Metagenomic Analysis of DNA Viruses with Targeted Sequence Capture of Canine Lobular Orbital Adenomas and Normal Conjunctiva.

Our study aims are: (1) to evaluate phenotypically normal canine conjunctival and orbital tissue and tissue from canine lobular orbital adenomas (CLOAs) for the presence of viral genomic material and (2) phylogenetically classify detected DNA viruses to determine if a DNA virus is associated with CLOAs. A total of 31 formalin fixed paraffin embedded CLOA tissue samples, 4 papillomas or sarcoid, and 10 fresh clinically normal conjunctival tissues were included in this study. Genomic DNA was isolated from all samples and sequencing libraries were prepared. The libraries were molecularly indexed and pooled and viral DNA was enriched via targeted sequence capture utilizing ViroCap. The libraries were sequenced on the Illumina HiSeq platform and compared to known viral DNA reference genomes to identify viral DNA. Carnivore parvovirus was identified in 6.4% and 20% of CLOA tissue and normal conjunctival samples, respectively. This study showed that conjunctival tissue from healthy dogs and CLOAs uncommonly harbor DNA viruses, and no DNA virus was associated with these tumors. Further studies are needed to evaluate the etiologic cause of CLOAs.

Virome Analysis and Association of Positive Coxsackievirus B Serology during Pregnancy with Congenital Heart Disease.

BACKGROUND: We have previously shown coxsackievirus B (CVB) to be a potent inducer of congenital heart disease (CHD) in mice. The clinical relevance of these findings in humans and the roles of other viruses in the pathogenesis of CHD remain unknown. METHODS: We obtained plasma samples, collected at all trimesters, from 89 subjects (104 pregnancies), 73 healthy controls (88 pregnancies), and 16 with CHD-affected birth (16 pregnancies), from the Perinatal Family Tissue Bank (PFTB). We performed CVB IgG/IgM serological assays on plasma. We also used ViroCap sequencing and PCR to test for viral nucleic acid in plasma, circulating leukocytes from the buffy coat, and in the media of a co-culture system. RESULTS: CVB IgG/IgM results indicated that prior exposure was 7.8 times more common in the CHD group (95% CI, 1.14-54.24, adj. p-value = 0.036). However, the CVB viral genome was not detected in plasma, buffy coat, or co-culture supernatant by molecular assays, although other viruses were detected. CONCLUSION: Detection of viral nucleic acid in plasma was infrequent and specifically no CVB genome was detected. However, serology demonstrated that prior CVB exposure is higher in CHD-affected pregnancies. Further studies are warranted to understand the magnitude of the contribution of the maternal blood virome to the pathogenesis of CHD.

The plasma virome in longitudinal samples from pregnant patients.

Introduction: Nucleic acid from viruses is common in peripheral blood, even in asymptomatic individuals. How physiologic changes of pregnancy impact host-virus dynamics for acute, chronic, and latent viral infections is not well described. Previously we found higher viral diversity in the vagina during pregnancy associated with preterm birth (PTB) and Black race. We hypothesized that higher diversity and viral copy numbers in the plasma would show similar trends. Methods: To test this hypothesis, we evaluated longitudinally collected plasma samples from 23 pregnant patients (11 term and 12 preterm) using metagenomic sequencing with ViroCap enrichment to enhance virus detection. Sequence data were analyzed with the ViroMatch pipeline. Results: We detected nucleic acid from at least 1 virus in at least 1 sample from 87% (20/23) of the maternal subjects. The viruses represented 5 families: Herpesviridae, Poxviridae, Papillomaviridae, Anelloviridae, and Flaviviridae. We analyzed cord plasma from 18 of the babies from those patients and found nucleic acid from viruses in 33% of the samples (6/18) from 3 families: Herpesviridae, Papillomaviridae, and Anelloviridae. Some viral genomes were found in both maternal plasma and cord plasma from maternal-fetal pairs (e.g. cytomegalovirus, anellovirus). We found that Black race associated with higher viral richness (number of different viruses detected) in the maternal blood samples (P=0.003), consistent with our previous observations in vaginal samples. We did not detect associations between viral richness and PTB or the trimester of sampling. We then examined anelloviruses, a group of viruses that is ubiquitous and whose viral copy numbers fluctuate with immunological state. We tested anellovirus copy numbers in plasma from 63 pregnant patients sampled longitudinally using qPCR. Black race associated with higher anellovirus positivity (P<0.001) but not copy numbers (P=0.1). Anellovirus positivity and copy numbers were higher in the PTB group compared to the term group (P<0.01, P=0.003, respectively). Interestingly, these features did not occur at the time of delivery but appeared earlier in pregnancy, suggesting that although anelloviruses were biomarkers for PTB they were not triggering parturition. Discussion: These results emphasize the importance of longitudinal sampling and diverse cohorts in studies of virome dynamics during pregnancy.

