Persistent low-level variants in a subset of viral genes are highly predictive of poor outcome in immunocompromised patients with cytomegalovirus infection.

BACKGROUND: Human cytomegalovirus is the most common and serious opportunistic infection after solid organ and haematopoietic stem cell transplantation. In this study, we used whole-genome cytomegalovirus data to investigate viral factors associated with the clinical outcome. METHODS: We sequenced cytomegalovirus samples from 16 immunocompromised paediatric patients with persistent viraemia. 8/16 patients died of complications due to cytomegalovirus infection. We also sequenced samples from 35 infected solid organ adult recipients of whom one died with cytomegalovirus infection. RESULTS: We showed that samples from both groups have fixed variants at resistance sites and mixed infections. NGS sequencing also revealed non-fixed variants at resistance sites in most of the patients who died (6/9). A machine learning approach identified 10 genes with non-fixed variants in these patients. These genes formed a viral signature which discriminated patients with cytomegalovirus infection who died from those that survived with high accuracy (AUC=0.96). Lymphocyte numbers for a subset of patients showed no recovery post-transplant in the patients who died. CONCLUSIONS: We hypothesise that the viral signature identified in this study may be a useful biomarker for poor response to antiviral drug treatment and indirectly for poor T cell function, potentially identifying early, those patients requiring non-pharmacological interventions.

Socioeconomic and Demographic Risk Factors for SARS-CoV-2 Seropositivity Among Healthcare Workers in a UK Hospital: A Prospective Cohort Study.

BACKGROUND: To protect healthcare workers (HCWs) from the consequences of disease due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is necessary to understand the risk factors that drive exposure and infection within hospitals. Insufficient consideration of key socioeconomic variables is a limitation of existing studies that can lead to bias and residual confounding of proposed risk factors for infection. METHODS: The Co-STARs study prospectively enrolled 3679 HCWs between April 2020 and September 2020. We used multivariate logistic regression to comprehensively characterize the demographic, occupational, socioeconomic, and environmental risk factors for SARS-CoV-2 seropositivity. RESULTS: After adjusting for key confounders, relative household overcrowding (odds ratio [OR], 1.4 [95% confidence interval {CI}, 1.1-1.9]; P = .006), Black, Black British, Caribbean, or African ethnicity (OR, 1.7 [95% CI, 1.2-2.3]; P = .003), increasing age (ages 50-60 years: OR, 1.8 [95% CI, 1.3-2.4]; P < .001), lack of access to sick pay (OR, 1.8 [95% CI, 1.3-2.4]; P < .001). CONCLUSIONS: Socioeconomic and demographic factors outside the hospital were the main drivers of infection and exposure to SARS-CoV-2 during the first wave of the pandemic in an urban pediatric referral hospital. Overcrowding and out-of-hospital SARS-CoV-2 contact are less amenable to intervention. However, lack of access to sick pay among externally contracted staff is more easily rectifiable. Our findings suggest that providing easier access to sick pay would lead to a decrease in SARS-CoV-2 transmission and potentially that of other infectious diseases in hospital settings. CLINICAL TRIALS REGISTRATION: NCT04380896.

Favipiravir induces HuNoV viral mutagenesis and infectivity loss with clinical improvement in immunocompromised patients.

Chronic human norovirus (HuNoV) infections in immunocompromised patients result in severe disease, yet approved antivirals are lacking. RNA-dependent RNA polymerase (RdRp) inhibitors inducing viral mutagenesis display broad-spectrum in vitro antiviral activity, but clinical efficacy in HuNoV infections is anecdotal and the potential emergence of drug-resistant variants is concerning. Upon favipiravir (and nitazoxanide) treatment of four immunocompromised patients with life-threatening HuNoV infections, viral whole-genome sequencing showed accumulation of favipiravir-induced mutations which coincided with clinical improvement although treatment failed to clear HuNoV. Infection of zebrafish larvae demonstrated drug-associated loss of viral infectivity and favipiravir treatment showed efficacy despite occurrence of RdRp variants potentially causing favipiravir resistance. This indicates that within-host resistance evolution did not reverse loss of viral fitness caused by genome-wide accumulation of sequence changes. This off-label approach supports the use of mutagenic antivirals for treating prolonged RNA viral infections and further informs the debate surrounding their impact on virus evolution.

