Plasmon-Enhanced Digital Fluoroimmunoassay for Subfemtomolar Detection of Protein Biomarkers.

Rapid and accurate quantification of low-abundance protein biomarkers in biofluids can transform the diagnosis of a range of pathologies, including infectious diseases. Here, we harness ultrabright plasmonic fluors as "digital nanolabels" and demonstrate the detection and quantification of subfemtomolar concentrations of human IL-6 and SARS-CoV-2 alpha and variant proteins in clinical nasopharyngeal swab and saliva samples from COVID-19 patients. The resulting digital plasmonic fluor-linked immunosorbent assay (digital p-FLISA) enables detection of SARS-CoV-2 nucleocapsid protein, both in solution and in live virions. Digital p-FLISA outperforms the "gold standard" enzyme-linked immunosorbent assay (ELISA), having a nearly 7000-fold lower limit-of-detection, and outperforms a commercial antigen test, having over 5000-fold improvement in analytical sensitivity. Detection and quantification of very low concentrations of target proteins holds potential for early detection of pathological conditions, treatment monitoring, and personalized medicine.

Oral Valganciclovir Initiated Beyond 1 Month of Age as Treatment of Sensorineural Hearing Loss Caused by Congenital Cytomegalovirus Infection: A Randomized Clinical Trial.

OBJECTIVE: The objective of this study was to determine if valganciclovir initiated after 1 month of age improves congenital cytomegalovirus-associated sensorineural hearing loss. STUDY DESIGN: We conducted a randomized, double-blind, placebo-controlled phase 2 trial of 6 weeks of oral valganciclovir at US (n = 12) and UK (n = 9) sites. Patients of ages 1 month through 3 years with baseline sensorineural hearing loss were enrolled. The primary outcome was change in total ear hearing between baseline and study month 6. Secondary outcome measures included change in best ear hearing and reduction in cytomegalovirus viral load in blood, saliva, and urine. RESULTS: Of 54 participants enrolled, 35 were documented to have congenital cytomegalovirus infection and were randomized (active group: 17; placebo group: 18). Mean age at enrollment was 17.8 ± 15.8 months (valganciclovir) vs 19.5 ± 13.1 months (placebo). Twenty (76.9%) of the 26 ears from subjects in the active treatment group did not have worsening of hearing, compared with 27 (96.4%) of 28 ears from subjects in the placebo group (P = .09). All other comparisons of total ear or best ear hearing outcomes were also not statistically significant. Saliva and urine viral loads decreased significantly in the valganciclovir group but did not correlate with change in hearing outcome. CONCLUSIONS: In this randomized controlled trial, initiation of antiviral therapy beyond the first month of age did not improve hearing outcomes in children with congenital cytomegalovirus-associated sensorineural hearing loss. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT01649869.

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.

Azithromycin therapy in infants hospitalized for respiratory syncytial virus bronchiolitis: Airway matrix metalloproteinase-9 levels and subsequent recurrent wheeze.

BACKGROUND: Early life respiratory syncytial virus (RSV) bronchiolitis is a significant risk factor for childhood asthma. In vitro and in vivo studies suggested that decreasing levels of airway matrix metalloproteinase (MMP)-9 during RSV bronchiolitis may be associated with clinical benefits. OBJECTIVE: To investigate whether azithromycin therapy during severe RSV bronchiolitis reduces upper airway MMP-9 levels, whether upper airway MMP-9 levels correlate with upper airway interleukin IL-8 levels, and whether MMP-9 level reduction is associated with reduced post-RSV recurrent wheeze (RW). METHODS: A total of 200 otherwise healthy 1- to 18-month-old infants hospitalized with RSV bronchiolitis were randomized into a double-blind, placebo-controlled trial of oral azithromycin (10 mg/kg daily for 7 days followed by 5 mg/kg daily for 7 days) or placebo. Infants were followed for 2 to 4 years for the outcome of RW (3 or more wheezing episodes). Nasal lavage samples for MMP-9 levels were obtained at baseline, day 14 (end of the study treatment), and after 6 months. RESULTS: Upper airway MMP-9 levels were highly correlated with IL-8 levels at all 3 time points: randomization, day 14, and 6 months (r = 0.80; P < .0001 for all time points). MMP-9 levels were similar between treatment groups at randomization, were lower on day 14 among children treated with azithromycin (P = .0085), but no longer different after 6 months. MMP-9 levels at baseline and change from baseline to day 14 were not associated with the development of RW (P = .49, .39, respectively). CONCLUSION: Azithromycin therapy in children hospitalized with RSV bronchiolitis had a short-term anti-inflammatory effect in reducing upper airway MMP-9 levels. However, the reduction in MMP-9 levels did not relate to subsequent RW post-RSV. TRIAL REGISTRATION: This study is a secondary analysis of the Azithromycin to Prevent Wheezing following severe RSV bronchiolitis-II clinical trial registered at Clinicaltrials.gov (NCT02911935).