Pathogen and Antibody Identification in Children with Encephalitis in Myanmar.

OBJECTIVE: Prospective studies of encephalitis are rare in regions where encephalitis is prevalent, such as low middle-income Southeast Asian countries. We compared the diagnostic yield of local and advanced tests in cases of pediatric encephalitis in Myanmar. METHODS: Children with suspected subacute or acute encephalitis at Yangon Children's Hospital, Yangon, Myanmar, were prospectively recruited from 2016-2018. Cohort 1 (n = 65) had locally available diagnostic testing, whereas cohort 2 (n = 38) had advanced tests for autoantibodies (ie, cell-based assays, tissue immunostaining, studies with cultured neurons) and infections (ie, BioFire FilmArray multiplex Meningitis/Encephalitis multiplex PCR panel, metagenomic sequencing, and pan-viral serologic testing [VirScan] of cerebrospinal fluid). RESULTS: A total of 20 cases (13 in cohort 1 and 7 in cohort 2) were found to have illnesses other than encephalitis. Of the 52 remaining cases in cohort 1, 43 (83%) had presumed infectious encephalitis, of which 2 cases (4%) had a confirmed infectious etiology. Nine cases (17%) had presumed autoimmune encephalitis. Of the 31 cases in cohort 2, 23 (74%) had presumed infectious encephalitis, of which one (3%) had confirmed infectious etiology using local tests only, whereas 8 (26%) had presumed autoimmune encephalitis. Advanced tests confirmed an additional 10 (32%) infections, 4 (13%) possible infections, and 5 (16%) cases of N-methyl-D-aspartate receptor antibody encephalitis. INTERPRETATION: Pediatric encephalitis is prevalent in Myanmar, and advanced technologies increase identification of treatable infectious and autoimmune causes. Developing affordable advanced tests to use globally represents a high clinical and research priority to improve the diagnosis and prognosis of encephalitis. ANN NEUROL 2023;93:615-628.

A Case of Persistent Human Pegivirus Infection in Two Separate Pregnancies of a Woman.

Human pegivirus (HPgV) is best known for persistent, presumably non-pathogenic, infection and a propensity to co-infect with human immunodeficiency virus or hepatitis C virus. However, unique attributes, such as the increased risk of malignancy or immune modulation, have been recently recognized for HPgV. We have identified a unique case of a woman with high levels HPgV infection in two pregnancies, which occurred 4 years apart and without evidence of human immunodeficiency virus or hepatitis C virus infection. The second pregnancy was complicated by congenital heart disease. A high level of HPgV infection was detected in the maternal blood from different trimesters by RT-PCR and identified as HPgV type 1 genotype 2 in both pregnancies. In the second pregnancy, the decidua and intervillous tissue of the placenta were positive for HPgV by PCR but not the chorion or cord blood (from both pregnancies), suggesting no vertical transmission despite high levels of viremia. The HPgV genome sequence was remarkably conserved over the 4 years. Using VirScan, sera antibodies for HPgV were detected in the first trimester of both pregnancies. We observed the same anti-HPgV antibodies against the non-structural NS5 protein in both pregnancies, suggesting a similar non-E2 protein humoral immune response over time. To the best of our knowledge, this is the first report of persistent HPgV infection involving placental tissues with no clear indication of vertical transmission. Our results reveal a more elaborate viral-host interaction than previously reported, expand our knowledge about tropism, and opens avenues for exploring the replication sites of this virus.

