Evaluating assembly and variant calling software for strain-resolved analysis of large DNA viruses.

Infection with human cytomegalovirus (HCMV) can cause severe complications in immunocompromised individuals and congenitally infected children. Characterizing heterogeneous viral populations and their evolution by high-throughput sequencing of clinical specimens requires the accurate assembly of individual strains or sequence variants and suitable variant calling methods. However, the performance of most methods has not been assessed for populations composed of low divergent viral strains with large genomes, such as HCMV. In an extensive benchmarking study, we evaluated 15 assemblers and 6 variant callers on 10 lab-generated benchmark data sets created with two different library preparation protocols, to identify best practices and challenges for analyzing such data. Most assemblers, especially metaSPAdes and IVA, performed well across a range of metrics in recovering abundant strains. However, only one, Savage, recovered low abundant strains and in a highly fragmented manner. Two variant callers, LoFreq and VarScan2, excelled across all strain abundances. Both shared a large fraction of false positive variant calls, which were strongly enriched in T to G changes in a 'G.G' context. The magnitude of this context-dependent systematic error is linked to the experimental protocol. We provide all benchmarking data, results and the entire benchmarking workflow named QuasiModo, Quasispecies Metric determination on omics, under the GNU General Public License v3.0 (https://github.com/hzi-bifo/Quasimodo), to enable full reproducibility and further benchmarking on these and other data.

Infection control of COVID-19 in pediatric tertiary care hospitals: challenges and implications for future pandemics.

BACKGROUND: More than 2 years into the COVID-19 pandemic, SARS-CoV-2 still impacts children's health and the management of pediatric hospitals. However, it is unclear which hygiene and infection control measures are effective and useful for pediatric hospitals. Here, we report infection control measures implemented at a tertiary care children's hospital. We evaluated frequency of SARS-CoV-2 detection in admitted patients, in-hospital transmission and infection related findings. Furthermore, we aimed to capture perspectives of health-care workers and caregivers on effectiveness and burden of infection control measures. Knowledge gained can inform management of the ongoing and future pandemics. METHODS: We designed a retrospective observational study and survey at a pediatric tertiary care referral center. Local infection control measures and respective guidelines regarding COVID-19 were reviewed. Three thousand seven hundred sixteen children under 18 years were tested for SARS-CoV-2 at the University Children's Hospital Tuebingen and data on SARS-CoV-2 transmission were retrieved from internal records. Two surveys were conducted among 219 staff members and 229 caregivers. RESULTS: Local infection control measures comprised the formation of a task force, triage, protective hygiene measures and an adaptable SARS-CoV-2 test strategy. Between January 2020 and March 2021, SARS-CoV-2 infection was detected in 37 children presenting to our hospital, 21 of these were admitted. One hospital-acquired infection occurred. About 90% of health-care staff perceived the majority of measures as effective and appropriate. However, visitor restrictions and cancellation of scheduled treatments were perceived least effective by hospital staff and as a particular burden for patients and their caregivers. Visits at the pediatric emergency department significantly decreased during the pandemic. We drafted a pandemic action plan by ranking infection control measures according to local transmission stages. CONCLUSIONS: SARS-CoV-2 infection control measures implemented in our tertiary care children's hospital were evaluated by health-care workers as mostly effective and appropriate. In particular, good communication, transparency of decision-making as well as universal masking and infection screening were assessed as successful measures of infection control management. Visitor restrictions and cancellation of routine appointments, in contrast, were perceived as a particular burden on patient care and should be avoided. An established pandemic action plan may guide children's hospitals in the future.

Acute interstitial nephritis due to flecainide therapy in the 38(th) week of pregnancy.