Long-Term Persistence of Spike Protein Antibody and Predictive Modeling of Antibody Dynamics After Infection With Severe Acute Respiratory Syndrome Coronavirus 2.

BACKGROUND: Antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been shown to neutralize the virus in vitro and prevent disease in animal challenge models on reexposure. However, the current understanding of SARS-CoV-2 humoral dynamics and longevity is conflicting. METHODS: The COVID-19 Staff Testing of Antibody Responses Study (Co-Stars) prospectively enrolled 3679 healthcare workers to comprehensively characterize the kinetics of SARS-CoV-2 spike protein (S), receptor-binding domain, and nucleoprotein (N) antibodies in parallel. Participants screening seropositive had serial monthly serological testing for a maximum of 7 months with the Meso Scale Discovery Assay. Survival analysis determined the proportion of seroreversion, while 2 hierarchical gamma models predicted the upper and lower bounds of long-term antibody trajectory. RESULTS: A total of 1163 monthly samples were provided from 349 seropositive participants. At 200 days after symptoms, >95% of participants had detectable S antibodies, compared with 75% with detectable N antibodies. S antibody was predicted to remain detectable in 95% of participants until 465 days (95% confidence interval, 370-575 days) using a "continuous-decay" model and indefinitely using a "decay-to-plateau" model to account for antibody secretion by long-lived plasma cells. S-antibody titers were correlated strongly with surrogate neutralization in vitro (R2 = 0.72). N antibodies, however, decayed rapidly with a half-life of 60 days (95% confidence interval, 52-68 days). CONCLUSIONS: The Co-Stars data presented here provide evidence for long-term persistence of neutralizing S antibodies. This has important implications for the duration of functional immunity after SARS-CoV-2 infection. In contrast, the rapid decay of N antibodies must be considered in future seroprevalence studies and public health decision-making. This is the first study to establish a mathematical framework capable of predicting long-term humoral dynamics after SARS-CoV-2 infection. CLINICAL TRIALS REGISTRATION: NCT04380896.

Evolution of viral variants in remdesivir-treated and untreated SARS-CoV-2-infected pediatrics patients.

Detailed information on intrahost viral evolution in SARS-CoV-2 with and without treatment is limited. Sequential viral loads and deep sequencing of SARS-CoV-2 from the upper respiratory tract of nine hospitalized children, three of whom were treated with remdesivir, revealed that remdesivir treatment suppressed viral load in one patient but not in a second infected with an identical strain without any evidence of drug resistance found. Reduced levels of subgenomic RNA during treatment of the second patient, suggest an additional effect of remdesivir on viral replication. Haplotype reconstruction uncovered persistent SARS-CoV-2 variant genotypes in four patients. These likely arose from within-host evolution, although superinfection cannot be excluded in one case. Although our dataset is small, observed sample-to-sample heterogeneity in variant frequencies across four of nine patients suggests the presence of discrete viral populations in the lung with incomplete population sampling in diagnostic swabs. Such compartmentalization could compromise the penetration of remdesivir into the lung, limiting the drugs in vivo efficacy, as has been observed in other lung infections.

A genome-wide meta-analysis of nodular sclerosing Hodgkin lymphoma identifies risk loci at 6p21.32.