Comparison of Metagenomic Sequencing and the NanoString nCounter Analysis System for the Characterization of Bacterial and Viral Communities in Vaginal Samples.

DNA sequencing assays have been used to characterize the vaginal microbiome and to identify associations with clinical outcomes. The purpose of this study was to evaluate the utility of the NanoString nCounter platform, a more efficient assay compared to sequencing, for the characterization of vaginal microbial communities. A panel of NanoString nCounter probes was designed to detect common vaginal bacteria and viruses with relevance to reproductive health. A defined synthetic community of microbes and 43 clinical samples were interrogated with NanoString nCounter assays and compared to known compositions or metagenomic shotgun sequencing (MSS) results. The NanoString nCounter platform and MSS were able to distinguish closely related microbes. In clinical samples, the relative abundance of bacterial species was similar between the two assays. The assays sometimes disagreed when targets were present at low abundance. More viruses were detected by MSS than by nCounter. However, the nCounter assays are able to provide results in about 30 h with minimal hands-on time, whereas MSS requires at least 138 to 178 h with extensive hands-on time. The reagent cost for the two assays was similar, but the overall cost of the nCounter was lower due to the minimal hands-on time. MSS can be used to inform the design of a targeted multiplex panel for the assessment of vaginal microbial communities, thereby allowing for more cost-effective and rapid screening of patient samples for research studies. The sensitivity for low abundance microbes could be improved, possibly by adding additional target amplification cycles before nCounter assessment. This approach has potential as an assay with both research and clinical applications. IMPORTANCE Metagenomic shotgun sequencing can inform the design of a targeted multiplex panel by which the NanoString nCounter platform can assess vaginal microbial communities, thereby allowing for more cost-effective and rapid screening of patient samples.

Association of the human semen DNA virome with successful in vitro fertilization.

OBJECTIVE: To comprehensively characterize the DNA virome in semen samples collected for in vitro fertilization (IVF). DESIGN: A descriptive clinical study. SETTING: Single academic fertility center. PATIENT(S): Twenty-four male partners from couples undergoing IVF. INTERVENTION(S): Couples were randomized to receive 1 g of azithromycin (standard of care) or no azithromycin at the time of baseline IVF assessment. Semen samples were collected at the time of the female partners' egg retrieval, and 100 µL of the sample was used for the virome analysis. MAIN OUTCOME MEASURE(S): Detection of viruses by ViroCap enrichment of viral nucleic acid and sequencing. Association between the virome, semen parameters, and pregnancy outcomes. RESULT(S): We detected viruses in 58% of the participants. Viruses included polyomaviruses, papillomaviruses, herpesviruses, and anelloviruses. Viromes detected in semen had little overlap with the viromes detected in vaginal samples from their female partners collected at the time of embryo transfer, which were analyzed in a previous study. A lower viral diversity in semen samples was positively associated with pregnancy (Hodges-Lehmann estimate of difference, 1; 95% confidence interval, 2-0.00003). There was no association between viral diversity and sperm concentration, motility, or fertilization rates. CONCLUSION(S): This comprehensive characterization of the DNA virome in semen reveals an association between virome diversity and pregnancy in couples undergoing IVF. However, no association was found with specific semen parameters or fertilization rates, suggesting that viral exposure may negatively affect pregnancy after fertilization. Future studies should be undertaken to evaluate the associations between the semen virome with IVF outcomes in larger cohorts.

Longitudinal Dynamics of Skin Bacterial Communities in the Context of Staphylococcus aureus Decolonization.

