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OBJECTIVE: The diagnosis of urinary tract infection (UTI) currently requires precise specimen collection, handling infectious human waste, controlled urine storage, and timely transportation to modern laboratory equipment for analysis. Here we investigate holographic lens free imaging (LFI) to show its promise for enabling automatic urine analysis at the patient bedside. METHODS: We introduce an LFI system capable of resolving important urine clinical biomarkers such as red blood cells, white blood cells, crystals, and casts in 2 mm thick urine phantoms. RESULTS: This approach is sensitive to the particulate concentrations relevant for detecting several clinical urine abnormalities such as hematuria and pyuria, linearly correlating to ground truth hemacytometer measurements with R(2) = 0.9941 and R(2) = 0.9973, respectively. We show that LFI can estimate E. coli concentrations of 10(3) to 10(5) cells/mL by counting individual cells, and is sensitive to concentrations of 10(5) cells/mL to 10(8) cells/mL by analyzing hologram texture. Further, LFI measurements of blood cell concentrations are relatively insensitive to changes in bacteria concentrations of over seven orders of magnitude. Lastly, LFI reveals clear differences between UTI-positive and UTI-negative urine from human patients. CONCLUSION: LFI is sensitive to clinically-relevant concentrations of bacteria, blood cells, and other sediment in large urine volumes. SIGNIFICANCE: Together, these results show promise for LFI as a tool for urine screening, potentially offering early, point-of-care detection of UTI and other pathological processes.

Dysfunctional immune activation accumulates during chronic viral infection and contributes to disease pathogenesis. In HIV-1, immune activation is exacerbated by concurrent infection with hepatitis C virus (HCV), accelerating depletion of CD4+ T cells. HIV-1 suppression with antiretroviral therapy (ART) generally reconstitutes CD4+ T cell counts, while also reducing the proportion that is activated. Whether this immune reconstitution also reduces the complexity of the CD4+ T cell population is unknown. We sought to characterize the relationship between activated CD4+ T cell repertoire diversity and immune reconstitution following ART in HIV-1/HCV coinfection. We extracted T cell receptor (TCR) sequences from RNA sequencing data obtained from activated CD4+ T cells of HIV-1/HCV coinfected individuals before and after treatment with ART (clinical trial NCT01285050). There was notable heterogeneity in both the extent of CD4+ T cell reconstitution and in the change in activated CD4+ TCR repertoire diversity following ART. Decreases in activated CD4+ TCR repertoire diversity following ART were predictive of the degree of CD4+ T cell reconstitution. The association of decreased activated CD4+ TCR repertoire diversity and improved CD4+ T cell reconstitution may represent loss of nonspecifically activated TCR clonotypes, and possibly selective expansion of specifically activated CD4+ clones. These results provide insight into the dynamic relationship between activated CD4+ TCR diversity and CD4+ T cell recovery of HIV-1/HCV coinfected individuals after suppression of HIV-1 viremia.

Inhibitors of leucine-rich repeat kinase 2 (LRRK2) and mutants, such as G2019S, have potential utility in Parkinson's disease treatment. Fragment hit-derived pyrrolo[2,3-d]pyrimidines underwent optimization using X-ray structures of LRRK2 kinase domain surrogates, based on checkpoint kinase 1 (CHK1) and a CHK1 10-point mutant. (2R)-2-Methylpyrrolidin-1-yl derivative 18 (LRRK2 G2019S cKi 0.7 nM, LE 0.66) was identified, with increased potency consistent with an X-ray structure of 18/CHK1 10-pt. mutant showing the 2-methyl substituent proximal to Ala147 (Ala2016 in LRRK2). Further structure-guided elaboration of 18 gave the 2-[(1,3-dimethyl-1H-pyrazol-4-yl)amino] derivative 32. Optimization of 32 afforded diastereomeric oxolan-3-yl derivatives 44 and 45, which demonstrated a favorable in vitro PK profile, although they displayed species disconnects in the in vivo PK profile, and a propensity for P-gp- and/or BCRP-mediated efflux in a mouse model. Compounds 44 and 45 demonstrated high potency and exquisite selectivity for LRRK2 and utility as chemical probes for the study of LRRK2 inhibition.

The serine/threonine kinase DYRK1A has been implicated in regulation of a variety of cellular processes associated with cancer progression, including cell cycle control, DNA damage repair, protection from apoptosis, cell differentiation, and metastasis. In addition, elevated-level DYRK1A activity has been associated with increased severity of symptoms in Down's syndrome. A selective inhibitor of DYRK1A could therefore be of therapeutic benefit. We have used fragment and structure-based discovery methods to identify a highly selective, well-tolerated, brain-penetrant DYRK1A inhibitor which showed in vivo activity in a tumor model. The inhibitor provides a useful tool compound for further exploration of the effect of DYRK1A inhibition in models of disease.

SARS-CoV-2 transmissions among healthcare personnel (HCP) and hospitalized patients are challenging to confirm. Investigation of infected persons often reveals multiple potential risk factors for viral acquisition. We combined exposure investigation with genomic analysis confirming two hospital-based clusters. Prolonged close contact with unmasked, unrecognized infectious, individuals was a common risk.

