Database on natural polymorphisms and resistance-related non-synonymous mutations in thymidine kinase and DNA polymerase genes of herpes simplex virus types 1 and 2.

The use of genotypic resistance testing of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) is increasing because the rapid availability of results significantly improves the treatment of severe infections, especially in immunocompromised patients. However, an essential precondition is a broad knowledge of natural polymorphisms and resistance-associated mutations in the thymidine kinase (TK) and DNA polymerase (pol) genes, of which the DNA polymerase (Pol) enzyme is targeted by the highly effective antiviral drugs in clinical use. Thus, this review presents a database of all non-synonymous mutations of TK and DNA pol genes of HSV-1 and HSV-2 whose association with resistance or natural gene polymorphism has been clarified by phenotypic and/or functional assays. In addition, the laboratory methods for verifying natural polymorphisms or resistance mutations are summarized. This database can help considerably to facilitate the interpretation of genotypic resistance findings in clinical HSV-1 and HSV-2 strains.

Hyperdiploidy with 58-66 chromosomes in childhood B-acute lymphoblastic leukemia is highly curable: 58951 CLG-EORTC results.

The aim of our study was to analyze the factors contributing to heterogeneity of prognosis in patients with hyperdiploidy>50 chromosomes (HD>50), a group of B-cell precursor acute lymphoblastic leukemia with favorable outcome. The 541 HD>50 patients registered prospectively in the 58951 European Organisation for Research and Treatment of Cancer (EORTC) Children's Leukemia Group (CLG) trial, identified by karyotype (446 patients) and by DNA index (DI) (490 patients), had a 6-year event-free survival (EFS) of 89.0% (standard error [SE] = 1.5%) and a 6-year overall survival (OS) of 95.9% (SE = 0.9%). The strongest prognostic factor was the modal number of chromosomes (MNC): the 6-year EFS of 51-53, 54-57, and 58-66 MNC groups were 80%, 89%, and 99%, respectively (P < .0001). Ploidy assessed by DI was also a favorable factor: the higher the DI, the better the outcome. The 6-year EFS of the 3 subgroups of DI < 1.16/≥1.16-<1.24/≥1.24 were 83%, 90%, and 95%, respectively (P = .009). All usual combinations of trisomies (chromosomes 4, 10, 17, 18) were significant favorable factors but had lower EFS when MNC was lower than 58. In multivariate analysis, MNC remained the strongest factor. Consequently, the best indicator for excellent outcome was ploidy assessed by karyotype because patients with 58-66 chromosomes stood every chance of being cured (OS of 100% at 6-year follow-up) with less-intensive therapy. This trial was registered at www.clinicaltrials.gov as #NCT00003728. Registered: http://www.eortc.org/, http://clinicaltrials.gov/show/NCT00003728.

Severe leptospirosis complicated by Epstein-Barr Virus reactivation.

INTRODUCTION: Weil's disease is a severe, potentially fatal illness following Leptospira interrogans infection. The reported case of a patient suffering from acute renal failure, jaundice, thrombocytopenia, rhabdomyolysis and encephalitis syndrome highlights the clinical challenge in reference to Weil syndrome complicated by Epstein-Barr Virus (EBV) reactivation. MATERIALS AND METHODS: The diagnosis of leptospirosis was performed using four different diagnostic methods. Sera were analyzed with an in-house IgM and IgG enzyme-linked immunosorbent assay (ELISA) and indirect haemagglutination assay (IHA). Microscopic agglutination test (MAT) was done using 17 reference strains comprising 14 serogroups and 17 serovars. Polyvalent EBV-IgG analysis, EBV-IgG/IgM/IgA western blot analysis as well as quantitative EBV polymerase chain reaction (PCR) were performed. RESULTS: Leptospira IHA showed an initial titer of 1:640 (cut-off 1:320), leptospiral IgG was negative, but IgM was positive. MAT was negative at that time for all 17 strains analyzed. One week later, leptospirosis IHA titer increased to 1:20,480. Leptospiral IgG was now positive, -IgM remained positive and urine was tested negative for leptospiral DNA. The MAT showed positive results for L. interrogans serovar Bataviae, serovar Copenhageni, serovar Pyrogenes and L. borgpetersenii serovar Serjoe. During follow-up examinations, both the leptospiral IgM and IgG remained positive and MAT showed positive results for L. interrogans of different serovars. EBV IgA immunoblot taken at admission was positive for VCA-p18, quantitative EBV-PCR showed an EBV viral load of 2.8E3 copies/ml indicating acute EBV-reactivation. CONCLUSION: Leptospirosis represents a neglected and re-emerging disease which is difficult to diagnose since Leptospira-PCR from whole blood or urine is frequently negative in the case of early empiric antibiotic treatment. EBV-reactivation might represent a severe complication in Weil's disease which potentially aggravates clinical manifestations of leptospirosis including hepatitis, nephritis, and rhabdomyolysis. Thus, there might be a need for peripheral blood EBV-PCR and EBV blotting in patients suffering from complicated Weil syndrome, also in terms of the choice of antibiotic treatment.