Decolonization with topical antimicrobials is frequently prescribed in health care and community settings to prevent Staphylococcus aureus infection. However, effects on commensal skin microbial communities remains largely unexplored. Within a household affected by recurrent methicillin-resistant S. aureus skin and soft tissue infections (SSTI), skin swabs were collected from the anterior nares, axillae, and inguinal folds of 14 participants at 1- to 3-month intervals over 24 months. Four household members experienced SSTI during the first 12-months (observational period) and were prescribed a 5-day decolonization regimen with intranasal mupirocin and bleach water baths at the 12-month study visit. We sequenced the 16S rRNA gene V1-V2 region and compared bacterial community characteristics between the pre- and post-intervention periods and between younger and older subjects. The median Shannon diversity index was stable during the 12-month observational period at all three body sites. Bacterial community characteristics (diversity, stability, and taxonomic composition) varied with age. Among all household members, not exclusively among the four performing decolonization, diversity was unstable throughout the year post-intervention. In the month after decolonization, bacterial communities were changed. Although communities largely returned to their baseline states, relative abundance of some taxa remained changed throughout the year following decolonization (e.g., more abundant Bacillus; less abundant Cutibacterium). This 5-day decolonization regimen caused disruption of skin bacteria, and effects differed in younger and older subjects. Some effects were observed throughout the year post-intervention, which emphasizes the need for better understanding of the collateral effects of decolonization for S. aureus eradication. IMPORTANCE Decolonization with topical antimicrobials is frequently prescribed to prevent Staphylococcus aureus infection, but the effects on commensal skin bacteria are undetermined. We found that decolonization with mupirocin and bleach water baths leads to sustained disruption of bacterial communities.

Fecal microbiome and bile acid metabolome in adult short bowel syndrome.

Loss of functional small bowel surface area causes short bowel syndrome (SBS), intestinal failure, and parenteral nutrition (PN) dependence. The gut adaptive response following resection may be difficult to predict, and it may take up to 2 yr to determine which patients will wean from PN. Here, we examined features of gut microbiota and bile acid (BA) metabolism in determining adaptation and ability to wean from PN. Stool and sera were collected from healthy controls and from patients with SBS (n = 52) with ileostomy, jejunostomy, ileocolonic, and jejunocolonic anastomoses fed with PN plus enteral nutrition or who were exclusively enterally fed. We undertook 16S rRNA gene sequencing, BA profiling, and 7α-hydroxy-4-cholesten-3-one (C4) quantitation with LC-MS/MS and serum amino acid analyses. Patients with SBS exhibited altered gut microbiota with reduced gut microbial diversity compared with healthy controls. We observed differences in the microbiomes of patients with SBS with ileostomy versus jejunostomy, jejunocolonic versus ileocolonic anastomoses, and PN dependence compared with those who weaned from PN. Stool and serum BA composition and C4 concentrations were also altered in patients with SBS, reflecting adaptive changes in enterohepatic BA cycling. Stools from patients who were weaned from PN were enriched in secondary BAs including deoxycholic acid and lithocholic aicd. Shifts in gut microbiota and BA metabolites may generate a favorable luminal environment in select patients with SBS, promoting the ability to wean from PN. Proadaptive microbial species and select BA may provide novel targets for patient-specific therapies for SBS.NEW & NOTEWORTHY Loss of intestinal surface area causes short bowel syndrome, intestinal failure, and parenteral nutrition dependence. We analyzed the gut microbiota and bile acid metabolome of a large cohort of short bowel syndrome adult patients with different postsurgical anatomies. We report a novel analysis of the microbiome of patients with ileostomy and jejunostomy. Enrichment of specific microbial and bile acid species may be associated with the ability to wean from parenteral nutrition.

Association of vaginal bacterial communities and reproductive outcomes with prophylactic antibiotic exposure in a subfertile population undergoing in vitro fertilization: a prospective exploratory study.