There is an urgent need for innovative methods to reduce transmission of bloodborne pathogens like HIV and HCV among people who inject drugs (PWID). We investigate if PWID who acquire non-pathogenic bloodborne viruses like anelloviruses and pegiviruses might be at greater risk of acquiring a bloodborne pathogen. PWID who later acquire HCV accumulate more non-pathogenic viruses in plasma than matched controls who do not acquire HCV infection. Additionally, phylogenetic analysis of those non-pathogenic virus sequences reveals drug use networks. Here we find first in Baltimore and confirm in San Francisco that the accumulation of non-pathogenic viruses in PWID is a harbinger for subsequent acquisition of pathogenic viruses, knowledge that may guide the prioritization of the public health resources to combat HIV and HCV., Spread of bloodborne infections, such as HCV and HIV, is a problem, particularly amongst people who inject drugs (PWID). Here, the authors describe and then confirm in observational PWID cohorts that those with more non-pathogenic viruses in plasma were more likely later to acquire HCV than PWID who had fewer of these non-pathogenic viruses.

Dysfunctional immune activation accumulates during chronic viral infection and contributes to disease pathogenesis. In HIV-1, immune activation is exacerbated by concurrent infection with hepatitis C virus (HCV), accelerating depletion of CD4(+) T cells. HIV-1 suppression with antiretroviral therapy (ART) generally reconstitutes CD4(+) T cell counts, while also reducing the proportion that is activated. Whether this immune reconstitution also reduces the complexity of the CD4(+) T cell population is unknown. We sought to characterize the relationship between activated CD4(+) T cell repertoire diversity and immune reconstitution following ART in HIV-1/HCV coinfection. We extracted T cell receptor (TCR) sequences from RNA sequencing data obtained from activated CD4(+) T cells of HIV-1/HCV coinfected individuals before and after treatment with ART (clinical trial NCT01285050). There was notable heterogeneity in both the extent of CD4(+) T cell reconstitution and in the change in activated CD4(+) TCR repertoire diversity following ART. Decreases in activated CD4(+) TCR repertoire diversity following ART were predictive of the degree of CD4(+) T cell reconstitution. The association of decreased activated CD4(+) TCR repertoire diversity and improved CD4(+) T cell reconstitution may represent loss of nonspecifically activated TCR clonotypes, and possibly selective expansion of specifically activated CD4(+) clones. These results provide insight into the dynamic relationship between activated CD4(+) TCR diversity and CD4(+) T cell recovery of HIV-1/HCV coinfected individuals after suppression of HIV-1 viremia.

Background: Uganda’s population, though, largely characterized by young people, has seen the number of people aged 60 and over grow from 686,000 twenty years ago, to 1,433,596 in 2014. Effective caring for the well-being of this population requires strategic and deliberate planning that involves quality of life (QoL) assessments. QoL assessments among the elderly are important in evaluating the efficacy of strategies, such as health interventions, welfare programs, health care, and well-being of the elderly. However, elderly in Uganda face several challenges, ranging from loneliness, poor housing, lack of social and financial support, and poor health. These may negatively affect older persons’ quality of life and consequently their perceptions and attitudes towards aging. Methods: The study was carried out in 2019 in the communities of Nansana and Busukuma town councils in Wakiso district, Uganda. The participants were 380 people 60 years and older. To establish the association between perceptions of ageing and QoL, this study utilized a locally adapted version of the Older Person’s Quality of Life Questionnaire (OPQOL) and the Brief Ageing Perceptions Questionnaire (B-APQ). The OPQOL assesses three domains of QoL: Health QoL (HQoL); Social economic QoL (SQoL); and Psychosocial QoL (PQoL). The B-APQ assesses perceptions about physical age, participation in social activities, and perceptions about ability to regulate emotions as one ages. Pearson’s Chi-square tests were used to characterize the relationship between the perceptions and quality of life.Results: The majority of the respondents, 61% (95%CI 56.7-64.8), had negative perceptions towards ageing. Eighty six percent had poor HQoL, 90% poor SQoL and 83% poor PQoL. There was a significant association between good HQoL and positive perception about participation in social activities (X2 = 7.3670, P = 0.007) as well as with positive perception on regulation of emotions (X2 = 18.1803, P2 = 5.3472, P = 0.021), as well with positive perception on regulation of emotions (X2 = 10.5128, P2 = 9.2414, P= 0.002).Conclusion: Positive perceptions of ageing are associated with good QoL. Directly addressing perceptions of ageing could be a low cost and effective strategy to improve the QoL of older persons in SSA

Early development of broadly neutralizing antibodies (bNAbs) targeting the hepatitis C virus (HCV) envelope glycoprotein E2 is associated with spontaneous clearance of infection, so induction of bNAbs is a major goal of HCV vaccine development. However, much remains to be learned at a molecular level about protective E2-reactive antibodies, since HCV infection persists in some individuals despite early development of broadly neutralizing plasma. To examine B cell repertoire features associated with broad neutralization and viral clearance, we performed RNA sequencing of the B cell receptors (BCRs) of HCV E2-reactive B cells of people with cleared or persistent HCV, including subjects with high or low plasma neutralizing breadth in both clearance and persistence groups. We identified many E2-reactive public BCR clonotypes, which are antibody clones with the same V and J-genes and identical CDR3 sequences, shared among subjects grouped by either clearance or neutralization status. The majority (89) of these public clonotypes were shared by two subjects with broad plasma neutralizing activity and cleared infection, but not found in subjects with high plasma neutralizing breadth and persistent infection. We cloned a potent, cross-reactive neutralizing monoclonal antibody (mAb) by pairing the most abundant public heavy and light chains from these two subjects, providing evidence that broadly E2-reactive public clonotypes arise in a subset of individuals with broadly neutralizing plasma and spontaneous clearance of infection. Further characterization of the molecular features and function of these antibodies can inform HCV vaccine development.