BACKGROUND: Acute interstitial nephritis (AIN) represents a frequent cause of acute kidney injury. While many etiologies of AIN have been recognized, the majority (60-70 %) are due to allergic reactions or drug exposure. Many different classes of drugs and several agents within a class can cause drug induced AIN. Flecainide, a class Ic antiarrhythmic drug, had thus far not been associated with the occurrence of AIN. CASE PRESENTATION: Here we describe a case of biopsy proven AIN after flecainide therapy in a pregnant patient. The 24-year old Caucasian woman was admitted to our university hospital for a planned c-section. She had been put on flecainide at a dose of 200 mg/d for supraventricular tachyarrhythmia of the fetus ten days earlier. The only fleaainide drug level was obtained 24 h after the last dose. At this time point the serum level was still in the therapeutic range (392 ng/mL). After hospital admission the patient underwent uneventful c-section and delivered a 3095 g baby girl with mild insufficiency of the tricuspid valve. In the hours following the c-section, a single dose of the non-steroidal anti-inflammatory drug (NSAID) ibuprofen (600 mg) as well as single dose of diclofenac (100 mg) was administered. Within 5 days after c-section her baseline creatinine of 30 µmol/L increased to 277 µmol/L. The serum creatinine continued to rise to 411 µmol/L on hospital day # 7. On renal ultrasound kidneys were enlarged and swollen. Urinary sediment at this point only revealed slight proteinuria (506 mg/g creatinine). A renal biopsy was performed showing acute interstitial nephritis. Within four days the renal function improved after discontinuation of flecainide and NSAIDs even without steroid therapy and the patient was discharged with a creatinine of 88 µmol/L after 13 days in the hospital. CONCLUSION: This case suggests that flecainide, at least in combination with NSAIDs, can cause AIN.

Virus-associated hemophagocytic syndrome as a major contributor to death in patients with 2009 influenza A (H1N1) infection.

INTRODUCTION: Virus-associated hemophagocytic syndrome (VAHS) is a severe complication of various viral infections often resulting in multiorgan failure and death. The purpose of this study was to describe baseline characteristics, development of VAHS, related treatments and associated mortality rate of consecutive critically ill patients with confirmed 2009 influenza A (H1N1) infection and respiratory failure. METHODS: We conducted a prospective observational study of 25 critically ill patients with 2009 influenza A (H1N1) infection at a single-center intensive care unit in Germany between 5 October 2009 and 4 January 2010. Demographic data, comorbidities, diagnosis of VAHS, illness progression, treatments and survival data were collected. The primary outcome measure was the development of VAHS and related mortality. Secondary outcome variables included duration of mechanical ventilation, support of extracorporeal membrane oxygenation and duration of viral shedding. RESULTS: VAHS developed in 9 (36%) of 25 critically ill patients with confirmed 2009 influenza A (H1N1) infection, and 8 (89%) of them died. In contrast, the mortality rate in the remaining 16 patients without VAHS was 25% (P = 0.004 for the survival difference in patients with or without VAHS by log-rank analysis). The patients were relatively young (median age, 45 years; interquartile range (IQR), 35 to 56 years of age); however, 18 patients (72%) presented with one or more risk factors for a severe course of illness. All 25 patients received mechanical ventilation for severe acute respiratory distress syndrome and refractory hypoxemia, with a median duration of mechanical ventilation of 19 days (IQR, 13 to 26 days). An additional 17 patients (68%) required extracorporeal membrane oxygenation for a median of 10 days (IQR, 6 to 19 days). CONCLUSIONS: The findings of this study raise the possibility that VAHS may be a frequent complication of severe 2009 influenza A (H1N1) infection and represents an important contributor to multiorgan failure and death.

Antibody Response against SARS-CoV-2 and Seasonal Coronaviruses in Nonhospitalized COVID-19 Patients.

The majority of infections with SARS-CoV-2 are asymptomatic or mild without the necessity of hospitalization. It is of importance to reveal if these patients develop an antibody response against SARS-CoV-2 and to define which antibodies confer virus neutralization. We conducted a comprehensive serological survey of 49 patients with a mild course of disease and quantified neutralizing antibody responses against a clinical SARS-CoV-2 isolate employing human cells as targets. Four patients (8%), even though symptomatic, did not develop antibodies against SARS-CoV-2, and two other patients (4%) were positive in only one of the six serological assays employed. For the remaining 88%, antibody response against the S protein correlated with serum neutralization whereas antibodies against the nucleocapsid were poor predictors of virus neutralization. None of the sera enhanced infection of human cells with SARS-CoV-2 at any dilution, arguing against antibody-dependent enhancement of infection in our system. Regarding neutralization, only six patients (12%) could be classified as high neutralizers. Furthermore, sera from several individuals with fairly high antibody levels had only poor neutralizing activity. In addition, employing a novel serological Western blot system to characterize antibody responses against seasonal coronaviruses, we found that antibodies against the seasonal coronavirus 229E might contribute to SARS-CoV-2 neutralization. Altogether, we show that there is a wide breadth of antibody responses against SARS-CoV-2 in patients that differentially correlate with virus neutralization. This highlights the difficulty to define reliable surrogate markers for immunity against SARS-CoV-2.IMPORTANCE There is strong interest in the nature of the neutralizing antibody response against SARS-CoV-2 in infected individuals. For vaccine development, it is especially important which antibodies confer protection against SARS-CoV-2, if there is a phenomenon called antibody-dependent enhancement (ADE) of infection, and if there is cross-protection by antibodies directed against seasonal coronaviruses. We addressed these questions and found in accordance with other studies that neutralization is mediated mainly by antibodies directed against the spike protein of SARS-CoV-2 in general and the receptor binding site in particular. In our test system, utilizing human cells for infection experiments, we did not detect ADE. However, using a novel diagnostic test we found that antibodies against the coronavirus 229E might be involved in cross-protection to SARS-CoV-2.