Nodular sclerosing Hodgkin lymphoma (NSHL) is a distinct, highly heritable Hodgkin lymphoma subtype. We undertook a genome-wide meta-analysis of 393 European-origin adolescent/young adult NSHL patients and 3315 controls using the Illumina Human610-Quad Beadchip and Affymetrix Genome-Wide Human SNP Array 6.0. We identified 3 single nucleotide polymorphisms (SNPs) on chromosome 6p21.32 that were significantly associated with NSHL risk: rs9268542 (P = 5.35 × 10(-10)), rs204999 (P = 1.44 × 10(-9)), and rs2858870 (P = 1.69 × 10(-8)). We also confirmed a previously reported association in the same region, rs6903608 (P = 3.52 × 10(-10)). rs204999 and rs2858870 were weakly correlated (r(2) = 0.257), and the remaining pairs of SNPs were not correlated (r(2) < 0.1). In an independent set of 113 NSHL cases and 214 controls, 2 SNPs were significantly associated with NSHL and a third showed a comparable odds ratio (OR). These SNPs are found on 2 haplotypes associated with NSHL risk (rs204999-rs9268528-rs9268542-rs6903608-rs2858870; AGGCT, OR = 1.7, P = 1.71 × 10(-6); GAATC, OR = 0.4, P = 1.16 × 10(-4)). All individuals with the GAATC haplotype also carried the HLA class II DRB1*0701 allele. In a separate analysis, the DRB1*0701 allele was associated with a decreased risk of NSHL (OR = 0.5, 95% confidence interval = 0.4, 0.7). These data support the importance of the HLA class II region in NSHL etiology.

Matching-adjusted indirect comparisons of axicabtagene ciloleucel to mosunetuzumab for the treatment of relapsed/refractory follicular lymphoma.

BACKGROUND: Axicabtagene ciloleucel (axi-cel) was the first chimeric antigen receptor (CAR) T-cell therapy approved for relapsed/refractory (R/R) follicular lymphoma (FL) patients, while mosunetuzumab was the first bispecific monoclonal antibody approved in this population. In the absence of head-to-head evidence, this study sought to conduct a matching-adjusted indirect comparison (MAIC) to estimate the comparative efficacy and safety of these treatments in 3rd line or higher (3L+) FL. METHODS: The evidence base consisted of individual patient data (IPD) of all enrolled patients, regardless of infusion status, from the single-arm axi-cel trial, ZUMA-5 (NCT03105336), and aggregate data from the mosunetuzumab FL trial (NCT02500407) from publications identified through a systematic review. Efficacy outcomes were progression-free survival (PFS), duration of response (DoR), objective response rate (ORR), complete response rate (CRR). Analyses used independent central review for both trials, where possible. Safety outcomes were cytokine release syndrome (CRS), neurological events (NE), and treatment-related adverse events (TRAEs). Unanchored MAICs were conducted to align ZUMA-5 to the patient characteristics of the mosunetuzumab trial. For each outcome, prognostic factors were identified a priori through quantitative analysis and clinical experts. For time-to-event outcomes, hazard ratios (HRs) were estimated using Cox regression using IPD from ZUMA-5 and pseudo-IPD extracted from Kaplan-Meier plots for mosunetuzumab. RESULTS: Patient characteristics were well-aligned between trials leading to large effective-sample sizes after matching, ranging from 93.4 to 115.5, for ZUMA-5 (n=127). In comparisons to mosunetuzumab (n=90), axi-cel was associated with improved PFS (HR: 0.39; 95% confidence interval [CI]: 0.24-0.62) and DoR (HR: 0.45; 95% CI: 0.27-0.76). Similarly, axi-cel led to higher ORR (OR: 3.87; 95% CI: 1.53-9.76) and CRR (OR: 2.80; 95% CI: 1.50-5.26). Although axi-cel was associated with a higher rate of all-grade CRS (OR: 5.54; 95% CI: 2.63-8.94) and NEs (OR: 3.54; 95% CI: 1.28-9.83), differences in grade ≥3 CRS and TRAEs were not statistically significant. CONCLUSIONS: Findings from this study show improved efficacy and more durable response for the treatment of 3L+ R/R FL with axi-cel relative to mosunetuzumab, with increased odds of all-grade CRS and NE, but not G3+ CRS and TRAEs.