Background and aims Implementing effective interventions for hepatitis C virus (HCV) requires detailed understanding of local transmission dynamics and geospatial spread. Little is known about HCV phylodynamics, particularly among high-burden populations such as people who inject drugs (PWID). Approach and results We used 483 HCV sequences and detailed individual-level data from PWID across four Indian cities. Bayesian phylogeographic analyses were used to evaluate transmission hotspots and geospatial diffusion of virus. Phylogenetic cluster analysis was performed to infer epidemiologic links and factors associated with clustering. A total of 492 HIV sequences were used to draw comparisons within the same population, and in the case of co-infections, evaluate molecular evidence for shared transmission pathways. Overall, 139/483 (28.8%) of HCV sequences clustered with a median cluster size of three individuals. Genetically linked participants with HCV were significantly younger and more likely to be infected with HCV subtype 3b, as well as live and inject close to one another. Phylogenetic evidence suggests likely ongoing HCV infection/reinfection with limited support for shared HIV/HCV transmission pathways. Phylogeographic analyses trace historic HCV spread back to Northeastern India and show diffusion patterns consistent with drug trafficking routes. Conclusions This study is among the first to characterize HCV phylodynamics among PWID in a low-and middle-income country setting. Heterogeneity and recent genetic linkage of HCV across geographically disparate Indian states suggest that targeted interventions could help prevent reimportation of virus via drug trafficking routes.

There is interest in understanding the mechanisms that underlie reports that patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain PCR positive many weeks after initial infection. The recent paper by Zhang et al. (1) suggests a potential explanation of this phenomenon by claiming that SARS-CoV-2 RNA can integrate into the genome of infected human cells. The authors also reanalyze RNA sequencing (RNA-seq) data and report that SARS-CoV-2−host chimeric reads are present in cells and patient tissues. Given the potential implications of this research on the long-term impacts of COVID-19, we feel that it’s necessary to scrutinize the evidence presented. To determine whether SARS-CoV-2 RNA might be retrotranscribed … [↵][1]1To whom correspondence may be addressed. Email: r.parry{at}uq.edu.au or lachlan.coin{at}unimelb.edu.au. [1]: #xref-corresp-1-1

End-organ cytomegalovirus (CMV) disease can be life threatening to solid organ transplant recipients. Diagnosis is often complicated by variation in amount of CMV DNA in plasma and the need for an invasive procedure to obtain a biopsy of the suspected affected organ, which can delay recognition and treatment. Several inflammatory cytokines are elevated in CMV disease, and the purpose of this study was to determine if they could be used to distinguish solid organ transplant recipients with CMV DNAemia alone from those with possible end-organ CMV disease. We found that regardless of pre-transplant CMV serostatus, plasma interleukin (IL)-18, tumor necrosis factor-α (TNF-α), and amount of CMV DNA in plasma were increased in possible end-organ CMV disease, with elevated IL-18 associated with increased odds of possible end-organ CMV disease even after adjusting for amount of CMV DNA. These findings highlight IL-18 and TNF-α as potential non-invasive markers of possible end-organ CMV disease regardless of transplanted organ or serostatus in solid organ transplant recipients.

Inhibitors of leucine-rich repeat kinase 2 (LRRK2) and mutants, such as G2019S, have potential utility in Parkinson's disease treatment. Fragment hit-derived pyrrolo[2,3

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants concerning for enhanced transmission, evasion of immune responses, or associated with severe disease have motivated the global increase in genomic surveillance. In the current study, large-scale whole-genome sequencing was performed between November 2020 and the end of March 2021 to provide a phylodynamic analysis of circulating variants over time. In addition, we compared the viral genomic features of March 2020 and March 2021. Methods A total of 1600 complete SARS-CoV-2 genomes were analyzed. Genomic analysis was associated with laboratory diagnostic volumes and positivity rates, in addition to an analysis of the association of selected variants of concern/variants of interest with disease severity and outcomes. Our real-time surveillance features a cohort of specimens from patients who tested positive for SARS-CoV-2 after completion of vaccination. Results Our data showed genomic diversity over time that was not limited to the spike sequence. A significant increase in the B.1.1.7 lineage (alpha variant) in March 2021 as well as a transient circulation of regional variants that carried both the concerning S: E484K and S: P681H substitutions were noted. Lineage B.1.243 was significantly associated with intensive care unit admission and mortality. Genomes recovered from fully vaccinated individuals represented the predominant lineages circulating at specimen collection time, and people with those infections recovered with no hospitalizations. Conclusions Our results emphasize the importance of genomic surveillance coupled with laboratory, clinical, and metadata analysis for a better understanding of the dynamics of viral spread and evolution., Genomic surveillance of severe acute respiratory syndrome coronavirus 2 in the US National Capital Region showed transient circulation of regional variants, emergence and predominance of lineage B.1.1.7, genomic diversity that is not limited to the spike sequence, and an association of lineage B.1.243 with severe disease.

A vaccine for hepatitis C virus (HCV) is urgently needed. Development of broadly neutralizing plasma antibodies during acute infection is associated with HCV clearance, but the viral epitopes of these plasma antibodies are unknown. Identifying these epitopes could define the specificity and function of neutralizing antibodies (NAbs) that should be induced by a vaccine. Here, we present the development and application of a high-throughput method that deconvolutes polyclonal anti-HCV NAbs in plasma, delineating the epitope specificities of anti-HCV NAbs in acute-infection plasma of 44 humans with subsequent clearance or persistence of HCV. Remarkably, we identified multiple broadly neutralizing antibody combinations that were associated with greater plasma neutralizing breadth and with HCV clearance. These studies have the potential to inform new strategies for vaccine development by identifying broadly neutralizing antibody combinations in plasma associated with the natural clearance of HCV, while also providing a high-throughput assay that could identify these responses after vaccination trials.