OBJECTIVE: To determine whether prophylactic azithromycin is associated with the vaginal bacterial microbiome and clinical outcomes in subfertile women undergoing in vitro fertilization (IVF). DESIGN: Prospective exploratory cohort study. SETTING: Single academic fertility center. PATIENTS: Subfertile women aged 18-43 years undergoing their first IVF cycle and fresh embryo transfer. INTERVENTION: Primary exposure to prophylactic azithromycin (1 g orally) once at baseline. MAIN OUTCOME MEASURES: The primary outcome was the effect of azithromycin on the vaginal microbiome compared with a no-azithromycin group at 3 time points throughout the IVF cycle (baseline, retrieval, and embryo transfer). The secondary outcomes were associations of vaginal bacterial communities with clinical outcomes. RESULTS: A planned a priori exploratory cohort of 27 subjects (12 in the azithromycin treatment group and 15 in the no-azithromycin group) contributed 79 vaginal swabs for the analysis as part of an ongoing randomized, controlled noninferiority trial. No specific taxa were associated with azithromycin or pregnancy at any time point. Azithromycin did not affect alpha diversity or community stability. Although there were trends of a lower bacterial load and higher percentage of Lactobacillus species in the azithromycin group at the time of transfer, these were not statistically significant. In women who did not become pregnant, the percentage of Lactobacillus species was lower (P = .048; Hodges-Lehmann estimate of difference, 0.41; 95% confidence interval, 0.08-0.65) and the change in community composition over time was higher. The percentage of Lactobacillus species at baseline was not predictive of the percentage of Lactobacillus species at the time of embryo transfer. CONCLUSIONS: Prophylactic azithromycin at baseline is not associated with changes in vaginal bacterial communities. Bacterial community features at the time of embryo transfer are associated with pregnancy. Bacterial community structures at baseline are not predictive of those at the time of embryo transfer. CLINICAL TRIAL REGISTRATION NUMBER: NCT03386227.

ViroMatch: A Computational Pipeline for the Detection of Viral Sequences from Complex Metagenomic Data.

ViroMatch is an automated pipeline that takes metagenomic sequencing reads as input and performs iterative nucleotide and translated nucleotide mapping to identify viral sequences. We provide a Docker image for ViroMatch, so that users will not have to install dependencies.

Chronic villitis of unknown etiology: Investigations into viral pathogenesis.

INTRODUCTION: Chronic villitis of unknown etiology (VUE) is a chronic inflammatory lesion of third trimester placenta, which contributes to major adverse obstetric outcomes. However, the inciting factors and mechanisms by which VUE contributes to adverse outcomes are poorly understood. This limits our ability to develop preventions or interventions. Our goals were to determine whether viruses can be detected in placental tissues with VUE and to determine whether gene expression profiles support an antiviral response. METHODS: We extracted RNA and DNA from 20 placentas with high-grade chronic villitis and 20 control placentas without inflammation. Viruses were assessed using ViroCap viral nucleic acid enrichment coupled with metagenomic sequencing. RNA sequencing was used to evaluate the inflammatory gene expression profiles in each placenta. RESULTS: We detected at least 1 virus in 50% of the samples tested. We found that herpesviruses, were found more frequently in cases compared with controls (P = 0.01). Antiviral pathways, including defense response to virus, interferon gamma response, and IFN alpha/beta response, were upregulated in cases. We observed two clusters of gene expression profiles in the VUE cases, suggesting multiple inflammatory profiles are associated with VUE. DISCUSSION: These data support a viral etiology for some cases of VUE. Furthermore, gene expression profiles suggest the possibility of more than one cause or manifestation of VUE. Viral mechanisms should be explored as potential targets for prevention or intervention in VUE.

A Protein Epitope Targeted by the Antibody Response to Kawasaki Disease.

BACKGROUND: Kawasaki disease (KD) is the leading cause of childhood acquired heart disease in developed nations and can result in coronary artery aneurysms and death. Clinical and epidemiologic features implicate an infectious cause but specific antigenic targets of the disease are unknown. Peripheral blood plasmablasts are normally highly clonally diverse but the antibodies they encode are approximately 70% antigen-specific 1-2 weeks after infection. METHODS: We isolated single peripheral blood plasmablasts from children with KD 1-3 weeks after onset and prepared 60 monoclonal antibodies (mAbs). We used the mAbs to identify their target antigens and assessed serologic response among KD patients and controls to specific antigen. RESULTS: Thirty-two mAbs from 9 of 11 patients recognize antigen within intracytoplasmic inclusion bodies in ciliated bronchial epithelial cells of fatal cases. Five of these mAbs, from 3 patients with coronary aneurysms, recognize a specific peptide, which blocks binding to inclusion bodies. Sera from 5/8 KD patients day ≥ 8 after illness onset, compared with 0/17 infant controls (P < .01), recognized the KD peptide antigen. CONCLUSIONS: These results identify a protein epitope targeted by the antibody response to KD and provide a means to elucidate the pathogenesis of this important worldwide pediatric problem.

