Virus-specific Th17 Cells Are Induced by Human Cytomegalovirus after Renal Transplantation.

CMV infection and Th17 cells are independently associated with increased risk for late allograft loss after renal transplantation. Although CMV-specific Th17 cells are detectable in animal models and nontransplant clinical populations, evidence linking CMV and Th17 cells after renal transplantation remains unclear. This prospective observational study evaluated a cohort of renal transplant recipients during 12 mo posttransplant to assess the presence of CMV-specific Th17 cells in peripheral blood and their relationship to pretransplant CMV serostatus and CMV DNAemia. CMV-specific Th17 cells were identified among CMV serostatus donor (D)+ and/or recipient (R)+ recipients and expanded during both primary (D+/R-) and reactivated (D+/R+, D-/R+) CMV DNAemia. A subset of CMV-specific Th17 cells coexpressed IFN-γ, indicating a Th1/17 phenotype. These Th17 and Th1/17 cells expressed CCR6, CCR5, activation and terminal differentiation markers (CD95, OX40, HLA-DR, CD57), and a central/effector memory phenotype. CMV-specific Th1/17 cells expressed activating/inhibitory receptors (CD57, 4-1BB, CD160, CTLA-4, PD-1) at higher frequencies than Th17 cells. In contrast, staphylococcal enterotoxin B-induced Th17 cells did not expand during CMV DNAemia, did not differ between CMV serostatus groups over time, expressed CCR6, predominantly coexpressed TNF-α, and had lower expression of activating and inhibitory receptors than pp65-specific Th17 and Th1/17 cells. These data show that CMV-specific Th17 cells expand during episodes of CMV DNAemia among renal transplant recipients, and that these virus-specific Th17 and Th1/17 cells have distinct phenotypes from global circulating Th(1)/17 cells. These results suggest a potential proinflammatory pathway by which CMV-induced Th17 cells may contribute to allograft injury, increasing risk for late allograft loss.

A safe and highly efficacious measles virus-based vaccine expressing SARS-CoV-2 stabilized prefusion spike.

The current pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights an urgent need to develop a safe, efficacious, and durable vaccine. Using a measles virus (rMeV) vaccine strain as the backbone, we developed a series of recombinant attenuated vaccine candidates expressing various forms of the SARS-CoV-2 spike (S) protein and its receptor binding domain (RBD) and evaluated their efficacy in cotton rat, IFNAR-/-mice, IFNAR-/--hCD46 mice, and golden Syrian hamsters. We found that rMeV expressing stabilized prefusion S protein (rMeV-preS) was more potent in inducing SARS-CoV-2-specific neutralizing antibodies than rMeV expressing full-length S protein (rMeV-S), while the rMeVs expressing different lengths of RBD (rMeV-RBD) were the least potent. Animals immunized with rMeV-preS produced higher levels of neutralizing antibody than found in convalescent sera from COVID-19 patients and a strong Th1-biased T cell response. The rMeV-preS also provided complete protection of hamsters from challenge with SARS-CoV-2, preventing replication in lungs and nasal turbinates, body weight loss, cytokine storm, and lung pathology. These data demonstrate that rMeV-preS is a safe and highly efficacious vaccine candidate, supporting its further development as a SARS-CoV-2 vaccine.

Murine cytomegalovirus promotes renal allograft inflammation via Th1/17 cells and IL-17A.

Human cytomegalovirus (HCMV) infection is associated with renal allograft failure. Allograft damage in animal models is accelerated by CMV-induced T helper 17 (Th17) cell infiltrates. However, the mechanisms whereby CMV promotes Th17 cell-mediated pathological organ inflammation are uncharacterized. Here we demonstrate that murine CMV (MCMV)-induced intragraft Th17 cells have a Th1/17 phenotype co-expressing IFN-γ and/or TNF-α, but only a minority of these cells are MCMV specific. Instead, MCMV promotes intragraft expression of CCL20 and CXCL10, which are associated with recruitment of CCR6+ CXCR3+ Th17 cells. MCMV also enhances Th17 cell infiltrates after ischemia-reperfusion injury, independent of allogeneic responses. Pharmacologic inhibition of the Th17 cell signature cytokine, IL-17A, ameliorates MCMV-associated allograft damage without increasing intragraft viral loads or reducing MCMV-specific Th1 cell infiltrates. Clinically, HCMV DNAemia is associated with higher serum IL-17A among renal transplant patients with acute rejection, linking HCMV reactivation with Th17 cell cytokine expression. In summary, CMV promotes allograft damage via cytokine-mediated Th1/17 cell recruitment, which may be pharmacologically targeted to mitigate graft injury while preserving antiviral T cell immunity.