CTOTC-08: A multicenter randomized controlled trial of rituximab induction to reduce antibody development and improve outcomes in pediatric lung transplant recipients.

We conducted a randomized, placebo-controlled, double-blind study of pediatric lung transplant recipients, hypothesizing that rituximab plus rabbit anti-thymocyte globulin induction would reduce de novo donor-specific human leukocyte antigen antibodies (DSA) development and improve outcomes. We serially obtained clinical data, blood, and respiratory samples for at least one year posttransplant. We analyzed peripheral blood lymphocytes by flow cytometry, serum for antibody development, and respiratory samples for viral infections using multiplex PCR. Of 45 subjects enrolled, 34 were transplanted and 27 randomized to rituximab (n = 15) or placebo (n = 12). No rituximab-treated subjects versus five placebo-treated subjects developed de novo DSA with mean fluorescence intensity >2000. There was no difference between treatment groups in time to the primary composite outcome endpoint (death, bronchiolitis obliterans syndrome [BOS] grade 0-p, obliterative bronchiolitis or listing for retransplant). A post-hoc analysis substituting more stringent chronic lung allograft dysfunction criteria for BOS 0-p showed no difference in outcome (p = .118). The incidence of adverse events including infection and rejection episodes was no different between treatment groups. Although the study was underpowered, we conclude that rituximab induction may have prevented early DSA development in pediatric lung transplant recipients without adverse effects and may improve outcomes (Clinical Trials: NCT02266888).

Auto-inflammation and auto-immunity pathways are associated with emergence of BOS in pediatric lung transplantation.

BACKGROUND: Long-term survival after lung transplantation (LTx) is limited by chronic lung allograft dysfunction (CLAD). METHODS: We report an analysis of cytokine profiles in bronchoalveolar lavage samples collected during a prospective multicenter non-interventional trial primarily designed to determine the impact of community-acquired respiratory viral infections (CARV) in outcomes after pediatric LTx. In this analysis, we identify potential biomarkers of auto-inflammation and auto-immunity associated with survival and risk of bronchiolitis obliterans (BOS) after LTx with cytokine analysis of bronchoalveolar lavage fluid (BALF) from 61 pediatric recipients. RESULTS: Higher IL-23 (p = .048) and IL-31 (p = .035) levels were associated with the risk of BOS, and lower levels of epithelial growth factor (EGF) (p = .041) and eotaxin (EOX) (p = .017) were associated with BOS. Analysis using conditional inference trees to evaluate cytokines at each visit associated with survival identified soluble CD30 (p < .001), pro-inflammatory cytokine IL-23 (p = .02), and sTNFRI (p = .01) below cutoff levels as associated with BOS-free survival. CONCLUSIONS: Our results indicate that post-LTx survival in children may be linked to activation of alternate pathways of the immune system that affect airway remodeling in addition to activation of "classical" pathways that have been described in adult LTx recipients. These may indicate pathways to target for intervention.

Epidemiology and persistence of rhinovirus in pediatric lung transplantation.

BACKGROUND: Infection with rhinovirus (HRV) occurs following pediatric lung transplantation. Prospective studies documenting frequencies, persistence, and progression of HRV in this at-risk population are lacking. METHODS: In the Clinical Trials in Organ Transplant in Children prospective observational study, we followed 61 lung transplant recipients for 2 years. We quantified molecular subtypes of HRV in serially collected nasopharyngeal (NP) and bronchoalveolar lavage (BAL) samples and correlated them with clinical characteristics. RESULTS: We identified 135 community-acquired respiratory infections (CARV) from 397 BAL and 480 NP samples. We detected 93 HRV events in 42 (68.8%) patients, 22 of which (23.4%) were symptomatic. HRV events were contiguous with different genotypes identified in 23 cases, but symptoms were not preferentially associated with any particular species. Nine (9.7%) HRV events persisted over multiple successive samples for a median of 36 days (range 18-408 days). Three persistent HRV were symptomatic. When we serially measured forced expiratory volume in one second (FEV1) in 23 subjects with events, we did not observe significant decreases in lung function over 12 months post-HRV. CONCLUSION: In conjunction with our previous reports, our prospectively collected data indicate that molecularly heterogeneous HRV infections occur commonly following pediatric lung transplantation, but these infections do not negatively impact clinical outcomes.