Background Men in Sub-Saharan Africa are less engaged than women in accessing HIV testing and treatment and, consequently, experience higher HIV-related mortality. Reaching men with HIV testing services is challenging, thus, increasing the need for innovative ways to engage men with low access and those at higher risk. In this study, we explore men’s perceptions of drivers and barriers of workplace-based HIV self-testing in Uganda. Methods An exploratory study involving men working in private security companies employing more than 50 men in two districts, in central and western Uganda. Focus group discussions and key informant interviews were conducted. Data were analyzed using inductive content analysis. Results Forty-eight (48) men from eight private security companies participated in 5 focus group discussions and 17 key informant interviews. Of the 48 men, 14(29.2%) were ages 26–35 years. The majority 31(64.6%) were security guards. The drivers reported for workplace-based HIV self-testing included convenience, autonomy, positive influence from work colleagues, the need for alternative access for HIV testing services, incentives, and involvement of employers. The barriers reported were the prohibitive cost of HIV tests, stigma, lack of testing support, the fear of discrimination and isolation, and concerns around decreased work productivity in the event of a reactive self-test. Conclusions We recommend the involvement of employers in workplace-based HIV self-testing to encourage participation by employees. There is need for HIV self-testing support both during and after the testing process. Both employers and employees recommend the use of non-monetary incentives, and regular training about HIV self-testing to increase the uptake and acceptability of HIV testing services at the workplace.

The early COVID-19 pandemic has been characterized by rapid global spread. In the United States National Capital Region, over 2,000 cases were reported within three weeks of its first detection in March 2020. We aimed to use genomic sequencing to understand the initial spread of SARS-CoV-2, the virus that causes COVID-19, in the region. By correlating genetic information to disease phenotype, we also aimed to gain insight into any correlation between viral genotype and case severity or transmissibility.We performed whole genome sequencing of clinical SARS-CoV-2 samples collected in March 2020 by the Johns Hopkins Health System. We analyzed these regional SARS-CoV-2 genomes alongside detailed clinical metadata and the global phylogeny to understand early establishment of the virus within the region.We analyzed 620 samples from the Johns Hopkins Health System collected between March 11-31, 2020, comprising 37.3% of the total cases in Maryland during this period. We selected 143 of these samples for sequencing, generating 114 complete viral genomes. These genomes belong to all five major Nextstrain-defined clades, suggesting multiple introductions into the region and underscoring the diversity of the regional epidemic. We also found that clinically severe cases had genomes belonging to all of these clades.We established a pipeline for SARS-CoV-2 sequencing within the Johns Hopkins Health system, which enabled us to capture the significant viral diversity present in the region as early as March 2020. Efforts to control local spread of the virus were likely confounded by the number of introductions into the region early in the epidemic and interconnectedness of the region as a whole.

Background The availability of effective, oral direct acting antivirals (DAAs) for hepatitis C virus (HCV) treatment has put elimination of HCV as a public health challenge within reach. However, little is known about the characteristics of transmission networks of people who inject drugs (PWID). Methods Sequencing of a segment of the HCV genome was performed on samples collected from a community-based cohort of PWID between August 2005 and December 2016. Phylogenetic trees were inferred, and clusters were identified (70% bootstrap threshold; 0.04 maximum genetic distance threshold). We describe sex, race, age difference, and HIV infection status of potential transmission partners. Logistic regression was used to assess factors associated with being in an HCV cluster. Results Of 508 HCV genotype 1 viremic PWID, 8% (n = 41) were grouped into 20 clusters, consisting of 19 pairs and 1 triad. In adjusted analyses, female sex (odds ratio [OR] 2.3 [95% confidence interval (CI) 1.2–4.5]) and HIV infection (OR 5.7 [CI 2.7–11.9]) remained independently associated with being in an HCV infection cluster. Conclusions Molecular epidemiological analysis reveals that, in this cohort of PWID in Baltimore, HIV infection and female sex were associated with HCV clustering. Combination HCV prevention interventions targeting HIV infected PWID and addressing HCV infection prevention needs of women have potential to advance HCV elimination efforts.

Multiple studies have shown loss of SARS-CoV-2 specific antibodies over time after infection, raising concern that humoral immunity against the virus is not durable. If immunity wanes quickly, millions of people may be at risk for reinfection after recovery from COVID-19. However, memory B cells (MBC) could provide durable humoral immunity even if serum neutralizing antibody titers decline. We performed multi-dimensional flow cytometric analysis of S protein receptor binding domain (S-RBD)-specific MBC in cohorts of ambulatory COVID-19 patients with mild disease, and hospitalized patients with moderate to severe disease, at a median of 54 (39–104) days after onset of symptoms. We detected S-RBD-specific class-switched MBC in 13 out of 14 participants, including 4 of the 5 participants with lowest plasma levels of anti-S-RBD IgG and neutralizing antibodies. Resting MBC (rMBC) made up the largest proportion of S-RBD-specific class-switched MBC in both cohorts. FCRL5, a marker of functional memory when expressed on rMBC, was dramatically upregulated on S-RBD-specific rMBC. These data indicate that most SARS-CoV-2-infected individuals develop S-RBD-specific, class-switched MBC that phenotypically resemble germinal center-derived B cells induced by effective vaccination against other pathogens, providing evidence for durable B cell-mediated immunity against SARS-CoV-2 after recovery from mild or severe COVID-19 disease., Graphical Abstract

SARS-CoV-2 infection induces severe disease in a subpopulation of patients, but the underlying mechanisms remain unclear. We demonstrate robust IgM autoantibodies that recognize angiotensin converting enzyme-2 (ACE2) in 18/66 (27%) patients with severe COVID-19, which are rare (2/52; 3.8%) in hospitalized patients who are not ventilated. The antibodies do not undergo class-switching to IgG, suggesting a T-independent antibody response. Purified IgM from anti-ACE2 patients activates complement. Pathological analysis of lung obtained at autopsy shows endothelial cell staining for IgM in blood vessels in some patients. We propose that vascular endothelial ACE2 expression focuses the pathogenic effects of these autoantibodies on blood vessels, and contributes to the angiocentric pathology observed in some severe COVID-19 patients. These findings may have predictive and therapeutic implications.One-sentence summaryACE2 autoantibodies in severe COVID-19 have features of a T-independent immune response, and may mediate vascular damage.