A phase 1 trial of oncolytic HSV-1, G207, given in combination with radiation for recurrent GBM demonstrates safety and radiographic responses.

G207, a mutant herpes simplex virus (HSV) type 1, is safe when inoculated into recurrent malignant glioma. We conducted a phase 1 trial of G207 to demonstrate the safety of stereotactic intratumoral administration when given 24 hours prior to a single 5 Gy radiation dose in patients with recurrent malignant glioma. Nine patients with progressive, recurrent malignant glioma despite standard therapy were included. Patients received one dose of G207 stereotactically inoculated into the multiple sites of the enhancing tumor margin and were then treated focally with 5 Gy radiation. Treatment was well tolerated, and no patient developed HSV encephalitis. The median interval between initial diagnosis and G207 inoculation was 18 months (mean: 23 months; range: 11-51 months). Six of the nine patients had stable disease or partial response for at least one time point. Three instances of marked radiographic response to treatment occurred. The median survival time from G207 inoculation until death was 7.5 months (95% confidence interval: 3.0-12.7). In conclusion, this study showed the safety and the potential for clinical response of single-dose oncolytic HSV therapy augmented with radiation in the treatment of malignant glioma patients. Additional studies with oncolytic HSV such as G207 in the treatment of human glioma are recommended.

Intensive consolidation therapy compared with standard consolidation and maintenance therapy for adults with acute myeloid leukaemia aged between 46 and 60 years: final results of the randomized phase III study (AML 8B) of the European Organization for Research and Treatment of Cancer (EORTC) and the Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto (GIMEMA) Leukemia Cooperative Groups.

The most effective post-remission treatment to maintain complete remission (CR) in adults aged between 46 and 60 years with acute myeloid leukaemia (AML) is uncertain. Previously untreated patients with AML in CR after induction chemotherapy with daunorubicin and cytarabine were randomized between two intensive courses of consolidation therapy containing high-dose cytarabine, combined with amsacrine or daunorubicin and a standard consolidation and maintenance therapy containing standard dose cytarabine and daunorubicin. One hundred fifty-eight CR patients were assigned to the intensive group and 157 patients to the standard group. After a median follow-up of 7.5 years, the 4-year survival rate was 32 % in the intensive group versus 34 % in the standard group (P = 0.29). In the intensive group, the 4-year relapse incidence was lower than in the standard group: 55 and 75 %, respectively (P = 0.0003), whereas treatment-related mortality incidence was higher: 22 versus 3 % (P < 0.0001). Two intensive consolidation courses containing high-dose cytarabine as post-remission treatment in patients with AML aged between 46 and 60 years old did not translate in better long-term outcome despite a 20 % lower relapse incidence. Better supportive care and prevention of treatment-related complications may improve the overall survival after intensified post-remission therapy in this age group.

New Safety Aspects in Corneal Donation-Studies on SARS-CoV-2-Positive Corneal Donors.