Host Gene Expression in Nose and Blood for the Diagnosis of Viral Respiratory Infection.

BACKGROUND: Recently there has been a growing interest in the potential for host transcriptomic analysis to augment the diagnosis of infectious diseases. METHODS: We compared nasal and blood samples for evaluation of the host transcriptomic response in children with acute respiratory syncytial virus (RSV) infection, symptomatic non-RSV respiratory virus infection, asymptomatic rhinovirus infection, and virus-negative asymptomatic controls. We used nested leave-one-pair-out cross-validation and supervised principal components analysis to define small sets of genes whose expression patterns accurately classified subjects. We validated gene classification scores using an external data set. RESULTS: Despite lower quality of nasal RNA, the number of genes detected by microarray in each sample type was equivalent. Nasal gene expression signal derived mainly from epithelial cells but also included a variable leukocyte contribution. The number of genes with increased expression in virus-infected children was comparable in nasal and blood samples, while nasal samples also had decreased expression of many genes associated with ciliary function and assembly. Nasal gene expression signatures were as good or better for discriminating between symptomatic, asymptomatic, and uninfected children. CONCLSUSIONS: Our results support the use of nasal samples to augment pathogen-based tests to diagnose viral respiratory infection.

Impact of Pretransplant Donor BK Viruria in Kidney Transplant Recipients.

BACKGROUND: BK virus (BKV) is a significant cause of nephropathy in kidney transplantation. The goal of this study was to characterize the course and source of BKV in kidney transplant recipients. METHODS: We prospectively collected pretransplant plasma and urine samples from living and deceased kidney donors and performed BKV polymerase chain reaction (PCR) and immunoglobulin G (IgG) testing on pretransplant and serially collected posttransplant samples in kidney transplant recipients. RESULTS: Among deceased donors, 8.1% (17/208) had detectable BKV DNA in urine prior to organ procurement. BK viruria was observed in 15.4% (6/39) of living donors and 8.5% (4/47) of deceased donors of recipients at our institution (P = .50). BKV VP1 sequencing revealed identical virus between donor-recipient pairs to suggest donor transmission of virus. Recipients of BK viruric donors were more likely to develop BK viruria (66.6% vs 7.8%; P < .001) and viremia (66.6% vs 8.9%; P < .001) with a shorter time to onset (log-rank test, P < .001). Though donor BKV IgG titers were higher in recipients who developed BK viremia, pretransplant donor, recipient, and combined donor/recipient serology status was not associated with BK viremia (P = .31, P = .75, and P = .51, respectively). CONCLUSIONS: Donor BK viruria is associated with early BK viruria and viremia in kidney transplant recipients. BKV PCR testing of donor urine may be useful in identifying recipients at risk for BKV complications.

Absence of evidence that respiratory viral infections influence pediatric lung transplantation outcomes: Results of the CTOTC-03 study.

Based on reports in adult lung transplant recipients, we hypothesized that community-acquired respiratory viral infections (CARVs) would be a risk factor for poor outcome after pediatric lung transplant. We followed 61 pediatric lung transplant recipients for 2+ years or until they met a composite primary endpoint including bronchiolitis obliterans syndrome/obliterative bronchiolitis, retransplant, or death. Blood, bronchoalveolar lavage, and nasopharyngeal specimens were obtained with standard of care visits. Nasopharyngeal specimens were obtained from recipients with respiratory viral symptoms. Respiratory specimens were interrogated for respiratory viruses by using multiplex polymerase chain reaction. Donor-specific HLA antibodies, self-antigens, and ELISPOT reactivity were also evaluated. Survival was 84% (1 year) and 68% (3 years). Bronchiolitis obliterans syndrome incidence was 20% (1 year) and 38% (3 years). The primary endpoint was met in 46% of patients. CARV was detected in 156 patient visits (74% enterovirus/rhinovirus). We did not find a relationship between CARV recovery from respiratory specimens and the primary endpoint (hazard ratio 0.64 [95% confidence interval: 0.25-1.59], P = .335) or between CARV and the development of alloimmune or autoimmune humoral or cellular responses. These findings raise the possibility that the immunologic impact of CARV following pediatric lung transplant is different than that observed in adults.

Ureaplasma urealyticum pyelonephritis presenting with progressive dysuria, renal failure, and neurologic symptoms in an immunocompromised patient.

Ureaplasma urealyticum is a bacterial species correlated with urethritis in healthy individuals and invasive infections in immunocompromised patients. We describe a 20-year-old female with a history of remote heart transplant on everolimus, mycophenolate, and rituximab presenting with progressive urinary tract symptoms, renal failure, and neurologic symptoms. An extensive workup ultimately identified U urealyticum infection, and the patient successfully recovered after a course of azithromycin and doxycycline.

