates that in cells HIV-1 capsid assembly occurs via a complex but transient series of steps requiring multiple viral-host interactions. To better understand the biochemistry of HIV assembly, our group established a cell-free system that faithfully reconstitutes HIV-1 Gag synthesis and post-translational events of capsid assembly using cellular extracts, albeit more slowly and less efficiently. This system allowed initial identification of interactions that occur very transiently in cells but can be tracked in the cell-free system. Analysis of the cell-free system revealed that Gag progresses sequentially through a step-wise, energy-dependent series of assembly intermediates containing cellular proteins. One of these cellular proteins, the ATPase ABCE1, has been shown to play a critical role in the assembly process. The existence of this energy-dependent assembly pathway was subsequently confirmed in cellular systems, further validating the cell-free HIV-1 capsid assembly system as an excellent tool for identifying mechanisms underlying HIV-1 capsid formation. Here we describe how to assemble immature HIV-1 capsids in a cell-free system and separate assembly intermediates by velocity sedimentation.

Diffuse leiomyomatosis is associated with the inherited kidney disease Alport syndrome, and characterized by visceral smooth muscle overgrowth within the respiratory, gastrointestinal and female reproductive tracts. Although partial deletions of the type IV collagen genes COL4A5 and COL4A6, paired head-to-head on chromosome Xq22, are known to cause diffuse leiomyomatosis, loss of function for type IV collagen does not explain smooth muscle overgrowth. To further clarify pathogenic mechanisms, we have characterized novel deletions in patients with Alport syndrome-diffuse leiomyomatosis or Alport syndrome alone. A 27.6-kb deletion, in a female with Alport syndrome-diffuse leiomyomatosis, is marked by the most proximal, i.e. most 5', COL4A5 breakpoint described to date. By comparing this deletion to others described here and previously, we have defined a minimal overlap region, only 4.2 kb in length and containing the COL4A5-COL4A6 proximal promoters, loss of which contributes to smooth muscle overgrowth. A novel deletion in a male with Alport syndrome alone is>1.4 Mb in length, encompassing COL4A5 and COL4A6 entirely, as well as neighboring genes. We postulate that loss of the 4.2-kb region in diffuse leiomyomatosis causes misregulation of neighboring genes, contributing to smooth muscle overgrowth. Deletion of the neighboring genes themselves may afford protection from this condition. © 2003 Wiley-Liss, Inc.