In the tissue donation field, to prevent pathogen transmission, all donors are screened by postmortem swabs for SARS-CoV-2 using qRT-PCR. Corneas from donors who tested positive for SARS-CoV-2 were subjected to further investigations. Corneal transplants and culture medium from positive donors were cultured under appropriate safety conditions for further analyses. Cornea tissue samples, including sclera/limbus/cornea, and culture media were taken at different time points for testing for SARS-CoV-2 using qRT-PCR, immunohistochemistry (IHC) and subgenomic RNA (sgRNA) analysis. Between January and May 2021, in four donors with initial negative premortem rapid tests, SARS-CoV-2 was detected post-mortem using qRT-PCR. In these cases, SARS-CoV-2 was observed at the beginning of cultivation in both tissue and culture medium using qRT-PCR and IHC. The virus was mainly localized in the limbus epithelial cells, with a stable detection level. Premortem rapid tests are potentially insufficient to exclude SARS-CoV-2 infection in corneal donors. While, for SARS-CoV-2, the risk of infection via transplants is considered low, a residual risk remains for presymptomatic new infections. However, our investigations provide the first indications that, with organ cultures, the risk of virus transmission is minimized due to the longer minimum culture period.

[West-Nile-Virus Infection acquired in Germany in a Kidney Transplant Recipient]. / In Deutschland erworbene West-Nil-Virusinfektion bei einem nierentransplantierten Patienten.

BACKGROUND: West-Nile-Virus (WNV) is a widely distributed flavivirus that is mainly transmitted between birds through different mosquito species (e. g. Culex, Aedes), but may also be transmitted to mammals including humans. WNV causes a spectrum of disease, ranging from asymptomatic infection to encephalitis in a minority of cases. Risk factors for severe disease are older age, cardiovascular disease and an immunocompromised state. MEDICAL HISTORY AND CLINICAL EXAMINATION: Here we report about a 60-year-old male patient who was referred to the University Hospital of Halle (Saale) with severe fever two years after kidney transplantation due to hypertensive nephropathy. No infection focus could be found and by day 6 in the course of his illness the patient developed neurologic symptoms and viral encephalitis was suspected. TREATMENT AND COURSE: The patient was initially treated with aciclovir. After initial reduction of immunosuppression, coincident graft dysfunction was treated with methylprednisolon. WNV-infection was suspected due to recent emerging human cases in the nearby area of the city of Leipzig. WNV lineage 2 was detected in the patient's urine by RT-PCR and seroconversion with presence of anti WNV IgM and IgG could be demonstrated. Consecutively, aciclovir treatment was stopped. The patient fully recovered and the transplanted kidney regained adequate function. Kidney biopsy did not reveal gross rejection of the transplant. CONCLUSION: This case highlights the need to consider rarer causes of illness like WNV-infection particularly in risk groups for more severe outcomes of infectious disease. WNV may be detected by PCR in the blood and cerebrospinal fluid early in the course of infection but it is also excreted for a prolonged period of time in the urine. Seroconversion to anti WNV IgG and IgM may be shown but serologic cross-reactivity among members of the flaviviridae family must be considered.

A herpes oncolytic virus can be delivered via the vasculature to produce biologic changes in human colorectal cancer.

Genetically engineered herpes simplex viruses (HSVs) can selectively infect and replicate in cancer cells, and are candidates for use as oncolytic therapy. This long-term report of a phase I trial examines vascular administration of HSV as therapy for cancer. Twelve subjects with metastatic colorectal cancer within the liver failing first-line chemotherapy were treated in four cohorts with a single dose (3 x 10(6) to 1 x 10(8) particles) of NV1020, a multimutated, replication-competent HSV. After hepatic arterial administration, subjects were observed for 4 weeks before starting intra-arterial chemotherapy. All patients exhibited progression of disease before HSV injection. During observation, levels of the tumor marker carcinoembryonic antigen (CEA) decreased (median % drop = 24%; range 13-74%; P < 0.02). One of three individuals at the 10(8) level showed a 39% radiologic decrease in tumor size by cross-section and 75% by volume. HSV infection was documented from liver tumor biopsies. After beginning regional chemotherapy, all patients demonstrated a further decrease in CEA (median 96%; range 50-98%; P < 0.008) and a radiologic partial response. Median survival for this group was 25 months. During follow-up, no signs of virus reactivation were found. Multimutated HSV can be delivered safely into the human bloodstream to produce selective infection of tumor tissues and biologic effects.