Severe SARS-CoV-2 disease in the context of a NF-κB2 loss-of-function pathogenic variant.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that emerged recently and has created a global pandemic. Symptomatic SARS-CoV-2 infection, termed coronavirus disease 2019 (COVID-19), has been associated with a host of symptoms affecting numerous organ systems, including the lungs, cardiovascular system, kidney, central nervous system, gastrointestinal tract, and skin, among others. OBJECTIVE: Although several risk factors have been identified as related to complications from and severity of COVID-19, much about the virus remains unknown. The host immune response appears to affect the outcome of disease. It is not surprising that patients with intrinsic or secondary immune compromise might be particularly susceptible to complications from SARS-CoV-2 infection. Pathogenic loss-of-function or gain-of-function heterozygous variants in nuclear factor-κB2 have been reported to be associated with either a combined immunodeficiency or common variable immunodeficiency phenotype. METHODS: We evaluated the functional consequence and immunologic phenotype of a novel NFKB2 loss of function variant in a 17-year-old male patient and describe the clinical management of SARS-CoV-2 infection in this context. RESULTS: This patient required a 2-week hospitalization for SARS-CoV-2 infection, including 7 days of mechanical ventilation. We used biologic therapies to avert potentially fatal acute respiratory distress syndrome and treat hyperinflammatory responses. The patient had an immunologic phenotype of B-cell dysregulation with decreased switched memory B cells. Despite the underlying immune dysfunction, he recovered from the infection with intense management. CONCLUSIONS: This clinical case exemplifies some of the practical challenges in management of patients with SARS-CoV-2 infection, especially in the context of underlying immune dysregulation.

Detection of Cytomegalovirus in Intestinal Tissue of Infants with Necrotizing Enterocolitis or Spontaneous Intestinal Perforation.

OBJECTIVE: To determine the frequency of detection of cytomegalovirus (CMV) in surgical or autopsy intestinal tissue from infants with necrotizing enterocolitis (NEC) or spontaneous intestinal perforation (SIP) of the small bowel. STUDY DESIGN: This was a retrospective cohort study of infants in the neonatal intensive care unit at Nationwide Children's Hospital, Columbus, Ohio, with NEC (Bell stage ≥2B) or SIP from 2000 to 2016. Paraffin-embedded surgical or autopsy intestinal tissues were examined for CMV by polymerase chain reaction (PCR) and immunohistochemistry (IHC), and clinical characteristics of CMV-positive vs CMV-negative cases were compared. RESULTS: CMV was detected by PCR or IHC in 7 (4%) of 178 infants with surgical or autopsy- confirmed NEC (n = 6) or SIP (n = 1). Among 143 NEC cases (123 surgical, 20 autopsy), CMV was detected in 6 (4%): 4 (2 surgical, 2 autopsy) by both PCR and IHC, and 2 (surgical) by PCR only. Among 35 SIP cases (32 surgical, 3 autopsy), 1 (3%) surgical case was positive, by PCR only. CMV-associated NEC cases had lower median gestational age (24 vs 28 weeks; P = .02), birth weight (649 vs 1121 g; P = .04), and platelet count (16 000/mm3 vs 50 000/mm3; P = .018) compared with CMV-negative cases, respectively. No association was found with receipt of maternal milk, age at NEC diagnosis, male sex, cholestasis, or mortality. CONCLUSIONS: CMV was detected in intestinal tissue from 4% of NEC or SIP cases (NEC, 4%; SIP, 3%). Lower gestational age, lower birth weight, and thrombocytopenia were significantly associated with detection of CMV in NEC or SIP cases.

Cytomegalovirus and Epstein-Barr virus infections among pediatric kidney transplant recipients at a center using universal Valganciclovir Prophylaxis.

