Torque teno virus (TTV) Infection in Patients with Encephalitis.

Torque teno virus (TTV) is a ssDNA orphan virus belonging to the Anelloviridae family, but some recent studies suggested its possible involvement in central nervous system (CNS) pathology. We analyzed serum and cerebrospinal fluid samples (CSF) from 109 patients with encephalitis for TTV infection using serological and molecular testing, virus quantitative measurement, and next-generation sequencing-based (NGS) phylogenetic analysis. TTV noncoding region (UTR) and/or open reading frame 1 (ORF-1) sequences were detected in serum of 86 (79%) patients and in nine (8%) patients in CSF. Five of the latter patients were coinfected with various entero- and herpesviruses. Anti-TTV-IgG were detected in 80 (73.4%) sera and in two (1.8%) CSF samples, while anti-TTV-IgM were present in three (2.8%) sera and in none of the CSFs. Phylogenic analysis of CSF-derived TTV ORF-1 sequences revealed the presence of three unique variants in one patient. TTV was quantified in five CSF-serum pairs: in two patients viral loads were similar, and in three serum TTV loads were approximately one log higher. Our results suggest at least an occasional replication of TTV in CNS. However, whether TTV could be the cause of encephalitis requires further studies.

mRNA vaccines: The future of prevention of viral infections?

Messenger RNA (mRNA) vaccines against COVID-19 are the first authorized biological preparations developed using this platform. During the pandemic, their administration has been proven to be a life-saving intervention. Here, we review the main advantages of using mRNA vaccines, identify further technological challenges to be met during the development of the mRNA platform, and provide an update on the clinical progress on leading mRNA vaccine candidates against different viruses that include influenza viruses, human immunodeficiency virus 1, respiratory syncytial virus, Nipah virus, Zika virus, human cytomegalovirus, and Epstein-Barr virus. The prospects and challenges of manufacturing mRNA vaccines in low-income countries are also discussed. The ongoing interest and research in mRNA technology are likely to overcome some existing challenges for this technology (e.g., related to storage conditions and immunogenicity of some components of lipid nanoparticles) and enhance the portfolio of vaccines against diseases for which classical formulations are already authorized. It may also open novel pathways of protection against infections and their consequences for which no safe and efficient immunization methods are currently available.

Metagenomic search of viral coinfections in herpes simplex encephalitis patients.

Little is known about concomitant central nervous system (CNS) infections by more than one virus. Current diagnostics are based on molecular tests for particular pathogens making it difficult to identify multi-viral infections. In the present study, we applied DNA- and RNA-based next-generation sequencing metagenomics (mNGS) to detect viruses in cerebrospinal fluids from 20 patients with herpes simplex encephalitis. Coinfection was detected in one patient: sequences in cerebrospinal fluids matched enterovirus A (2.660 reads; 4% of recovered genome) and enterovirus B (1.571 reads; 13% of recovered genome). Subsequent PCR combined with serotyping allowed to identify human echovirus 6, a representative of enterovirus B. Several other mNGS hits (human pegivirus, Merkel cell polyomavirus, human papillomavirus type 5) were not considered to represent a genuine signal as they could not be confirmed by specific RT-PCR/PCR. HSV DNA, while being detectable by PCR in every patient, was detected by mNGS in only one. In conclusion, contaminations and false signals may complicate mNGS interpretation; however, the method can be useful in diagnostics of viral coinfections in CNS, particularly in the case of rare pathogens.

Challenges of the Immunotherapy: Perspectives and Limitations of the Immune Checkpoint Inhibitor Treatment.

Immunotherapy is a quickly developing type of treatment and the future of therapy in oncology. This paper is a review of recent findings in the field of immunotherapy with an emphasis on immune checkpoint inhibitors. The challenges that immunotherapy might face in near future, such as primary and acquired resistance and the irAEs, are described in this article, as well as the perspectives such as identification of environmental modifiers of immunity and development of anti-cancer vaccines and combined therapies. There are multiple factors that may be responsible for immunoresistance, such as genomic factors, factors related to the immune system cells or to the cancer microenvironment, factors emerging from the host cells, as well as other factors such as advanced age, biological sex, diet, many hormones, existing comorbidities, and the gut microbiome.

Eosinophilic gastroenteritis and graft-versus-host disease induced by transmission of Norovirus with fecal microbiota transplant.