A virome sequencing approach to feline oral squamous cell carcinoma to evaluate viral causative factors.

Feline oral squamous cell carcinoma (FOSCC) may be the best naturally-occurring model of human head and neck squamous cell carcinoma (HNSCC). HNSCC can be broadly divided into human papillomavirus (HPV)-negative cancers and HPV-positive cancers where HPV is the causative agent. Previous studies in FOSCC have used both species-specific and species-nonspecific PCR primers that may be insensitive to the detection of PVs and other viruses that may be divergent from known sequences. ViroCap is a targeted capture and next generation sequencing tool that was designed to identify all known vertebrate DNA and RNA viruses. In this study we used a metagenomic approach using ViroCap for DNA viruses in 20 FOSCC, 9 normal feline oral mucosal, and 8 suspected PV positive control samples. We tested the hypothesis that viruses would be enriched in FOSCC compared to normal oral mucosa. The virome of the FOSCC and normal feline oral mucosa consisted of feline foamy virus in 7/20 and 2/9 (35% and 22%), feline torque teno virus in 2/20 and 0/9 (10% and 0%), alphaherpesvirus in 2/10 and 0/9 (10% and 0%), FIV (0% and 22%), Epstein-Barr virus in 1/20 and 0/9 (5% and 0%) and feline papillomavirus in 1/20 and 0/9 samples (5% and 0% respectively). Felis catus papillomavirus-3 was found in 1 of 20 FOSCC samples. A virus was not associated consistently with FOSCC. If PVs have a role in FOSCC it is at most a supplementary or uncommon role. FOSCC appears most closely related to HPV-negative HNSCC. Future research on FOSCC should focus on identifying genetic and environmental causes.

Genome Organization of Canada Goose Coronavirus, A Novel Species Identified in a Mass Die-off of Canada Geese.

The complete genome of a novel coronavirus was sequenced directly from the cloacal swab of a Canada goose that perished in a die-off of Canada and Snow geese in Cambridge Bay, Nunavut, Canada. Comparative genomics and phylogenetic analysis indicate it is a new species of Gammacoronavirus, as it falls below the threshold of 90% amino acid similarity in the protein domains used to demarcate Coronaviridae. Additional features that distinguish the genome of Canada goose coronavirus include 6 novel ORFs, a partial duplication of the 4 gene and a presumptive change in the proteolytic processing of polyproteins 1a and 1ab.

Impaired Chylomicron Assembly Modifies Hepatic Metabolism Through Bile Acid-Dependent and Transmissible Microbial Adaptations.