BACKGROUND: CMV is associated with adverse effects in renal transplant recipients. The objective of this study was to characterize the incidence and timing of CMV and EBV infections in relation to valGCV prophylaxis in a pediatric renal transplant cohort. METHODS: Retrospective cohort of pediatric renal transplant patients given universal valGCV prophylaxis and universal viral surveillance was evaluated. Demographics, prophylaxis, acute rejection, and CMV and EBV infections were abstracted. RESULTS: A total of 92 pediatric renal allograft recipients, 2008-2013, were included. One or more viral infections developed in 77/92 (83.7%) of the patients. EBV was the most common in 62/92 (67%) patients, irrespective of valGCV (82% of episodes occurring on valGCV). CMV DNAemia occurred in 30/92 (33%) patients, 14 episodes (47%) occurring on valGCV. Incidence of breakthrough CMV on prophylaxis was 15% and was associated with persistent DNAemia (OR 7.8, CI:1.6-32.9, P < 0.02). CMV tissue-invasive disease was not seen. CMV syndrome occurred in 10% of the cohort, only in CMV D+ patients, and only one symptomatic breakthrough infection required treatment. Out of 92, 21 (23%) had simultaneous co-infections with 2-3 viruses. CONCLUSIONS: Viral infections in pediatric renal transplant recipients receiving universal valGCV prophylaxis were common. EBV infections were not reduced by valGCV prophylaxis, and nearly half of CMV infections occurred on valGCV. Symptomatic CMV infection while on prophylaxis was rare. valGCV prophylaxis may prevent symptomatic CMV infection but not EBV infection, and frequent CMV surveillance in pediatric renal transplant recipients on prophylaxis may not be necessary.

NK cell and Th17 responses are differentially induced in murine cytomegalovirus infected renal allografts and vary according to recipient virus dose and strain.

Human cytomegalovirus (HCMV) donor positive (D+) serostatus with acute rejection is associated with renal allograft loss, but the impact of recipient positive (R+) serostatus is unclear. In an allogeneic renal transplant model, antiviral natural killer (NK) and CD8+ T cell memory responses in murine CMV (MCMV) D+/R+ transplants were compared to D-/R- and D+/R- transplants, with recipient infection varied by MCMV dose and strain. D+/R- transplants had high primary antiviral cytolytic (interferon-γ+) and cytotoxic (granzyme B+) NK responses, whereas NK memory responses were lower in D+/R+ recipients receiving a high primary MCMV dose. Despite MCMV immunity, D+/R+ recipients receiving a low MCMV dose showed primary-like high cytolytic and cytotoxic NK responses. D+/R+ transplants infected with different D/R strains had low cytolytic NK responses but high cytotoxic NK responses. NK memory also induced a novel TNF-α+ NK response among high-dose virus recipients. MCMV+ transplants had greater Th17 responses than MCMV-uninfected transplants and Th17 inhibition ameliorated graft injury. All MCMV+ recipients had similar CD8+ T cell responses. In sum, NK and Th17 responses, but not CD8+ T cells, varied according to conditions of primary recipient infection. This variability could contribute to variable graft outcomes in HCMV D+/R+ renal transplantation.

Racial and Ethnic Differences in the Prevalence of Congenital Cytomegalovirus Infection.

OBJECTIVE: To evaluate the impact of race and ethnicity upon the prevalence and clinical spectrum of congenital cytomegalovirus infection (cCMV). STUDY DESIGN: From 2007 to 2012, 100 332 infants from 7 medical centers were screened for cCMV while in the hospital. Ethnicity and race were collected and cCMV prevalence rates were calculated. RESULTS: The overall prevalence of cCMV in the cohort was 4.5 per 1000 live births (95% CI, 4.1-4.9). Black infants had the highest cCMV prevalence (9.5 per 1000 live births; 95% CI, 8.3-11.0), followed by multiracial infants (7.8 per 1000 live births; 95% CI, 4.7-12.0). Significantly lower prevalence rates were observed in non-Hispanic white infants (2.7 per 1000 live births; 95% CI, 2.2-3.3), Hispanic white infants (3.0 per 1000 live births; 95% CI, 2.4-3.6), and Asian infants (1.0 per 1000 live births; 95% CI, 0.3-2.5). After adjusting for socioeconomic status and maternal age, black infants were significantly more likely to have cCMV compared with non-Hispanic white infants (adjusted prevalence OR, 1.9; 95% CI, 1.4-2.5). Hispanic white infants had a slightly lower risk of having cCMV compared with non-Hispanic white infants (adjusted prevalence OR, 0.7; 95% CI, 0.5-1.0). However, no significant differences in symptomatic cCMV (9.6%) and sensorineural hearing loss (7.8%) were observed between the race/ethnic groups. CONCLUSIONS: Significant racial and ethnic differences exist in the prevalence of cCMV, even after adjusting for socioeconomic status and maternal age. Although once infected, the newborn disease and rates of hearing loss in infants are similar with respect to race and ethnicity.