BackgroundThe early COVID-19 pandemic has been characterized by rapid global spread. In the United States National Capital Region, over 2,000 cases were reported within three weeks of its first detection in March 2020. We aimed to use genomic sequencing to understand the initial spread of SARS-CoV-2, the virus that causes COVID-19, in the region. By correlating genetic information to disease phenotype, we also aimed to gain insight into any correlation between viral genotype and case severity or transmissibility.MethodsWe performed whole genome sequencing of clinical SARS-CoV-2 samples collected in March 2020 by the Johns Hopkins Health System. We analyzed these regional SARS-CoV-2 genomes alongside detailed clinical metadata and the global phylogeny to understand early establishment of the virus within the region.ResultsWe analyzed 620 samples from the Johns Hopkins Health System collected between March 11–31, 2020, comprising 37.3% of the total cases in Maryland during this period. We selected 143 of these samples for sequencing, generating 114 complete viral genomes. These genomes belong to all five major Nextstrain-defined clades, suggesting multiple introductions into the region and underscoring the diversity of the regional epidemic. We also found that clinically severe cases had genomes belonging to all of these clades.ConclusionsWe established a pipeline for SARS-CoV-2 sequencing within the Johns Hopkins Health system, which enabled us to capture the significant viral diversity present in the region as early as March 2020. Efforts to control local spread of the virus were likely confounded by the number of introductions into the region early in the epidemic and interconnectedness of the region as a whole.

Antibodies targeting the hepatitis C virus (HCV) envelope glycoprotein E2 are associated with delayed disease progression, and these antibodies can also facilitate spontaneous clearance of infection in some individuals. However, many infected people demonstrate low titer and delayed anti-E2 antibody responses. Since a goal of HCV vaccine development is induction of high titers of anti-E2 antibodies, it is important to define the mechanisms underlying these suboptimal antibody responses. By staining lymphocytes with a cocktail of soluble E2 (sE2) glycoproteins, we detected HCV E2-specific (sE2+) B cells directly ex vivo at multiple acute infection timepoints in 29 HCV-infected subjects with a wide range of anti-E2 IgG titers, including 17 persistently infected subjects and 12 subjects with spontaneous clearance of infection. We performed multi-dimensional flow cytometric analysis of sE2+ and E2-nonspecific (sE2-) class-switched B cells (csBC). In sE2+ csBC from both persistence and clearance subjects, frequencies of resting memory B cells (rMBC) were reduced, frequencies of activated MBC (actMBC) and tissue-like MBC (tlMBC) were increased, and expression of FCRL5, an IgG receptor, was significantly upregulated. Across all subjects, plasma anti-E2 IgG levels were positively correlated with frequencies of sE2+ rMBC and sE2+ actMBC, while anti-E2 IgG levels were negatively correlated with levels of FCRL5 expression on sE2+ rMBC and PD-1 expression on sE2+ actMBC. Upregulation of FCRL5 on sE2+ rMBC and upregulation of PD-1 on sE2+ actMBC may limit anti-E2 antibody production in vivo. Strategies that limit upregulation of these molecules could potentially generate higher titers of protective antibodies against HCV or other pathogens., Author summary Antiviral immunity relies on production of protective immunoglobulin G (IgG) by B cells, but many hepatitis C virus (HCV)-infected individuals have very low levels of HCV-specific IgG in their serum. Elucidating mechanisms underlying this suboptimal IgG expression remains paramount in guiding therapeutic and vaccine strategies. In this study, we developed a highly specific method to capture HCV-specific B cells and characterized their surface protein expression. Two proteins analyzed were Fc receptor-like protein 5 (FCRL5), a cell surface receptor for IgG, and programmed cell death protein-1 (PD-1), a marker of lymphocyte activation and exhaustion. We measured serum levels of anti-HCV IgG in these subjects and demonstrated that overexpression of FCRL5 and PD-1 on memory B cells was associated with reduced anti-E2 IgG levels. This study uses HCV as a viral model, but the findings may be applicable to many viral infections, and they offer new potential targets to enhance antiviral IgG production.

The availability of generic direct acting antivirals (DAAs) for hepatitis C virus (HCV) treatment has prompted many low-and-middle-income countries to launch HCV elimination programs. Because the efficacy of some of these generic DAAs varies by HCV viral subtype, information on subtype distribution can contribute important information to these elimination programs. We conducted a cross-sectional serosurvey to characterize HCV subtype diversity among HIV positive people who inject drugs (PWID) across 14 cities in India. Of 801 HIV positive PWID sampled, 639 tested HCV antibody positive (78.9%). Among 105 samples sequenced, genotype 3 (58.1%) was the most commonly observed followed by genotype 1 (36.2%) and genotype 6 (5.7%). Of the genotype 3 infections, 65% were subtype 3a and 35% were subtype 3b. Of the genotype 1 infections, 94% were subtype 1a and 6% were subtype 1b. All genotype 6 samples were subtype 6n. There was some variability in genotype diversity depending on geographic region and PWID epidemic stage with greater diversity observed in older PWID epidemics. One sequence, HY018, did not cluster with any known reference sequences in phylogenetic analysis. Nearly 80% of HIV infected PWID across India are co-infected with HCV, and subtype prevalence and genetic diversity varied by region and PWID epidemic stage. HCV elimination programs in India will need to consider HCV subtype. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13337-019-00553-2) contains supplementary material, which is available to authorized users.