Phase Ib trial of mutant herpes simplex virus G207 inoculated pre-and post-tumor resection for recurrent GBM.

We have previously demonstrated safety of G207, a doubly mutated (deletion of both gamma(1)34.5 loci, insertional inactivation of U(L)39) herpes simplex virus (HSV) for patients stereotactically inoculated in enhancing portions of recurrent malignant gliomas. We have now determined safety of two inoculations of G207, before and after tumor resection. Inclusion criteria were histologically proven recurrent malignant glioma, Karnofsky score >or=70, and ability to resect the tumor without ventricular system breach. Patients received two doses of G207 totaling 1.15 x 10(9) plaque-forming units with 13% of this total injected via a catheter placed stereotactically in the tumor. Two or five days later, tumor was resected en bloc with catheter in place. The balance of G207 dose was injected into brain surrounding the resection cavity. Six patients with recurrent glioblastoma multiforme were enrolled. Two days after the second G207 inoculation, one patient experienced transient fever, delirium, and hemiparesis, which entirely resolved on high-dose dexamethasone. No patient developed HSV encephalitis or required treatment with acyclovir. Radiographic and neuropathologic evidence suggestive of antitumor activity is reported. Evidence of viral replication was demonstrated. G207 appears safe for multiple dose delivery, including direct inoculation into the brain surrounding tumor resection cavity.

EBV miRNA expression profiles in different infection stages: A prospective cohort study.

The Epstein-Barr virus (EBV) produces different microRNAs (miRNA) with distinct regulatory functions within the infectious cycle. These viral miRNAs regulate the expression of viral and host genes and have been discussed as potential diagnostic markers or even therapeutic targets, provided that the expression profile can be unambiguously correlated to a specific stage of infection or a specific EBV-induced disorder. In this context, miRNA profiling becomes more important since the roles of these miRNAs in the pathogenesis of infections and malignancies are not fully understood. Studies of EBV miRNA expression profiles are sparse and have mainly focused on associated malignancies. This study is the first to examine the miRNA profiles of EBV reactivation and to use a correction step with seronegative patients as a reference. Between 2012 and 2017, we examined the expression profiles of 11 selected EBV miRNAs in 129 whole blood samples from primary infection, reactivation, healthy carriers and EBV seronegative patients. Three of the miRNAs could not be detected in any sample. Other miRNAs showed significantly higher expression levels and prevalence during primary infection than in other stages; miR-BHRF1-1 was the most abundant. The expression profiles from reactivation differed slightly but not significantly from those of healthy carriers, but a specific marker miRNA for each stage could not be identified within the selected EBV miRNA targets.

Value of the eazyplex® CSF direct assay in rapid diagnosis of invasive meningococcal disease - Case report.

There is a need for easy-to-use molecular assays for diagnosis of invasive meningococcal disease. Here, we report the rapid identification of Neisseria meningitidis in a cerebrospinal fluid sample from a patient with purulent meningitis using a commercially available loop-mediated isothermal amplification assay, resulting in a prompt de-escalation of the initial empiric antibiotic therapy.

Neuroborreliosis and acute encephalopathy: The use of CXCL13 as a biomarker in CNS manifestations of Lyme borreliosis.

We report the case of an 80-year-old patient with acute onset confusion initially suspected to reflect delirium in incipient Alzheimer's disease. Cerebrospinal fluid tests revealed an unusually severe form of neuroborreliosis, which resolved following antibiotic treatment. This was mirrored in the measurement of CXCL13, which is suggested as a complementary biomarker. Clinical implications for screening, differential diagnosis and treatment are discussed.

Vaccination trial with HPV16 L1E7 chimeric virus-like particles in women suffering from high grade cervical intraepithelial neoplasia (CIN 2/3).