Successful Treatment of Bloodstream Infection Due to Metallo-ß-Lactamase-Producing Stenotrophomonas maltophilia in a Renal Transplant Patient.

Stenotrophomonas maltophilia is an emerging multidrug-resistant (MDR) opportunistic pathogen for which new antibiotic options are urgently needed. We report our clinical experience treating a 19-year-old renal transplant recipient who developed prolonged bacteremia due to metallo-ß-lactamase-producing S. maltophilia refractory to conventional treatment. The infection recurred despite a prolonged course of colistimethate sodium (colistin) but resolved with the use of a novel drug combination with clinical efficacy against the patient's S. maltophilia isolate.

Cytomegalovirus enhances macrophage TLR expression and MyD88-mediated signal transduction to potentiate inducible inflammatory responses.

Circulating monocytes carrying human CMV (HCMV) migrate into tissues, where they differentiate into HCMV-infected resident macrophages that upon interaction with bacterial products may potentiate tissue inflammation. In this study, we investigated the mechanism by which HCMV promotes macrophage-orchestrated inflammation using a clinical isolate of HCMV (TR) and macrophages derived from primary human monocytes. HCMV infection of the macrophages, which was associated with viral DNA replication, significantly enhanced TNF-α, IL-6, and IL-8 gene expression and protein production in response to TLR4 ligand (LPS) stimulation compared with mock-infected LPS-stimulated macrophages during a 6-d in vitro infection. HCMV infection also potentiated TLR5 ligand-stimulated cytokine production. To elucidate the mechanism by which HCMV infection potentiated inducible macrophage responses, we show that infection by HCMV promoted the maintenance of surface CD14 and TLR4 and TLR5, which declined over time in mock-infected macrophages, and enhanced both the intracellular expression of adaptor protein MyD88 and the inducible phosphorylation of IκBα and NF-κB. These findings provide additional information toward elucidating the mechanism by which HCMV potentiates bacteria-induced NF-κB-mediated macrophage inflammatory responses, thereby enhancing organ inflammation in HCMV-infected tissues.

Saliva polymerase-chain-reaction assay for cytomegalovirus screening in newborns.

BACKGROUND: Congenital cytomegalovirus (CMV) infection is an important cause of hearing loss, and most infants at risk for CMV-associated hearing loss are not identified early in life because of failure to test for the infection. The standard assay for newborn CMV screening is rapid culture performed on saliva specimens obtained at birth, but this assay cannot be automated. Two alternatives--real-time polymerase-chain-reaction (PCR)-based testing of a liquid-saliva or dried-saliva specimen obtained at birth--have been developed. METHODS: In our prospective, multicenter screening study of newborns, we compared real-time PCR assays of liquid-saliva and dried-saliva specimens with rapid culture of saliva specimens obtained at birth. RESULTS: A total of 177 of 34,989 infants (0.5%; 95% confidence interval [CI], 0.4 to 0.6) were positive for CMV, according to at least one of the three methods. Of 17,662 newborns screened with the use of the liquid-saliva PCR assay, 17,569 were negative for CMV, and the remaining 85 infants (0.5%; 95% CI, 0.4 to 0.6) had positive results on both culture and PCR assay. The sensitivity and specificity of the liquid-saliva PCR assay were 100% (95% CI, 95.8 to 100) and 99.9% (95% CI, 99.9 to 100), respectively, and the positive and negative predictive values were 91.4% (95% CI, 83.8 to 96.2) and 100% (95% CI, 99.9 to 100), respectively. Of 17,327 newborns screened by means of the dried-saliva PCR assay, 74 were positive for CMV, whereas 76 (0.4%; 95% CI, 0.3 to 0.5) were found to be CMV-positive on rapid culture. Sensitivity and specificity of the dried-saliva PCR assay were 97.4% (95% CI, 90.8 to 99.7) and 99.9% (95% CI, 99.9 to 100), respectively. The positive and negative predictive values were 90.2% (95% CI, 81.7 to 95.7) and 99.9% (95% CI, 99.9 to 100), respectively. CONCLUSIONS: Real-time PCR assays of both liquid- and dried-saliva specimens showed high sensitivity and specificity for detecting CMV infection and should be considered potential screening tools for CMV in newborns. (Funded by the National Institute on Deafness and Other Communication Disorders.).