The mechanisms by which alterations in intestinal bile acid (BA) metabolism improve systemic glucose tolerance and hepatic metabolic homeostasis are incompletely understood. We examined metabolic adaptations in mice with conditional intestinal deletion of the abetalipoproteinemia (ABL) gene microsomal triglyceride transfer protein (Mttp-IKO), which blocks chylomicron assembly and impairs intestinal lipid transport. Mttp-IKO mice exhibit improved hepatic glucose metabolism and augmented insulin signaling, without weight loss. These adaptations included decreased BA excretion, increased pool size, altered BA composition, and increased fibroblast growth factor 15 production. Mttp-IKO mice absorb fructose normally but are protected against dietary fructose-induced hepatic steatosis, without weight loss or changes in energy expenditure. In addition, Mttp-IKO mice exhibit altered cecal microbial communities, both at baseline and following fructose feeding, including increased abundance of Bacteroides and Lactobacillus genera. Transplantation of cecal microbiota from chow-fed Mttp-IKO mice into antibiotic-treated wild-type recipients conferred transmissible protection against fructose-induced hepatic steatosis in association with a bloom in Akkermansia and increased Clostridium XIVa genera, whose abundance was positively correlated with fecal coprostanol and total neutral sterol excretion in recipient mice. However, antibiotic-treated Mttp-IKO mice were still protected against fructose-induced hepatic steatosis, suggesting that changes in microbiota are not required for this phenotype. Nevertheless, we found increased abundance of fecal Akkermansia from two adult ABL subjects with MTTP mutations compared to their heterozygous parents and within the range noted in six healthy control subjects. Furthermore, Akkermansia abundance across all subjects was positively correlated with fecal coprostanol excretion. Conclusion: The findings collectively suggest multiple adaptive pathways of metabolic regulation following blocked chylomicron assembly, including shifts in BA signaling and altered microbial composition that confer a transmissible phenotype.

Dynamic host immune response in virus-associated cancers.

Viruses drive carcinogenesis in human cancers through diverse mechanisms that have not been fully elucidated but include promoting immune escape. Here we investigated associations between virus-positivity and immune pathway alteration for 2009 tumors across six virus-related cancer types. Analysis revealed that for 3 of 72 human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSC) the HPV genome integrated in immune checkpoint genes PD-L1 or PD-L2, driving elevated expression in the corresponding gene. In addition to the previously described upregulation of the PD-1 immunosuppressive pathway in Epstein-Barr virus (EBV)-positive stomach tumors, we also observed upregulation of the PD-1 pathway in cytomegalovirus (CMV)-positive tumors. Furthermore, we found signatures of T-cell and B-cell response in HPV-positive HNSC and EBV-positive stomach tumors and HPV-positive HNSC patients were associated with better survival when T-cell signals were detected. Our work reveals that viral infection may recruit immune effector cells, and upregulate PD-1 and CTLA-4 immunosuppressive pathways.

Whole-Genome Sequencing of Klebsiella pneumoniae Isolates to Track Strain Progression in a Single Patient With Recurrent Urinary Tract Infection.

Klebsiella pneumoniae is an important uropathogen that increasingly harbors broad-spectrum antibiotic resistance determinants. Evidence suggests that some same-strain recurrences in women with frequent urinary tract infections (UTIs) may emanate from a persistent intravesicular reservoir. Our objective was to analyze K. pneumoniae isolates collected over weeks from multiple body sites of a single patient with recurrent UTI in order to track ordered strain progression across body sites, as has been employed across patients in outbreak settings. Whole-genome sequencing of 26 K. pneumoniae isolates was performed utilizing the Illumina platform. PacBio sequencing was used to create a refined reference genome of the original urinary isolate (TOP52). Sequence variation was evaluated by comparing the 26 isolate sequences to the reference genome sequence. Whole-genome sequencing of the K. pneumoniae isolates from six different body sites of this patient with recurrent UTI demonstrated 100% chromosomal sequence identity of the isolates, with only a small P2 plasmid deletion in a minority of isolates. No single nucleotide variants were detected. The complete absence of single-nucleotide variants from 26 K. pneumoniae isolates from multiple body sites collected over weeks from a patient with recurrent UTI suggests that, unlike in an outbreak situation with strains collected from numerous patients, other methods are necessary to discern strain progression within a single host over a relatively short time frame.

Carriage of the Toxic Shock Syndrome Toxin Gene by Contemporary Community-Associated Staphylococcus aureus Isolates.

We report here the prevalence of the tst-1 gene among 252 methicillin-susceptible Staphylococcus aureus (MSSA) isolates and 458 methicillin-resistant S aureus (MRSA) isolates collected from 531 subjects between 2008 and 2017, one of which was recovered from a child with MRSA toxic shock syndrome. tst-1 was encoded by 43 (6%) S aureus isolates overall: 42 (16.7%) MSSA isolates and 1 (0.2%) MRSA isolate (P < .001).