Persistent infection with human papillomaviruses (HPV) is a prerequisite for the development of cervical cancer. Vaccination with virus-like particles (VLP) has demonstrated efficacy in prophylaxis but lacks therapeutic potential. HPV16 L1E7 chimeric virus-like particles (CVLP) consist of a carboxy-terminally truncated HPV16L1 protein fused to the amino-terminal part of the HPV16 E7 protein and self-assemble by recombinant expression of the fusion protein. The CVLP are able to induce L1- and E7-specific cytotoxic T lymphocytes. We have performed a first clinical trial to gain information about the safety and to generate preliminary data on the therapeutic potential of the CVLP in humans. A randomized, double blind, placebo-controlled clinical trial has been conducted in 39 HPV16 mono-infected high grade cervical intraepithelial neoplasia (CIN) patients (CIN 2/3). Two doses (75 mug or 250 mug) of CVLP were applied. The duration of the study was 24 weeks with 2 optional visits after another 12 and 24 weeks. The vaccine showed a very good safety profile with only minor adverse events attributable to the immunization. Antibodies with high titers against HPV16 L1 and low titers against HPV16 E7 as well as cellular immune responses against both proteins were induced. Responses were equivalent for both vaccine concentrations. A trend for histological improvement to CIN 1 or normal was seen in 39% of the patients receiving the vaccine and only 25% of the placebo recipients. Fifty-six percent of the responders were also HPV16 DNA-negative by the end of the study. Therefore, we demonstrated evidence for safety and a nonsignificant trend for the clinical efficacy of the HPV16 L1E7 CVLP vaccine.

A double-blind randomized multicentre clinical trial to evaluate the efficacy and safety of two doses of etomoxir in comparison with placebo in patients with moderate congestive heart failure: the ERGO (etomoxir for the recovery of glucose oxidation) study.

Etomoxir is an inhibitor of mitochondrial CPT1 (carnitine palmitoyltransferase 1) and thereby switches energy metabolism from fatty acids to glucose oxidation. Such a metabolic change may be beneficial in CHF (congestive heart failure). The ERGO (etomoxir for the recovery of glucose oxidation) study was designed in which etomoxir was tested at a dose of 80 and 40 mg compared with placebo for a period of 6 months in patients with CHF. As the principle measure of efficacy, a maximal exercise tolerance test and a submaximal 6-min corridor walk test were used. Secondary end points were echocardiographical dimensions and quality-of-life assessment scores. A total of 350 patients were planned to be screened, with the expectation that end point data would be available from approx. 260 patients. However, the study had to be stopped prematurely, because unacceptably high liver transaminase levels were detected in four patients taking etomoxir. At the termination of the study, 121 patients were randomized to placebo, 118 to 40 mg of etomoxir and 108 to 80 mg of etomoxir. At that time, 21 patients in the placebo group, 16 in the 40 mg of etomoxir group and 14 patients in the 80 mg of etomoxir group had completed the study. The mean increases in exercise time were 3.3, 10.2 and 19.4 s for the placebo, 40 mg of etomoxir and 80 mg of etomoxir groups respectively (P value was not significant). No changes were obvious in the 6-min corridor walk test or in echocardiographical parameters from baseline. The number of patients that completed the study was too small to demonstrate significant effects on exercise time, although there was a tendency towards an increase in exercise time. Therefore, before rejecting the hypothesis that inhibition of fatty acid oxidation might be beneficial in CHF, similar studies have to be performed using different inhibitors of fatty acid oxidation targeting CPT1 and other enzymes in this metabolic pathway.

Chronic persistent parvovirus B19 bone marrow infection resulting in transfusion-dependent pure red cell aplasia in multiple myeloma after allogeneic haematopoietic stem cell transplantation and severe graft versus host disease.