Molecular testing for gastrointestinal pathogens in intestinal tissue of infants with necrotizing enterocolitis or spontaneous intestinal perforation.

OBJECTIVE: The objective of this study was to determine the frequency of common gastrointestinal bacterial, parasitic, and viral pathogen detection in necrotizing enterocolitis (NEC) or spontaneous intestinal perforation (SIP) -associated intestinal tissue. STUDY DESIGN: Retrospective cohort study examined formalin fixed, paraffin embedded (FFPE) surgical or autopsy intestinal tissue from NEC or SIP specimens. DNA and RNA were extracted and analyzed by multiplex PCR panel (GIFA Biofire). DNA or RNA from stool samples containing each pathogen were extracted for positive controls. RESULTS: The total number of intestinal tissue samples were 193 from 310 infants (156 NEC, 37 SIP). Six (3%) infants with stage III NEC tested positive for a target pathogen; 2, C. difficile; 3, Enteroaggregtive E. coli; and 1, Giardia. No gastrointestinal viral pathogens were detected. CONCLUSION: Molecular testing yielded few GI pathogens suggesting that these organisms are likely not major causes or facilitators of NEC or SIP.

T cell responses and clinical symptoms among infants with congenital cytomegalovirus infection.

BACKGROUNDCongenital cytomegalovirus (cCMV) infection can cause developmental impairment and sensorineural hearing loss (SNHL). To determine the relationship between immune responses to cCMV infection and neurologic sequelae, T cell responses were compared for their connection to clinical symptoms at birth and neurodevelopmental outcomes.METHODSThirty cCMV-infected and 15 uninfected infants were enrolled in a single-center prospective observational case-control study. T cell pp65-specific cytokine responses; CD57, CD28, and PD-1 expression; and memory subsets were compared.RESULTSInfected neonates (73% symptomatic at birth) lacked pp65-specific cytokine-secreting T cells, with elevated frequencies of CD57+, CD28-, and PD-1+CD8+ T cells and effector memory subsets. Though frequencies overlapped between cCMV symptom groups, asymptomatic infants had higher frequencies of CD57+PD-1+CD8+ T cells. Neonates with subsequent developmental delay lacked detectable CMV-specific T cell responses, with patterns resembling those of uninfected infants. Two children with progressive SNHL had high frequencies of PD-1+CD8+ T cells over the first year compared with children without progressive SNHL.CONCLUSIONSimilar to published reports, neonatal viral antigen-specific cytokine-secreting T cell responses were not detected, but overall patterns indicate that globally differentiated memory CD8+ T cell populations were induced by cCMV infection, with higher frequencies of terminally differentiated PD-1+CD8+ T cells potentially associated with asymptomatic infection. In this cohort, a lack of in utero T cell differentiation was associated with developmental delay, and high frequencies of PD-1+CD8+ T cells persisted only in children with progressive SNHL. Further work is needed to define the specificity of these T cells and their mechanistic connection to these outcomes.FUNDINGThis study was funded through an intramural research award at Nationwide Children's Hospital, the Pediatric Infectious Disease Society Fellowship Award funded by Stanley and Susan Plotkin and Sanofi Pasteur, the Abigail Wexner Research Institute at Nationwide Children's Hospital, and the Pichichero Family Foundation Vaccines for Children Initiative Research Award from the Pediatric Infectious Diseases Society Foundation.

Δγ1134.5 herpes simplex viruses encoding human cytomegalovirus IRS1 or TRS1 induce interferon regulatory factor 3 phosphorylation and an interferon-stimulated gene response.