Characterization of NOL7 gene point mutations, promoter methylation, and protein expression in cervical cancer.

NOL7 is a putative tumor suppressor gene localized to 6p23, a region with frequent loss of heterozygosity in a number of cancers, including cervical cancer (CC). We have previously demonstrated that reintroduction of NOL7 into CC cells altered the angiogenic phenotype and suppressed tumor growth in vivo by 95%. Therefore, to understand its mechanism of inactivation in CC, we investigated the genetic and epigenetic regulation of NOL7. NOL7 mRNA and protein levels were assessed in 13 CC cell lines and 23 consecutive CC specimens by real-time quantitative polymerase chain reaction, western blotting, and immunohistochemistry. Methylation of the NOL7 promoter was analyzed by bisulfite sequencing and mutations were identified through direct sequencing. A CpG island with multiple CpG dinucleotides spanned the 5' untranslated region and first exon of NOL7. However, bisulfite sequencing failed to identify persistent sites of methylation. Mutational sequencing revealed that 40% of the CC specimens and 31% of the CC cell lines harbored somatic mutations that may affect the in vivo function of NOL7. Endogenous NOL7 mRNA and protein expression in CC cell lines were significantly decreased in 46% of the CC cell lines. Finally, immunohistochemistry demonstrated strong NOL7 nucleolar staining in normal tissues that decreased with histologic progression toward CC. NOL7 is inactivated in CC in accordance with the Knudson 2-hit hypothesis through loss of heterozygosity and mutation. Together with evidence of its in vivo tumor suppression, these data support the hypothesis that NOL7 is the legitimate tumor suppressor gene located on 6p23.

Using Whole Genome Sequences to Investigate Adenovirus Outbreaks in a Hematopoietic Stem Cell Transplant Unit.

A recent surge in human mastadenovirus (HAdV) cases, including five deaths, amongst a haematopoietic stem cell transplant population led us to use whole genome sequencing (WGS) to investigate. We compared sequences from 37 patients collected over a 20-month period with sequences from GenBank and our own database of HAdVs. Maximum likelihood trees and pairwise differences were used to evaluate genotypic relationships, paired with the epidemiological data from routine infection prevention and control (IPC) records and hospital activity data. During this time period, two formal outbreaks had been declared by IPC, while WGS detected nine monophyletic clusters, seven were corroborated by epidemiological evidence and by comparison of single-nucleotide polymorphisms. One of the formal outbreaks was confirmed, and the other was not. Of the five HAdV-associated deaths, three were unlinked and the remaining two considered the source of transmission. Mixed infection was frequent (10%), providing a sentinel source of recombination and superinfection. Immunosuppressed patients harboring a high rate of HAdV positivity require comprehensive surveillance. As a consequence of these findings, HAdV WGS is being incorporated routinely into clinical practice to influence IPC policy contemporaneously.

Surgical peripheral nerve decompression for the treatment of painful diabetic neuropathy of the foot - A level 1 pragmatic randomized controlled trial.

AIMS: To assess the efficacy of surgical decompression of lower extremity nerves for the treatment of painful diabetic peripheral sensorimotor polyneuropathy (DPN). METHODS: People with painful diabetic neuropathy were randomized single-blind to a lower extremity decompression surgery (n = 12) or observation (n = 10) for 1 year. RESULTS: Pain was the primary outcome assessed with 2 measures. The McGill pain visual analogue scores over time changed within the groups (p for time < 0.0001), and changed differently over time within the groups (p for group × time = 0.0138). The NeuroQoL pain sensitivity analysis significantly changed from baseline to 12 months comparing intervention to control (p = 0.0079), and the joint effect of group and time on pain scores was statistically significant (p for group × time = 0.0009). At the study end-point of 12 months, intervention group participants had over 3 times the odds of rating their pain as "better" compared to "unchanged" or "worse" in the control group (p = 0.0177). CONCLUSIONS: Surgical decompression of lower limb nerves was an effective treatment for decreasing pain in patients with DPN and superimposed nerve compressions.

