Whole-genome approach to assessing human cytomegalovirus dynamics in transplant patients undergoing antiviral therapy

Human cytomegalovirus (HCMV) is the most frequent cause of opportunistic viral infection following transplantation. Viral factors of potential clinical importance include the selection of mutants resistant to antiviral drugs and the occurrence of infections involving multiple HCMV strains. These factors are typically addressed by analyzing relevant HCMV genes by PCR and Sanger sequencing, which involves independent assays of limited sensitivity. To assess the dynamics of viral populations with high sensitivity, we applied high-throughput sequencing coupled with HCMV-adapted target enrichment to samples collected longitudinally from 11 transplant recipients (solid organ, n=9, and allogeneic hematopoietic stem cell, n=2). Only the latter presented multiple-strain infections. Four cases presented resistance mutations (n=6), two (A594V and L595S) at high (100%) and four (V715M, 32 V781I, A809V and T838A) at low (<25%) frequency. One allogeneic hematopoietic stem cell transplant recipient presented up to four resistance mutations, each at low frequency. The use of high throughput sequencing to monitor mutations and strain composition in people at risk of HCMV disease is of potential value in helping clinicians implement the most appropriate therapy

Human cytomegalovirus genomes sequenced directly from clinical material: variation, multiple-strain infection, recombination and gene loss

The genomic characteristics of human cytomegalovirus (HCMV) strains sequenced directly from clinical pathology samples were investigated, focusing on variation, multiple-strain infection, recombination, and gene loss. A total of 207 datasets generated in this and previous studies using target enrichment and high-throughput sequencing were analyzed, in the process enabling the determination of genome sequences for 91 strains. Key findings were that (i) it is important to monitor the quality of sequencing libraries in investigating variation; (ii) many recombinant strains have been transmitted during HCMV evolution, and some have apparently survived for thousands of years without further recombination; (iii) mutants with nonfunctional genes (pseudogenes) have been circulating and recombining for long periods and can cause congenital infection and resulting clinical sequelae; and (iv) intrahost variation in single-strain infections is much less than that in multiple-strain infections. Future population-based studies are likely to continue illuminating the evolution, epidemiology, and pathogenesis of HCMV

Molecular phylogeny of a novel human adenovirus type 8 strain causing a prolonged, multi-state keratoconjunctivitis epidemic in Germany

The German infectious disease surveillance system revealed an increase of epidemic keratoconjunctivitis (EKC) from an average of 320 cases/year (2001 to 2010) up to 2146 and 1986 cases in 2012 and 2013, respectively. From November 2011 until December 2013 (epidemic period) 85% of typed isolates were human adenovirus type 8 (HAdV-D8), whereas only low level circulation (19%) of HAdV-D8 was observed outside the epidemic period. In order to investigate whether a novel monophyletic HAdV-D8 strain prevailed during the epidemic period, complete genomic sequences of 23 HAdV-D8 isolates were generated by deep sequencing and analyzed phylogenetically. For comparison, eight HAdV-D8 isolates from outside the epidemic period were sequenced. HAdV-D8 isolates of the epidemic period had a very high sequence identity of at least 99.9% and formed a monophyletic cluster with two subclusters. A single outlier was closely related to HAdV-D8 strains isolated prior to the epidemic period. Circulation of the epidemic strain was detected as early as 2010 but not after the epidemic period in 2014. In conclusion, molecular phylogeny of complete genomic sequences proved a monophyletic HAdV-D8 epidemic. However, co-circulation of other HAdV types as well as better reporting may have contributed to the huge increase of reported cases

Varicella Zoster Virus Meningitis in a Young Immunocompetent Adult without Rash: A Misleading Clinical Presentation

Meningitis caused by varicella zoster virus (VZV) is rare in healthy population. Predominantly immunocompromised patients are affected by reactivation of this virus with primary clinical features of rash and neurological symptoms. Here we report a young otherwise healthy man diagnosed with a VZV meningitis without rash. He complained of acute headache, nausea, and vomiting. The clinical examination did not show any neurological deficits or rash. Cerebrospinal fluid (CSF) analysis revealed a high leukocyte cell count of 1720 cells/µL and an elevated total protein of 1460 mg/L misleadingly indicating a bacterial infection. Further CSF analyses, including polymerase chain reaction (PCR) and detection of intrathecal synthesis of antibodies, showed a VZV infection. Clinical and CSF follow-up examinations proved the successful antiviral treatment. In conclusion, even young immunocompetent patients without rash might present with VZV meningitis. CSF examination is a key procedure in the diagnosis of CNS infections but in rare cases the standard values cell count and total protein might misleadingly indicate a bacterial infection. Thus, virological analyses should be considered even when a bacterial infection is suspected

A case series of children and young people admitted to a tertiary care hospital in Germany with COVID-19