The chimeric herpes simplex viruses (HSV) are Δγ134.5 vectors encoding the human cytomegalovirus (HCMV) IRS1 or TRS1 genes. They are capable of late viral protein synthesis and are superior to Δγ134.5 HSVs in oncolytic activity. The interferon (IFN) response limits efficient HSV gene expression and replication. HCMV TRS1 and IRS1 restore one γ134.5 gene function: evasion of IFN-inducible protein kinase R, allowing late viral protein synthesis. Here we show that, unlike wild-type HSV, the chimeric HSV do not restore another γ134.5 function, the suppression of early IFN signaling mediated by IFN regulatory factor 3 (IRF3).

TGF-ß2 suppresses macrophage cytokine production and mucosal inflammatory responses in the developing intestine.

BACKGROUND & AIMS: Premature neonates are predisposed to necrotizing enterocolitis (NEC), an idiopathic, inflammatory bowel necrosis. We investigated whether NEC occurs in the preterm intestine due to incomplete noninflammatory differentiation of intestinal macrophages, which increases the risk of a severe mucosal inflammatory response to bacterial products. METHODS: We compared inflammatory properties of human/murine fetal, neonatal, and adult intestinal macrophages. To investigate gut-specific macrophage differentiation, we next treated monocyte-derived macrophages with conditioned media from explanted human fetal and adult intestinal tissues. Transforming growth factor-ß (TGF-ß) expression and bioactivity were measured in fetal/adult intestine and in NEC. Finally, we used wild-type and transgenic mice to investigate the effects of deficient TGF-ß signaling on NEC-like inflammatory mucosal injury. RESULTS: Intestinal macrophages in the human preterm intestine (fetus/premature neonate), but not in full-term neonates and adults, expressed inflammatory cytokines. Macrophage cytokine production was suppressed in the developing intestine by TGF-ß, particularly the TGF-ß(2) isoform. NEC was associated with decreased tissue expression of TGF-ß(2) and decreased TGF-ß bioactivity. In mice, disruption of TGF-ß signaling worsened NEC-like inflammatory mucosal injury, whereas enteral supplementation with recombinant TGF-ß(2) was protective. CONCLUSIONS: Intestinal macrophages progressively acquire a noninflammatory profile during gestational development. TGF-ß, particularly the TGF-ß(2) isoform, suppresses macrophage inflammatory responses in the developing intestine and protects against inflammatory mucosal injury. Enterally administered TGF-ß(2) protected mice from experimental NEC-like injury.

Human cytomegalovirus induces TGF-ß1 activation in renal tubular epithelial cells after epithelial-to-mesenchymal transition.

Human cytomegalovirus (HCMV) infection is associated epidemiologically with poor outcome of renal allografts due to mechanisms which remain largely undefined. Transforming growth factor-ß1 (TGF-ß1), a potent fibrogenic cytokine, is more abundant in rejecting renal allografts that are infected with either HCMV or rat CMV as compared to uninfected, rejecting grafts. TGF-ß1 induces renal fibrosis via epithelial-to-mesenchymal transition (EMT) of renal epithelial cells, a process by which epithelial cells acquire mesenchymal characteristics and a migratory phenotype, and secrete molecules associated with extracellular matrix deposition and remodeling. We report that human renal tubular epithelial cells infected in vitro with HCMV and exposed to TGF-ß1 underwent morphologic and transcriptional changes of EMT, similar to uninfected cells. HCMV infected cells after EMT also activated extracellular latent TGF-ß1 via induction of MMP-2. Renal epithelial cells transiently transfected with only the HCMV IE1 or IE2 open reading frames and stimulated to undergo EMT also induced TGF-ß1 activation associated with MMP-2 production, suggesting a role for these viral gene products in MMP-2 production. Consistent with the function of these immediate early gene products, the antiviral agents ganciclovir and foscarnet did not inhibit TGF-ß1 production after EMT by HCMV infected cells. These results indicate that HCMV infected renal tubular epithelial cells can undergo EMT after exposure to TGF-ß1, similar to uninfected renal epithelial cells, but that HCMV infection by inducing active TGF-ß1 may potentiate renal fibrosis. Our findings provide in vitro evidence for a pathogenic mechanism that could explain the clinical association between HCMV infection, TGF-ß1, and adverse renal allograft outcome.