Fecal microbiota transplantation (FMT) was performed to decolonize gastrointestinal tract from antibiotic-resistant bacteria before allogeneic hematopoietic cells transplantation (alloHCT). AlloHCT was complicated by norovirus gastroenteritis, acute graft-versus-host disease, and eosinophilic pancolitis. Norovirus was identified in samples from FMT material. Symptoms resolved after steroids course and second norovirus-free FMT from another donor.

Comprehensive Analysis of Correlations in the Expression of miRNA Genes and Immune Checkpoint Genes in Bladder Cancer Cells.

Personalised medicine is the future and hope for many patients, including those with cancers. Early detection, as well as rapid, well-selected treatment, are key factors leading to a good prognosis. MicroRNA mediated gene regulation is a promising area of development for new diagnostic and therapeutic methods, crucial for better prospects for patients. Bladder cancer is a frequent neoplasm, with high lethality and lacking modern, advanced therapeutic modalities, such as immunotherapy. MicroRNAs are involved in bladder cancer pathogenesis, proliferation, control and response to treatment, which we summarise in this perspective in response to lack of recent review publications in this field. We further performed a correlation-based analysis of microRNA and gene expression data in bladder cancer (BLCA) TCGA dataset. We identified 27 microRNAs hits with opposite expression profiles to genes involved in immune response in bladder cancer, and 24 microRNAs hits with similar expression profiles. We discuss previous studies linking the functions of these microRNAs to bladder cancer and assess if they are good candidates for personalised medicine therapeutics and diagnostics. The discussed functions include regulation of gene expression, interplay with transcription factors, response to treatment, apoptosis, cell proliferation and angiogenesis, initiation and development of cancer, genome instability and tumour-associated inflammatory reaction.

Coronaviruses - a new old menace.

The current pandemic caused by novel coronavirus SARS-CoV-2 pandemic has been described as a global health emergency. The outbreak of this virus has raised a number of questions: what exactly is SARS-CoV-2? How transmissible the novel coronavirus is? How severely affected are patients infected with SARS-CoV-2? What are the risk factors for COVID-19? What are the differences between this novel coronavirus and other coronaviruses? To answer these questions, a comparative study of three pathogenic coronaviruses that primarily invade the human respiratory system and may cause death, namely, severe acute respiratory syndrome (SARS-CoV-1), Middle East respiratory syndrome (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Middle East respiratory syndrome coronavirus (MERS-CoV). This review describes the source of origin, transmission, and pathogenicity of these viruses. Prevention of SARS-CoV-2 spreading entails home isolation of suspected cases and those with mild illnesses and strict infection control measures at hospitals that include contact and droplet precautions. The novel coronavirus spreads faster than its two predecessors - the SARS-CoV-1 and MERS-CoV - but has lower fatality rate. The global impact of this new pandemic is still uncertain, but it is a challenge to healthcare systems around the world.

Inhibitory Effects of Bacteriophage Preparations on Adenoviral Replication.

BACKGROUND/AIMS: Bacteriophages (phages) are viruses of bacteria. Escherichia coli phage (T4) can potentially interfere with adsorption of HAdV-5 to cellular integrins by its KGD motif, while staphylococcal A5/80 phage does not possess this structure. The objective of this study was to investigate the effects of T4 and A5/80 phage preparations on type 5 human adenovirus (HAdV-5) DNA synthesis and the expression of HAdV-5 genes. METHODS: Experiments were performed on the A549 cell line. HAdV-5 DNA synthesis was investigated with real-time PCR. Expression of HAdV-5 early (DBP) and late (hexon) genes was determined by quantitative real-time PCR in preincubation and coincubation experiments. RESULTS: While both phage preparations significantly reduced the expression of HAdV-5 genes, synthesis of HAdV-5 DNA was inhibited only by T4. CONCLUSION: Phage preparations show promise as novel antiviral agents. However, further studies are required to investigate their antiviral effects.

Human Pegivirus in Patients with Encephalitis of Unclear Etiology, Poland.

Human pegivirus (HPgV), previously called hepatitis G virus or GB virus C, is a lymphotropic virus with undefined pathology. Because many viruses from the family Flaviviridae, to which HPgV belongs, are neurotropic, we studied whether HPgV could infect the central nervous system. We tested serum and cerebrospinal fluid samples from 96 patients with a diagnosis of encephalitis for a variety of pathogens by molecular methods and serology; we also tested for autoantibodies against neuronal antigens. We found HPgV in serum and cerebrospinal fluid from 3 patients who had encephalitis of unclear origin; that is, all the markers that had been tested were negative. Single-strand confirmation polymorphism and next-generation sequencing analysis revealed differences between the serum and cerebrospinal fluid-derived viral sequences, which is compatible with the presence of a separate HPgV compartment in the central nervous system. It is unclear whether HPgV was directly responsible for encephalitis in these patients.