We investigated longitudinal viral clustering among and within subjects in a highly networked cohort of people who inject drugs (PWID). All subjects had estimated dates of infection and two or more E1 sequences (bp 943-1288 relative to H77) with 1 to 14 years of follow up. Two methods (HIV-TRACE and PhyloPart) were used to determine clusters. Genetic distance thresholds were determined by comparing intra-and inter-host distances. Additional phylogenetic analysis was performed on subjects with complicated viral histories. At the optimal threshold of 3.9%, HIV-TRACE found 77 clusters and PhyloPart found 63 clusters, of which 27 and 32 contained multiple subjects, respectively. Furthermore, 1/3 of the subjects had sequences in different clusters over the course of the study, including some cases in which a later-sampled sequence matched a cluster detected much earlier in the infection, despite being separated by RNA-negative lab visit and detection of sequences in different clusters. A detailed phylogenetic analysis of four subjects with such patterns showed that in all four cases, the earlier and later variants grouped closely on the tree, and did not group with concurrent sequences from any other subject. These observations suggest that subjects are either experiencing rapid and recurring infection-clearance-reinfection cycles from the same source, or a single transmission event produces a chronic infection that may go undetected and/or co-circulate with different viruses from separate transmission events. Furthermore, our results show the utility of using longitudinal sampling to obtain a more comprehensive view of the viral linkages in high-risk populations.

Hepatitis C infection affects close to 150 million people worldwide. In the US, it is estimated that as many as 5 million people have been infected with the hepatitis C virus (HCV),7 many of whom are unaware of their infection. Some individuals clear the infection on their own, but the overwhelming majority of patients (approximately 75%) develop chronic infection, which can be asymptomatic for many years. Ultimately, chronic HCV infection can lead to cirrhosis, and potentially liver failure and hepatocellular carcinoma. In the US, HCV is now the most common indication for liver transplantation, and it accounts for more deaths each year than all other reportable infectious diseases combined, including HIV. The latest generation of direct-acting antivirals (DAA) has the potential to cure the disease in at least 95% of patients. Although the cost of these medications is slowly declining, they are still expensive (∼$30000–$100 000 per case), and states have varying health-care coverage limitations as to who can be treated. That said, the first step in eliminating the virus is identifying those with infection. To this end, the CDC has recommended one-time baby-boomer cohort screening as the prevalence of infection is highest in those born between 1945–1965. More recently, we have seen an increase in the rate of infection in individuals 20–40 years of age, which has been associated with increased injection drug use in the current US opioid epidemic. The currently recommended laboratory testing algorithm involves first screening for HCV antibodies (HCV Ab) via immunoassay. For those patients with positive HCV Ab results, follow-up HCV RNA testing is necessary to differentiate those who have spontaneously cleared the infection from those who have become chronically infected and need further medical care. Studies have shown that, even with a successful screening program, many patients do not get the necessary …

Background Human cytomegalovirus (CMV) continues to cause significant morbidity and mortality in solid organ transplant (SOT) recipients despite prophylaxis. Tissue invasive CMV disease (TI-CMV) can lead to end-organ damage and graft loss. Diagnosing TI-CMV can be challenging as CMV viral load in the blood does not always correlate with episodes of TI-CMV and therefore definitive diagnosis often requires an invasive procedure such as bronchoscopy or colonoscopy. The purpose of this study was to determine if proinflammatory cytokines, including IL-18, are elevated in SOT recipients with probably TI-CMV as a way to identify patients at risk for this severe form of CMV disease. Methods The electronic medical record was searched for adult SOT recipients who were tested for CMV via blood qPCR during an 11-month period.Twenty-nine SOT recipients were identified that had episodes of CMV DNAemia without other concomitant infections during this time period. Patients were divided into those that had probable TI-CMV and those with CMV DNAemia alone, by chart review. Inflammatory cytokines (IFNγ, TNFα, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-18, and IL-1RA) were measured in residual plasma from these patients using a commercially available multiplex assay for at least two time points during the study period. Wilcoxon-Rank-Sum, logistic regression, and principal component analysis was performed comparing patients with and without probable TI-CMV. Results Patients with probable TI-CMV had significantly higher IL-18, TNFα, and IL-1β than patients with CMV DNAemia alone (p < 0.001, < 0.001, and < 0.05 respectively). When adjusting for transplant type and CMV recipient serostatus, elevations in TNFα (OR 1.43, 95% CI 1.07-1.92) and IL-18 (OR 2.00, 95% CI 1.06-3.75) were associated with increased odds of having probable TI-CMV. In principal component analysis the combination of CMV viral load, IL-18, TNFa, and IL-1β accounted for 80% of the variance in the data. Conclusion TNFα and IL-18 in combination with CMV viral load may be useful in predicting likelihood of TI-CMV. This is important in situations where tissue biopsies are not feasible, and adds to our diagnostic capability for TI-CMV in SOT recipients. Disclosures Stuart C. Ray, MD, miDiagnostics, Inc. (Board Member, Research Grant or Support) Robin K. Avery, MD, Aicuris (Scientific Research Study Investigator)Astellas (Scientific Research Study Investigator)Chimerix (Scientific Research Study Investigator)Merck (Grant/Research Support, Scientific Research Study Investigator)Oxford Immunotec (Scientific Research Study Investigator)Qiagen (Scientific Research Study Investigator)Takeda/Shire (Scientific Research Study Investigator)