Enterovirus D68 Surveillance, St. Louis, Missouri, USA, 2016.

A fall 2016 outbreak of enterovirus D68 infection in St. Louis, Missouri, USA, had less effect than a fall 2014 outbreak on hospital census, intensive care unit census, and hospitalization for a diagnosis of respiratory illness. Without ongoing surveillance and specific testing, these cases might have been missed.

Heartland Virus and Hemophagocytic Lymphohistiocytosis in Immunocompromised Patient, Missouri, USA.

Heartland virus is a suspected tickborne pathogen in the United States. We describe a case of hemophagocytic lymphohistiocytosis, then death, in an immunosuppressed elderly man in Missouri, USA, who was infected with Heartland virus.

Valganciclovir for symptomatic congenital cytomegalovirus disease.

BACKGROUND: The treatment of symptomatic congenital cytomegalovirus (CMV) disease with intravenous ganciclovir for 6 weeks has been shown to improve audiologic outcomes at 6 months, but the benefits wane over time. METHODS: We conducted a randomized, placebo-controlled trial of valganciclovir therapy in neonates with symptomatic congenital CMV disease, comparing 6 months of therapy with 6 weeks of therapy. The primary end point was the change in hearing in the better ear ("best-ear" hearing) from baseline to 6 months. Secondary end points included the change in hearing from baseline to follow-up at 12 and 24 months and neurodevelopmental outcomes, with each end point adjusted for central nervous system involvement at baseline. RESULTS: A total of 96 neonates underwent randomization, of whom 86 had follow-up data at 6 months that could be evaluated. Best-ear hearing at 6 months was similar in the 6-month group and the 6-week group (2 and 3 participants, respectively, had improvement; 36 and 37 had no change; and 5 and 3 had worsening; P=0.41). Total-ear hearing (hearing in one or both ears that could be evaluated) was more likely to be improved or to remain normal at 12 months in the 6-month group than in the 6-week group (73% vs. 57%, P=0.01). The benefit in total-ear hearing was maintained at 24 months (77% vs. 64%, P=0.04). At 24 months, the 6-month group, as compared with the 6-week group, had better neurodevelopmental scores on the Bayley Scales of Infant and Toddler Development, third edition, on the language-composite component (P=0.004) and on the receptive-communication scale (P=0.003). Grade 3 or 4 neutropenia occurred in 19% of the participants during the first 6 weeks. During the next 4.5 months of the study, grade 3 or 4 neutropenia occurred in 21% of the participants in the 6-month group and in 27% of those in the 6-week group (P=0.64). CONCLUSIONS: Treating symptomatic congenital CMV disease with valganciclovir for 6 months, as compared with 6 weeks, did not improve hearing in the short term but appeared to improve hearing and developmental outcomes modestly in the longer term. (Funded by the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT00466817.).

Enhanced virome sequencing using targeted sequence capture.

Metagenomic shotgun sequencing (MSS) is an important tool for characterizing viral populations. It is culture independent, requires no a priori knowledge of the viruses in the sample, and may provide useful genomic information. However, MSS can lack sensitivity and may yield insufficient data for detailed analysis. We have created a targeted sequence capture panel, ViroCap, designed to enrich nucleic acid from DNA and RNA viruses from 34 families that infect vertebrate hosts. A computational approach condensed ∼1 billion bp of viral reference sequence into <200 million bp of unique, representative sequence suitable for targeted sequence capture. We compared the effectiveness of detecting viruses in standard MSS versus MSS following targeted sequence capture. First, we analyzed two sets of samples, one derived from samples submitted to a diagnostic virology laboratory and one derived from samples collected in a study of fever in children. We detected 14 and 18 viruses in the two sets, comprising 19 genera from 10 families, with dramatic enhancement of genome representation following capture enrichment. The median fold-increases in percentage viral reads post-capture were 674 and 296. Median breadth of coverage increased from 2.1% to 83.2% post-capture in the first set and from 2.0% to 75.6% in the second set. Next, we analyzed samples containing a set of diverse anellovirus sequences and demonstrated that ViroCap could be used to detect viral sequences with up to 58% variation from the references used to select capture probes. ViroCap substantially enhances MSS for a comprehensive set of viruses and has utility for research and clinical applications.

Detection of Viruses in Clinical Samples by Use of Metagenomic Sequencing and Targeted Sequence Capture.