Histopathological and clinical findings in renal transplants with Banff type II and III acute cellular rejection without tubulointerstitial infiltrates.

According to the Banff guidelines for renal transplants, pure endothelialitis without any tubulointerstitial infiltrates (with the Banff components v ≥ 1, i0, t0) has to be called acute cellular rejection (ACR). The pathophysiology of this rare lesion abbreviated as v_only is currently unclear, as well as its clinical, serological, and prognostic implications. Therefore, we conducted this retrospective comparative study. We compared all 23 biopsies with v_only from Hannover Medical School between 2003 and 2010 with 23 matched biopsies with the Banff components v ≥ 1, i ≥ 1, and t ≥ 1 (v_plus) and 23 biopsies with v0, i0, and t0 (v0i0t0). Serological (available in 10, 11, and 14 patients, respectively), histological, and clinical data were compared. Of all biopsies, 0.4 % had findings of v_only. v_only, v_plus, and v0i0t0 only showed minimal differences in the Banff components apart from the cohort-defining components. Endothelialitis in v_only more frequently involved the arcuate arteries than the smaller preglomerular vessels compared to v_plus and vice versa. Combining histopathological data and serological data, v_only more frequently showed criteria for acute humoral rejection than v0i0t0 (albeit not persistent after the Bonferroni-Holm correction in pairwise comparisons), while there was no difference between v_only and v_plus. No difference could be demonstrated regarding clinical presentation at biopsy or outcome. Our results show minimal differences regarding clinical presentation, outcome, and histological features between v_only and v_plus. Patients with v_only should be thoroughly investigated for evidence of acute humoral rejection.

Glomerular mRNA expression of prothrombotic and antithrombotic factors in renal transplants with thrombotic microangiopathy.

BACKGROUND: Thrombotic microangiopathy (TMA) in renal transplants (rTx-TMA) is a serious complication and is usually either recurrent TMA (RecTMA) due to humoral rejection (HR-TMA) or due to calcineurin inhibitor toxicity (CNI-TMA). Although the triggers are known, our knowledge about the thrombogenic transcriptome changes in the microvessels is rudimentary. METHODS: We examined the expression of several prothrombotic and antithrombotic genes in 25 biopsies with rTx-TMA (6 RecTMA, 9 HR-TMA, and 10 CNI-TMA) and 8 controls. RNA from microdissected glomeruli of paraffin-embedded tissue was isolated and mRNA transcripts were quantified with real-time polymerase chain reaction after preamplification. Results were correlated with clinicopathologic parameters. RESULTS: Glomerular mRNA expression of KLF2, KLF4, and tPA was lower and that of PAI-1 was higher in rTx-TMA than in the controls. Glomerular mRNA expression of KLF2 and KLF4 correlated with that of tPA and inversely with that of PAI-1 in rTx-TMA. The mRNA expression of complement regulators CD46 and CD59 were higher in rTx-TMA than in the controls. Only in HR-TMA were glomerular ADAMTS13 and CD55 down-regulated. CONCLUSIONS: The glomerular capillary bed seems to contribute to all subtypes of rTx-TMA by down-regulation of the endothelial transcription factors KLF2 and KLF4, indicating dedifferentiation with subsequent up-regulation of PAI-1 and down-regulation of tPA, resulting in inhibition of local fibrinolysis. Decreased glomerular expression of ADAMTS13 and CD55 could be an additional pathway toward microthrombosis exclusively in HR-TMA.