Correlation and clinical utility of pp65 antigenemia and quantitative polymerase chain reaction assays for detection of cytomegalovirus in pediatric renal transplant patients.

qPCR and pp65 antigenemia assays are used to monitor CMV infection in renal transplant recipients, but correlation of assays in a pediatric population has not been evaluated. Paired CMV real-time qPCR and pp65 antigenemia tests from 882 blood samples collected from 115 pediatric renal transplant recipients were analyzed in this retrospective cohort study for the strength of association and clinical correlates. The assays correlated well in detecting infection (κ = 0.61). Higher qPCR values were demonstrated with increasing levels of antigenemia (p < 0.01). Discordant test results were associated with antiviral treatment (OR 4.33, p < 0.01) and low-level viremia, with odds of concordance increasing at higher qPCR values (OR 3.67, p < 0.01), and no discordance occurring above 8500 genomic equivalents/mL. Among discordant samples, neither test preceded the other in detecting initial infection or in returning to negative while on treatment. Only two cases of disease occurred during the two-yr study period. With strong agreement in the detection of CMV infection, either qPCR or pp65 antigenemia assays can be used effectively for monitoring pediatric renal transplant patients for both detection and resolution of infection.

Antibody Titers Against Human Cytomegalovirus gM/gN and gB Among Pregnant Women and Their Infants.

Congenital CMV (cCMV) infection can affect infants born to mothers with preconceptional seroimmunity. To prevent cCMV due to nonprimary maternal infection, vaccines eliciting responses exceeding natural immunity may be required. Anti-gM/gN antibodies have neutralizing capacity in-vitro and in animal models, but anti-gM/gN antibodies have not been characterized among seroimmune pregnant women. Paired maternal and infant cord sera from 92 CMV seropositive mothers and their full-term or preterm infants were tested for anti-gM/gN antibody titers in comparison with anti-gB titers and neutralizing activity. Anti-gM/gN titers were significantly lower than anti-gB titers for all groups and did not correlate with serum neutralizing capacity. Further study is needed to determine if higher anti-gM/gN antibody titers might enhance serum neutralizing capacity among seropositive adults.

Cerebrospinal Fluid Analysis for Viruses by Metagenomic Next-Generation Sequencing in Pediatric Encephalitis: Not Yet Ready for Prime Time?

BACKGROUND: Metagenomic next-generation sequencing offers an unbiased approach to identifying viral pathogens in cerebrospinal fluid of patients with meningoencephalitis of unknown etiology. METHODS: In an 11-month case series, we investigated the use of cerebrospinal fluid metagenomic next-generation sequencing to diagnose viral infections among pediatric hospitalized patients presenting with encephalitis or meningoencephalitis of unknown etiology. Cerebrospinal fluid from patients with known enterovirus meningitis were included as positive controls. Cerebrospinal fluid from patients with primary intracranial hypertension were included to serve as controls without known infections. RESULTS: Cerebrospinal fluid metagenomic next-generation sequencing was performed for 37 patients. Among 27 patients with encephalitis or meningoencephalitis, 4 were later diagnosed with viral encephalitis, 6 had non-central nervous system infections with central nervous system manifestations, 6 had no positive diagnostic tests, and 11 were found to have a noninfectious diagnosis. Metagenomic next-generation sequencing identified West Nile virus (WNV) in the cerebrospinal fluid of 1 immunocompromised patient. Among the 4 patients with known enterovirus meningitis, metagenomic next-generation sequencing correctly identified enteroviruses and characterized the viral genotype. No viral sequences were detected in the cerebrospinal fluid of patients with primary intracranial hypertension. Metagenomic next-generation sequencing also identified sequences of nonpathogenic torque Teno virus in cerebrospinal fluid specimens from 13 patients. CONCLUSIONS: Our results showed viral detection by cerebrospinal fluid metagenomic next-generation sequencing only in 1 immunocompromised patient and did not offer a diagnostic advantage over conventional testing. Viral phylogenetic characterization by metagenomic next-generation sequencing could be used in epidemiologic investigations of some viral pathogens, such as enteroviruses. The finding of torque Teno viruses in cerebrospinal fluid by metagenomic next-generation sequencing is of unknown significance but may merit further exploration for a possible association with noninfectious central nervous system disorders.