Metagenomic shotgun sequencing (MSS) is a revolutionary approach to viral diagnostic testing that allows simultaneous detection of a broad range of viruses, detailed taxonomic assignment, and detection of mutations associated with antiviral drug resistance. To enhance sensitivity for virus detection, we previously developed ViroCap, a targeted sequence capture panel designed to enrich nucleic acid from a comprehensive set of eukaryotic viruses prior to sequencing. To demonstrate the utility of MSS with targeted sequence capture for detecting clinically important viruses and characterizing clinically important viral features, we used ViroCap to analyze clinical samples from a diagnostic virology laboratory containing a broad range of medically relevant viruses. From 26 samples, MSS with ViroCap detected all of the expected viruses and 30 additional viruses. Comparing sequencing after capture enrichment with standard MSS, we detected 13 viruses only with capture enrichment and observed a consistent increase in the number and percentage of viral sequence reads as well as the breadth and depth of coverage of the viral genomes. Compared with clinical testing, MSS enhanced taxonomic assignment for 15 viruses, and codons associated with antiviral drug resistance in influenza A virus, herpes simplex virus (HSV), human immunodeficiency virus (HIV), and hepatitis C virus (HCV) could be analyzed. Overall, in clinical samples, MSS with targeted sequence capture provides enhanced virus detection and information of clinical and epidemiologic relevance compared with clinical testing and MSS without targeted sequence capture.

Viral DNAemia and Immune Suppression in Pediatric Sepsis.

OBJECTIVES: Demonstrate that DNA viremia is common in pediatric sepsis and quantitate its associations with host immune function and secondary infection risk. DESIGN: Retrospective analysis of a prospective cohort study. PATIENTS: Seventy-three children admitted with sepsis-induced organ failure. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: This study was performed as an ancillary investigation to a single-center prospective study of children with severe sepsis. Longitudinally collected, batched, frozen plasma was examined using real time-polymerase chain reaction for the presence of cytomegalovirus, Epstein-Barr virus, herpes simplex virus, human herpes virus-6, torque teno virus, and adenovirus DNA. Innate immune function was also measured longitudinally via quantification of ex vivo lipopolysaccharide -induced tumor necrosis factor-α production capacity. Viral DNAemia with a virus other than torque teno virus was detected in 28 of 73 subjects (38%) and included cytomegalovirus 5%, Epstein-Barr virus 11%, herpes simplex virus 4%, human herpes virus-6 8%, and adenovirus 26%. In addition, torque teno virus was detected in 89%. Epstein-Barr virus DNAemia was associated with preexisting immune suppression (p = 0.007) Viral DNAemia was associated with preexisting immune suppression and high risk for the subsequent development of secondary infection (p < 0.05 for both). Subjects with viral DNAemia had lower innate immune function over time compared with those who were virus negative (p < 0.05). CONCLUSIONS: DNAemia from multiple viruses can be detected in septic children and is strongly associated with preexisting immune suppression and secondary infection risk. The role of DNA viruses in the perpetuation of impaired host defense in this setting should be the subject of prospective study.

Polyomavirus Reactivation and Immune Responses to Kidney-Specific Self-Antigens in Transplantation.

Humoral immune responses against donor antigens are important determinants of long-term transplant outcomes. Reactivation of the polyomavirus BK has been associated with de novo antibodies against mismatched donor HLA antigens in kidney transplantation. The effect of polyomavirus reactivation (BK viremia or JC viruria) on antibodies to kidney-specific self-antigens is unknown. We previously reported excellent 5-year outcomes after minimization of immunosuppression for BK viremia and after no intervention for JC viruria. Here, we report the 10-year results of this trial (n=193) along with a nested case-control study (n=40) to explore associations between polyomavirus reactivation and immune responses to the self-antigens fibronectin (FN) and collagen type-IV (Col-IV). Consistent with 5-year findings, subjects taking tacrolimus, compared with those taking cyclosporin, had less acute rejection (11% versus 22%, P=0.05) and graft loss (9% versus 22%, P=0.01) along with better transplant function (eGFR 65±19 versus 50±24 ml/min per 1.73 m2, P<0.001) at 10 years. Subjects undergoing immunosuppression reduction for BK viremia had 10-year outcomes similar to those without viremia. In the case-control study, antibodies to FN/Col-IV were more prevalent during year 1 in subjects with polyomavirus reactivation than in those without reactivation (48% versus 11%, P=0.04). Subjects with antibodies to FN/Col-IV had more acute rejection than did those without these antibodies (38% versus 8%, P=0.02). These data demonstrate the long-term safety and effectiveness of minimizing immunosuppression to treat BK viremia. Furthermore, these results indicate that polyomavirus reactivation associates with immune responses to kidney-specific self-antigens that may increase the risk for acute rejection through unclear mechanisms.