Influence of structural variation on nuclear localization of DNA-binding polyamide-fluorophore conjugates.

A pivotal step forward in chemical approaches to controlling gene expression is the development of sequence-specific DNA-binding molecules that can enter live cells and traffic to nuclei unaided. DNA-binding polyamides are a class of programmable, sequence-specific small molecules that have been shown to influence a wide variety of protein-DNA interactions. We have synthesized over 100 polyamide-fluorophore conjugates and assayed their nuclear uptake profiles in 13 mammalian cell lines. The compiled dataset, comprising 1300 entries, establishes a benchmark for the nuclear localization of polyamide-dye conjugates. Compounds in this series were chosen to provide systematic variation in several structural variables, including dye composition and placement, molecular weight, charge, ordering of the aromatic and aliphatic amino-acid building blocks and overall shape. Nuclear uptake does not appear to be correlated with polyamide molecular weight or with the number of imidazole residues, although the positions of imidazole residues affect nuclear access properties significantly. Generally negative determinants for nuclear access include the presence of a beta-Ala-tail residue and the lack of a cationic alkyl amine moiety, whereas the presence of an acetylated 2,4-diaminobutyric acid-turn is a positive factor for nuclear localization. We discuss implications of these data on the design of polyamide-dye conjugates for use in biological systems.

A variant at 9p21.3 functionally implicates CDKN2B in paediatric B-cell precursor acute lymphoblastic leukaemia aetiology.

Paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) is the most common cancer of childhood, yet little is known about BCP-ALL predisposition. In this study, in 2,187 cases of European ancestry and 5,543 controls, we discover and replicate a locus indexed by rs77728904 at 9p21.3 associated with BCP-ALL susceptibility (Pcombined=3.32 × 10(-15), OR=1.72) and independent from rs3731217, the previously reported ALL-associated variant in this region. Of correlated SNPs tagged by this locus, only rs662463 is significant in African Americans, suggesting it is a plausible causative variant. Functional analysis shows that rs662463 is a cis-eQTL for CDKN2B, with the risk allele associated with lower expression, and suggests that rs662463 influences BCP-ALL risk by regulating CDKN2B expression through CEBPB signalling. Functional analysis of rs3731217 suggests it is associated with BCP-ALL by acting within a splicing regulatory element determining CDKN2A exon 3 usage (P=0.01). These findings provide new insights into the critical role of the CDKN2 locus in BCP-ALL aetiology.

Buffered lidocaine and bupivacaine mixture - the ideal local anesthetic solution?

The use of injectable local anesthetic solutions to facilitate pain-free surgery is an integral component of many procedures performed by the plastic surgeon. In many instances, a solution that has both rapid onset and prolonged duration of analgesia is optimal. A combination of lidocaine and bupivacaine, plain or with epinephrine, is readily available in most Canadian health care settings where such procedures are performed, and fulfills these criteria. However, commercially available solutions of both medications are acidic and cause a burning sensation on injection. Buffering to neutral pH with sodium bicarbonate is a practical method to mitigate the burning sensation, and has the added benefit of increasing the fraction of nonionized lipid soluble drug available. The authors report on the proportions of the three drugs to yield a neutral pH, and the results of an initial survey regarding the use of the combined solution with epinephrine in hand surgery.