Background!#!While our knowledge about COVID-19 in adults has rapidly increased, data on the course of disease and outcome in children with different comorbidities is still limited.!##!Methods!#!Prospective, observational study at a tertiary care children's hospital in southern Germany. Clinical and virology data from all paediatric patients admitted with SARS-CoV-2 infection at our hospital were prospectively assessed.!##!Results!#!Between March and November 2020, 14 patients were admitted with COVID-19. One patient was admitted a second time with COVID-19 6 months after initial disease. Among seven patients with severe underlying comorbidities, three developed multisystem inflammatory syndrome (MIS-C), two were admitted to the paediatric intensive care unit. One patient needed invasive ventilation. Another patient died shortly after discharge of COVID-19-related complications.!##!Conclusions!#!While COVID-19 generally causes mild disease in children, severe respiratory illness and MIS-C occur, in some cases with fatal outcome. Children with underlying diseases might be at special risk for severe disease

Molecular Evolution of Human Adenovirus (HAdV) Species C

Currently, 88 different Human Adenovirus (HAdV) types are grouped into seven HAdV species A to G. Most types (57) belong to species HAdV-D. Recombination between capsid genes (hexon, penton and fiber) is the main factor contributing to the diversity in species HAdV-D. Noteworthy, species HAdV-C contains so far only five types, although species HAdV-C is highly prevalent and clinically significant in immunosuppressed patients. Therefore, the evolution of species HAdV-C was studied by generating 51 complete genome sequences from circulating strains. Clustering of the whole genome HAdV-C sequences confirmed classical typing results (fifteen HAdV-C1, thirty HAdV-C2, four HAdV-C5, two HAdV-C6). However, two HAdV-C2 strains had a novel penton base sequence and thus were re-labeled as the novel type HAdV-C89. Fiber and early gene region 3 (E3) sequences clustered always with the corresponding prototype sequence but clustering of the E4 region indicated recombination events in 26 out of the 51 sequenced specimens. Recombination of the E1 gene region was detected in 16 circulating strains. As early gene region sequences are not considered in the type definition of HAdVs, evolution of HAdV-C remains on the subtype level. Nonetheless, recombination of the E1 and E4 gene regions may influence the virulence of HAdV-C strains.Peer Reviewe

Comparison of the performance of the Panther Fusion respiratory virus panel to R-Gene and laboratory developed tests for diagnostic and hygiene screening specimens from the upper and lower respiratory tract

Introduction. Diagnosis of acute respiratory infections (ARIs) can be facilitated by the Panther Fusion (PF) automatic, random access PCR system for the detection of influenzavirus A (Flu A) and B (Flu B), parainfluenzavirus (Paraflu), respiratory syncytial virus (RSV), human metapneumovirus (hMPV), rhinovirus (RV) and human adenovirus (AdV) in nasopharyngeal swabs. Aim. To evaluate the performance of PF in comparison with established methods, including subsets of (1) lower respiratory tract (LRT) specimens and (2) upper respiratory tract (URT) hygiene screening specimens of patients without ARI symptoms. Methodology. The performance characteristics of PF were compared with bioMerieux R-Gene and laboratory-developed PCR tests (LDTs). Overall, 1544 specimens with 6658 individual diagnostic requests were analysed. Results. The overall concordances of PF and LDTs for Flu A, Flu B and AdV were 98.4, 99.9 and 96.1%, respectively; by re-testing of discrepant specimens concordances increased to 99.4, 99.9 and 98.0%, respectively. Initial concordances of PF and R--Gene assays for RSV, Paraflu, hMPV and RV were 98.4, 96.3, 99.3 and 96.0%, respectively, and retest concordances were 99.7, 97.9, 99.9 and 98.9%, respectively. No differences to the overall performance were found for the subgroups of LRT and hygiene screening specimens. PCR cycle threshold (Ct) values correlated very well between methods, indicating that a semi-quantitative diagnostic approach using Ct values (e.g. highly vs. weakly positive) could augment the diagnostic information. Conclusion. PF performed similar to R-Gene and LDTs not only for its intended use but also for LRT and hygiene screening specimens with shorter hands-on and turnaround times

Cerebrospinal fluid features in adults with enteroviral nervous system infection

Objectives: The aim of this study was to investigate the clinical and laboratory features of adults with nervous system infections caused by enteroviruses, with special emphasis on cerebrospinal fluid (CSF). Methods: The data of 46 patients who were PCR-positive for enteroviruses in the CSF between 2002 and 2017 were evaluated. Results: Meningitis was the most common clinical manifestation (89%), followed by encephalitis (7%) and isolated cranial nerve involvement (4%). Twenty percent of patients reported a sudden onset of severe headache that led to the initial suspected diagnosis of subarachnoid haemorrhage. General signs of infection, such as fever, elevated C-reactive protein, and an elevated white blood cell count, were found in only 61%. Most patients exhibited consistent inflammatory CSF changes, with elevated cell counts (85%) and blood–CSF barrier dysfunction (83%). Patients with normal CSF cell counts were significantly older, less frequently presented with meningitis, and exhibited lower peripheral white blood cell counts. Sequencing revealed species Enterovirus B in all patients, with most sequences related to echovirus 30. Conclusions: The absence of CSF pleocytosis, isolated cranial nerve involvement, and only infrequent general signs of infection may impede the diagnosis of enteroviral nervous system infections. A thorough CSF analysis including PCR is essential for a reliable diagnosis. Keywords: Cerebrospinal fluid, Cranial nerve, Encephalitis, Enterovirus, Headache, Meningiti