A Cluster of Fatal Tick-borne Encephalitis Virus Infection in Organ Transplant Setting.

Background: Tick-borne encephalitis virus (TBEV) infection has become a major health problem in Europe and is currently a common cause of viral brain infection in many countries. Encephalitis in transplant recipients, althrough rare, is becoming a recognized complication. Our study provides the first description of transmission of TBEV through transplantation of solid organs. Methods: Three patients who received solid organ transplants from a single donor (2 received kidney, and 1 received liver) developed encephalitis 17-49 days after transplantation and subsequently died. Blood and autopsy tissue samples were tested by next-generation sequencing (NGS) and reverse transcription polymerase chain reaction (RT-PCR). Results: All 3 recipients were first analyzed in autopsy brain tissue samples and/or cerebrospinal fluid by NGS, which yielded 24-52 million sequences per sample and 9-988 matched TBEV sequences in each patient. The presence of TBEV was confirmed by RT-PCR in all recipients and in the donor, and direct sequencing of amplification products corroborated the presence of the same viral strain. Conclusions: We demonstrated transmission of TBEV by transplantation of solid organs. In such a setting, TBEV infection may be fatal, probably due to pharmacological immunosuppression. Organ donors should be screened for TBEV when coming from or visiting endemic areas.

Fecal Microbiota Transplantation in Patients With Blood Disorders Inhibits Gut Colonization With Antibiotic-Resistant Bacteria: Results of a Prospective, Single-Center Study.

Background: Patients with blood disorders colonized with antibiotic-resistant bacteria (ARB) are prone to systemic infections that are difficult to treat. Reintroduction of commensal bacteria in a murine model of enterococcal colonization of the gut can lead to eradication of enterococci. We hypothesized that fecal microbiota transplantation (FMT) could be used to eradicate ARB in humans. Methods: Participants colonized with ARB were treated with intraduodenal FMT according to a prospective protocol (NCT02461199). The primary endpoint was complete ARB decolonization at 1 month after FMT. Secondary endpoints included safety assessment and partial ARB decolonization. Microbiome sequencing was performed to investigate the influence of microbial composition of the transplanted material on the outcome of FMT. Results: Twenty-five FMTs were performed in 20 participants (including 40% who had neutropenia) who were colonized by a median of 2 (range, 1-4) strains of ARB. The primary endpoint was reached in 15/25 (60%) of the FMTs and more frequently in cases in which there was no periprocedural use of antibiotics (79% vs 36%, P < .05). Among participants, 15/20 (75%) experienced complete ARB decolonization. There were no severe adverse events, and partial ARB decolonization was observed in 20/25 (80%) of the FMTs. The microbiota composition analysis revealed higher abundance of Barnesiella spp., Bacteroides, and Butyricimonas and greater bacterial richness in the fecal material, resulting in eradication of Klebsiella pneumoniae compared with nonresponders. Conclusions: FMT in patients with blood disorders is safe and promotes eradication of ARB from the gastrointestinal tract. Clinical Trials Registration: NCT02461199.

[Serological and molecular methods in diagnostics of primary acute hepatitis caused with human herpesvirus type 6 - a case report].

INTRODUCTION: HHV-6 has been identified as the etiologic agent of exanthema subitum in infants and acute febrile illness in young children, although primary HHV-6 infection in immunocompetent adolescents and adults is very rare. We report the case of acute hepatitis in an 18-year-old man without any immunological dysfunctions. MATERIAL AND METHODS: Full virological examination of sera samples was performed. Specimens were tested for the presence of specific anti-EBV and anti-HHV-6 antibodies and also for the presence of DNA of other herpesviruses, using qPCR assays. RESULTS: Obtained results demonstrated the presence of HHV-6 DNA in all examined sera samples and IgM-class antibodies against HHV-6 in the second specimen. No other etiologic agents were found that are known to induce hepatitis. Hence, patient was diagnosed as having acute hepatitis triggered by HHV-6. CONCLUSIONS: We found that qPCR is a very useful tool for the detection of different herpesvirus infections, especially with low-copy viraemia in clinical specimens.