L'injection d'anesthésiques injectables locaux pour favoriser une opération sans douleur fait partie intégrante des nombreuses interventions réalisées par le plasticien. Dans bien des cas, une solution analgésique à action longue et rapide est optimale. Une bithérapie de lidocaïne et de bupivacaïne, seule ou accompagnée d'adrénaline, disponible dans la plupart des milieux de santé canadiens où de telles interventions sont effectuées, respecte ce critère. Les solutions commerciales de ces deux médicaments sont toutefois acides et provoquent une sensation de brûlure à l'injection. L'utilisation d'un tampon de bicarbonate de sodium pour obtenir un pH neutre est pratique pour limiter la sensation de brûlure et a l'avantage supplémentaire d'accroître la fraction de médicament liposoluble non ionisé disponible. Les auteurs rendent compte des proportions des trois médicaments nécessaires pour obtenir un pH neutre et des résultats d'un sondage initial sur l'utilisation de la bithérapie combinée à l'adrénaline pour la chirurgie de la main.

Variants at 6q21 implicate PRDM1 in the etiology of therapy-induced second malignancies after Hodgkin's lymphoma.

Survivors of pediatric Hodgkin's lymphoma are at risk for radiation therapy-induced second malignant neoplasms (SMNs). We identified two variants at chromosome 6q21 associated with SMNs in survivors of Hodgkin's lymphoma treated with radiation therapy as children but not as adults. The variants comprise a risk locus associated with decreased basal expression of PRDM1 (encoding PR domain containing 1, with ZNF domain) and impaired induction of the PRDM1 protein after radiation exposure. These data suggest a new gene-exposure interaction that may implicate PRDM1 in the etiology of radiation therapy-induced SMNs.

Mycobacterium smegmatis infection of the hand.

We report a case of Mycobacterium smegmatis granuloma in the soft tissues of the first web space of the left hand in a 67-year-old Caucasian woman. She was in good systematic health; there was no recollection of trauma to the hand. A combined regime of prolonged antibiotic therapy and surgical debridement was necessary to ultimately eradicate the infection. The natural history, microbiology, and treatment of this rare hand infection are discussed.

Post-traumatic stenosing flexor tenosynovitis.

Two cases of post-traumatic stenosing flexor tenosynovitis (flexor tendon entrapment or trigger digit) are presented. The medical literature contains few reports of this uncommon etiology for a problem that afflicts at least 2% of the adult population.

Voici deux cas de ténosynovite sténosante, post-traumatique du fléchisseur (compression du tendon fléchisseur ou doigt à ressort). La documentation scientifique traite peu de cette cause inhabituelle d'inflammation, qui touche au moins 2 % de la population adulte.

Nuclear localization of pyrrole-imidazole polyamide-fluorescein conjugates in cell culture.

A series of hairpin pyrrole-imidazole polyamide-fluorescein conjugates were synthesized and assayed for cellular localization. Thirteen cell lines, representing 11 human cancers, one human transformed kidney cell line, and one murine leukemia cell line, were treated with 5 microM polyamide-fluorescein conjugates for 10-14 h, then imaged by confocal laser scanning microscopy. A conjugate containing a beta-alanine residue at the C terminus of the polyamide moiety showed no nuclear localization, whereas an analogous compound lacking the beta-alanine residue was strongly localized in the nuclei of all cell lines tested. The localization profiles of several other conjugates suggest that pyrrole-imidazole sequence and content, dye choice and position, linker composition, and molecular weight are determinants of nuclear localization. The attachment of fluorescein to the C terminus of a hairpin polyamide results in an approximately 10-fold reduction in DNA-binding affinity, with no loss of binding specificity with reference to mismatch binding sites.

Design of artificial transcriptional activators with rigid poly-L-proline linkers.

Typical eukaryotic transcriptional activators are composed of distinct functional domains, including a DNA binding domain and an activating domain. Artificial transcription factors have been designed wherein the DNA binding domain is a minor groove DNA binding hairpin polyamide linked by a flexible tether to short activating peptides, typically 16-20 residues in size. In this study, the linker between the polyamide and the peptide was altered in an incremental fashion using rigid oligoproline "molecular rulers" in the 18-45 A length range. We find that there is an optimal linker length which separates the DNA and the activation region for transcription activation.