INTRODUCTION: We report a chronic persistent Parvovirus B19 (PVB19) infection despite long-term immunoglobulin substitution intravenous immunoglobulin (IVIG) and tapering of immune-suppressive therapy in a 41-year-old patient after allogeneic haematopoietic stem cell transplantation (alloHSCT) and long-term immune-suppressive therapy due to a steroid-refractory graft versus host disease (GvHD). CLINICAL COURSE: More than 18 month after alloHSCT the patient acquired a de novo transfusion-dependent pure red cell aplasia (PRCA) due to a PVB19 infection. Despite prompt tapering of GvHD-directed therapy and application of various IVIG regimens, transfusion-dependent anaemia (fourerythrocyte concentrates a month) persisted, and a high PVB19 replication is still evident for more than 3.5 years. Virological analysis at different time points showed a very high PVB19 load in the blood (range: 6.79E9-1.56E11), as well as highly elevated PVB19-IgG (range: 1.95-3.34) and -IgM (range: 1.97-9.74) levels in serology testing. Other virological parameters were not significantly elevated. After 30 months, a bone marrow (BM) examination still revealed a highly dysplastic erythropoiesis without any cellular maturation, and a high-grade expression of PVB19 within the dysplastic erythropoietic progenitor cells, consistent with a PRCA due to a PVB19 infection of the BM. We suggest that PRCA was most probably caused by a primary PVB19 infection of unknown source following alloHSCT with a PVB19-negative donor. CONCLUSION: PRCA due a PVB19 infection of the BM may persist over a long-time, despite prolonged administration of various IVIG regimen and tapering of GvHD-directed therapy. The case emphasizes the importance of PVB19 monitoring in heavily pre-treated haematological patients. Currently, PVB19-directed treatment options are extremely limited and optimized therapeutic strategies are urgently needed.

Phase I, open-label, dose-escalating study of a genetically engineered herpes simplex virus, NV1020, in subjects with metastatic colorectal carcinoma to the liver.

Current regimens of systemic chemotherapy result in only modest lengthening of survival in patients with advanced stage, liver-dominant, metastatic colorectal cancer who have failed first-line chemotherapy. The objective of this study was to investigate the safety and tolerability of NV1020, a replication-competent, attenuated, genetically engineered herpes simplex virus type 1 (HSV-1), in patients with hepatic colorectal metastases refractory to first-line chemotherapy. A phase I, open-label, dose-escalating study of a single 10-min hepatic arterial infusion of NV1020 in four cohorts. Three patients in each cohort received doses of 3 x 10(6), 1 x 10(7), 3 x 10(7), and 1 x 10(8) plaque-forming units. Adverse events were either mild or moderate in severity, and self-limiting. Only three serious adverse events (one transient rise in serum y-glutamyltransferase, one diarrhea, and one leukocytosis) experienced by three patients were considered to be possibly or probably related to NV1020. There were no deaths during the study, and there was no evidence of disseminated herpes infection. Viral presence was detected in only one saliva sample and two serum samples from one asymptomatic patient in the highest dose cohort. In the first week after viral administration only rare and minor increases were noted for tumor necrosis factor-alpha (six samples; three patients; peak, 40 pg/ml), interleukin (IL)-1 (two samples; two patients; peak, 28 pg/ml), and interferon-y (four samples; two subjects; peak, 54 pg/ml). No IL-2 was detected. Mild liver enzyme elevations were self-limiting and not associated with clinical symptoms. We conclude that NV1020, a genetically engineered but replication-competent HSV-1 oncolytic virus, can be safely administered into the hepatic artery without significant effects on normal liver function.

Draft Genome Sequence of the Vaccination Strain Mycobacterium bovis BCG S4-Jena.

Here, we present the draft genome sequence of ITALIC! Mycobacterium bovisBCG S4-Jena, a tuberculosis vaccine strain. The genome of S4-Jena is represented by 48 scaffolds, consisting of 132 scaffolded contigs and amounting to a size of about 4.2 Mb. New genes potentially encoding a phage fragment were identified in the genome.

Antibiotic treatment algorithm development based on a microarray nucleic acid assay for rapid bacterial identification and resistance determination from positive blood cultures.

Rapid diagnosis of bloodstream infections remains a challenge for the early targeting of an antibiotic therapy in sepsis patients. In recent studies, the reliability of the Nanosphere Verigene Gram-positive and Gram-negative blood culture (BC-GP and BC-GN) assays for the rapid identification of bacteria and resistance genes directly from positive BCs has been demonstrated. In this work, we have developed a model to define treatment recommendations by combining Verigene test results with knowledge on local antibiotic resistance patterns of bacterial pathogens. The data of 275 positive BCs were analyzed. Two hundred sixty-three isolates (95.6%) were included in the Verigene assay panels, and 257 isolates (93.5%) were correctly identified. The agreement of the detection of resistance genes with subsequent phenotypic susceptibility testing was 100%. The hospital antibiogram was used to develop a treatment algorithm on the basis of Verigene results that may contribute to a faster patient management.