Mutations in leucine-rich repeat kinase 2 (LRRK2), such as G2019S, are associated with an increased risk of developing Parkinson’s disease. Surrogates for the LRRK2 kinase domain based on checkpoint kinase 1 (CHK1) mutants were designed, expressed in insect cells infected with baculovirus, purified, and crystallized. X-ray structures of the surrogates complexed with known LRRK2 inhibitors rationalized compound potency and selectivity. The CHK1 10-point mutant was preferred, following assessment of surrogate binding affinity with LRRK2 inhibitors. Fragment hit-derived arylpyrrolo[2,3-b]pyridine LRRK2 inhibitors underwent structure-guided optimization using this crystallographic surrogate. LRRK2-pSer935 HEK293 IC50 data for 22 were consistent with binding to Ala2016 in LRRK2 (equivalent to Ala147 in CHK1 10-point mutant structure). Compound 22 was shown to be potent, moderately selective, orally available, and brain-penetrant in wild-type mice, and confirmation of target engagement was demonstrated, with LR...

High integrity data retention and curation are critical for preserving the scientific record and informing future discovery.1 However, these steps are often neglected or inadequate because of lack of a tractable, easily operated approach. We offer general guidelines and an exemplar method that is applicable to many, but by no means all, laboratories. Data generated from National Institutes of Health funding should be stored for 3 years after the end of the last competitive renewal. In some cases, data related to patients and patents has longer storage obligations. Data storage rules are in evolution and may differ among various funding agencies, institutions, and journals. The data belong to the host institution, but the responsibility for storage (ie, stewardship) is typically transferred to individual investigators, many of whom have insufficient understanding of or infrastructure for this important role.2 While authors are routinely asked to affirm their accountability for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved,3 there is no standard infrastructure for sharing all primary data among all authors concurrently during drafting of an article, much less after publication. If you have worked in a laboratory for much time, you will know it is sometimes difficult to locate original data. Some of these challenges are magnified by moving laboratory locations, storing data on proprietary and outmoded platforms, lack of a clear paper trail after laboratory personnel move on, or multiple collaborators generating data at various sites. In general, older data are harder to follow compared with newer data. These truths became starkly evident to me (M.E. Anderson) after a former laboratory member was discovered to have engaged in scientific misconduct. While there was no doubt about his transgressions involving manipulated …

The availability of effective, oral direct acting antivirals for hepatitis C virus (HCV) treatment has fueled optimism for HCV elimination through treatment as prevention (TasP) among people who inject drugs (PWID). Identifying characteristics of individuals in transmission networks would provide critical information for the development and implementation of effective, targeted HCV TasP strategies. The AIDS linked to the IntraVenous Experience (ALIVE) cohort has followed PWID in Baltimore since 1988. Sequencing of the HCV core/E1 region (342 nucleotides) was performed on HCV viremic samples from the most recent study visit attended by ALIVE participants between August, 2005 and December, 2016. Outgroup sequences were retrieved from GenBank through a BLAST search for HCV sequences similar to study sequences to support identification of ‘local clusters’ and were aligned to study sequences using Clustal O. Phylogenetic trees were inferred for each of HCV subtype 1a and 1b separately through maximum likelihood analysis implemented in the MEGA X software using the Tamura-Nei model with gamma distribution and invariant sites. Nucleotide substitution model selection was based on the corrected Akaike information criterion scores of various models in MEGA. Robustness of the resulting tree was assessed by bootstrapping with 1,000 replicates. Clusters were identified using ClusterPicker software (70% bootstrap threshold and 0.05 maximum genetic distance threshold). Sensitivity analyses were performed by varying the genetic distance threshold between 0.025 and 0.05 to determine the effect on identification of factors associated with clustering. HCV infection clustering was defined as > 2 participants with HCV genome sequences satisfying 70 per cent bootstrap and 0.05 genetic threshold distance requirement for sequence similarity. Logistic regression was used to assess sociodemographic factors associated with being in an HCV cluster. Among 512 HCV genotype 1 viremic PWID, HCV subtype prevalence was 83 per cent genotype 1a and 17 per cent genotype 1b. The median age of participants was 54 years, 68 per cent male, 87 per cent Black, and 38 per cent HIV infected. Overall, 9 per cent (n = 44) were grouped into 21 clusters, consisting of 20 pairs and 1 triad. Of the 425 genotype 1a and 87 genotype 1b samples evaluated, 8 per cent (n = 33) and 13 per cent (n = 11) respectively, were in clusters. In unadjusted analyses, membership in a cluster, was associated with younger age (odds ratio (OR) 1.5 [95% confidence interval (CI) 1.1–2.1] per 10 year age decrease); female sex (OR 2.8 [95% CI 1.5–5.3]), HIV infection (OR 4.9 [95% CI 2.5–9.9]), and living in East Baltimore (versus outside East Baltimore, OR 2.0 [95% CI 1.0–3.9]). In adjusted analyses, female sex (OR 2.0 [95% CI 1.0–3.9] and HIV infection (OR 5.4 [95% CI 2.6–11.1] remained independently associated with being in an HCV infection cluster. HIV-infected PWID and their networks should be prioritized for HCV treatment and prevention interventions given an increased likelihood of transmission in these groups.

We describe our work to establish structure- and fragment-based drug discovery to identify small molecules that inhibit the anti-apoptotic activity of the proteins Mcl-1 and Bcl-2. This identified hit series of compounds, some of which were subsequently optimized to clinical candidates in trials for treating various cancers. Many protein constructs were designed to identify protein with suitable properties for different biophysical assays and structural methods. Fragment screening using ligand-observed NMR experiments identified several series of compounds for each protein. The series were assessed for their potential for subsequent optimization using 1H and 15N heteronuclear single-quantum correlation NMR, surface plasmon resonance, and isothermal titration calorimetry measurements to characterize and validate binding. Crystal structures could not be determined for the early hits, so NMR methods were developed to provide models of compound binding to guide compound optimization. For Mcl-1, a benzodioxane/benzoxazine series was optimized to a Kd of 40 μM before a thienopyrimidine hit series was identified which subsequently led to the lead series from which the clinical candidate S 64315 (MIK 665) was identified. For Bcl-2, the fragment-derived series were difficult to progress, and a compound derived from a published tetrahydroquinone compound was taken forward as the hit from which the clinical candidate (S 55746) was obtained. For both the proteins, the work to establish a portfolio of assays gave confidence for identification of compounds suitable for optimization.

A complete blood count (CBC) is the most commonly ordered laboratory test for patient diagnosis and treatment.1 Clinical decisions are often made on the basis of a single result which is rarely repeated. The availability of more rapid tests for CBCs that are accessible and approved for Clinical Laboratory Improvement Amendments (CLIA)-waived use at the point of need increases the likelihood that serial testing may occur over short periods of time.2 Understanding natural CBC variation in healthy individuals may help clinicians interpret serial test results which may have a range of values. We sought to evaluate CBCs from blood drawn over a short time period and assessed in separate, accredited labs to understand biological and laboratory-based variability. Ten healthy adult volunteers with a self-report of being healthy were recruited by a study nurse and signed informed consent between April and May 2017. At baseline, 1 hour and 2 hours, six 3 mL tubes were drawn each time and sent as routine tests in duplicate to three College of American Pathologist certified clinical core laboratories for CBC: Lab 1 used a Beckman Coulter LH750 (Beckman Coulter, Atlanta, GA), Lab 2 used a Sysmex XE2100 (Sysmex America, Lincolnshire, IL) and Lab 3 used a Sysmex XN9000 (Sysmex America). The total time range covered by this study was 120 min, consistent …

Lens-free holographic imaging is important in many biomedical applications, as it offers a wider field of view, more mechanical robustness and lower cost than traditional microscopes. In many cases, it is important to be able to detect biological objects, such as blood cells, in microscopic images. However, state-of-the-art object detection methods are not designed to work on holographic images. Typically, the hologram must first be reconstructed into an image of the specimen, given a priori knowledge of the distance between the specimen and sensor, and standard object detection methods can then be used to detect objects in the reconstructed image. This paper describes a method for detecting objects directly in holograms while jointly reconstructing the image. This is achieved by assuming a sparse convolutional model for the objects being imaged and modeling the diffraction process responsible for generating the recorded hologram. This paper also describes an unsupervised method for training the convolutional templates, shows that the proposed method produces promising results for detecting white blood cells in holographic images, and demonstrates that the proposed object detection method is robust to errors in estimated focal depth.

In this paper we consider the task of detecting platelets in images of diluted whole blood taken with a lens-free microscope. Despite having several advantages over traditional microscopes, lens-free imaging systems have the significant challenge that the resolution of the system is typically limited by the pixel dimensions of the image sensor. As a result of this limited resolution, detecting platelets is very difficult to do even by manual inspection of the images due to the fact that platelets occupy just a few pixels of the reconstructed image. To address this challenge, we develop an optical model of diluted whole blood to generate physically realistic simulated holograms which we then use to train a convolutional neural network (CNN) for platelet detection. We validate our approach by collecting both lens-free and fluorescent microscopy images of the same field of view of diluted whole blood samples with fluorescently labeled platelets.

Hepatitis-C Virus (HCV) sequences are often used to establish networks of people who inject drugs (PWID). However, the degree to which within-host evolutionary dynamics affect those inferences has not been carefully studied. Here, we analyzed 702 longitudinally-sampled HCV E1 sequences from 88 HCV+ people who inject drugs (PWID) in the Baltimore Before and After Acute Study of Hepatitis (BBAASH) cohort. Individuals were tested for HCV RNA over multiple visits to the clinic, and the HCV E1 gene was sequenced for HCV+ samples. Genetic clustering was performed on the full set of sequences using a 3% genetic distance threshold to define epidemiological linkage. Maximum-likelihood (ML) phylogenies were inferred to assess evolutionary relationships. We found 22 clusters containing sequences sampled over five or more years (long-term clusters, LTC), of which 17 had >1 subject. In six of the multi-subject LTC, one subject had a sequence sampled >3 years earlier or later than the next-closest subject in the cluster (time-gap LTC). ML trees showed that, in three of the time-gap LTC, two subjects had identical sequences despite 7–10 years separating the sampling times. In four of the time-gap LTC for whom additional data were available, the subject with the later detected shared variant had both different variants and visits with no detectable HCV RNA (RNA-) prior to the appearance of the shared variant. In the subject with the earlier detection of the shared variant, different variants and RNA- visits were also detected in multiple cases subsequent to appearance of the shared variant. Complex patterns of shared viral variation among PWID reflect on-going re-infection, multiple transmission partners, and/or inconsistent detection of viral variants. Our results suggest that transmission events are currently underestimated by analysis of sequences at a single point in time.