[Frequency of detection of soluble antigens of Candida spp. and Aspergillus spp. using serological assays in body fluids of patients of Central Clinical Hospital in Warsaw].

INTRODUCTION: Immunodeficient patients, e.g. transplant recipients, patients treated with corticosteroids, people with AIDS and individuals undergoing prolonged antibiotic therapy are at high risk of invasive fungal infections, especially invasive aspergillosis. Basic method for detection of organ/systemic fungal infection is serological monitoring in body fluids, first of all in serum, bu also in broncho-alveolar lavages (BALF). Proven invasive fungal infection should be diagnosed by culture of the pathogen or histopathological examination of infected tissues, however the detection of soluble fungal antigens in body fluids gives enough information for diagnosis of probable fungal infection, according to European Organization for Research and Treatment of Cancer recommendations, what allows introduction of antifungal therapy. Aim of the study was to asses the frequency of detection of circulation soluble fungal antigens with use of immunoenzymatic techniques in patients hospitalized between 2010 and 2015 in Independent Public Central Clinical Hospital (IPCCH) in Warsaw. Methods: In IPCCH, between 2010 and 2015, 6475 serum samples, taken from 2096 patients, was tested for Candida spp. mannan antigen, and 7745 sera from 2243 patients were tested for Candida spp. mannan antigen, and 7745 sera from 2243 patients were tested for galactomannan antigen of Aspergillus spp, as well as 64 samples of BALF. Material was collected mainly from haematopoietic stem cell transplant recipients, hospitalized in Haematology and Oncology Clinics, during their routine pos-transplant monitoring. Testing was performed with use of quantitative (Candida antigen) or semiquantitative (Aspergillus antigen) immunoenzymatic methods (BioRad-Platelia), according to respective protocols. Results: During examined period, increase in number of examinations was observed, starting from 1311 tests performed in 2010, up to 3052 examination in 2015. In 2015 testing for Aspergillus antigen in BALF samples was also introduced, resulting in 64 samples tested. Candida spp. antigen was detected in 171 samples (2,7% of all tested samples), and Aspergillus galactomannan was detected in 645 serum samples (8,4%) and 8 BALF samples (12,5%). Majority of examinations was performed for patients hospitalized in Haematology and Oncology Clinics (72,7%), Blood Vessel Surgery and Transplantology Clinics (3,8%), as well as in patients under care of post-transplantation (8,3%) and haematology (4,2%) out-patients clinics. Conclusions: (i) In the 2015-2015 visible increase in number of fungal antigens examinations was observed, (ii) significant number of examinations was performed in onco-haematological patients (88,7%), what also indicates main risk group, (iii) 8,3% of fungal antigen testing was performed in solid organ transplant recipients, the second risk group for invasive fungal infection.

[Prevalence of CI-HHV-6 in patients with hematological diseases and healthy donors in Poland - preliminary study].

of specific anti-HHV-6 antibodies in IgM and IgG classes and viral DNA. Quantitative real-time PCR assay was also used to determine viral load in alloHSCT recipients in plasma samples. Results: All individuals from studied group have not IgM antibodies against-HHV-6 prior transplantation. Specific IgG-class anti-HHV-6 antibodies were detected in 38 of 54 (70%) donors and in 47 of 54 recipients before HSCT (870/o), respectively. High load of HHV-6 DNA (>1x10A6 copies/ml) was detected in plasma samples taken only from one person (1,9%) of the 54 recipients. Conclusions: There is a high frequency specific anti-HHV-6 antibody in studied Polish patients; otherwise CI-HHV-6 was rare detected. Nonetheless, we urge careful observation of individuals with hematological malignances supposed to have CI-HHV-6. Further research on larger study group is needed to determine the clear role of CI-HHV-6 in alloHSCT recipients.

[Development of TaqMan-based real-time PCR assay for detection of Epstein-Barr virus DNA]. / Zaprojektowanie i optymalizacja metody PCR z detekcja w czasie rzeczywistym do wykrywania DNA wirusa Epsteina-Barr z wykorzystaniem fluorescencyjnej sondy typu TaqMan.

INTRODUCTION: Epstein-Barr virus (EBV) is a human herpesvirus which infects almost all of the world's population subclinically during childhood and thereafter remains for a life. Immunocompromised persons often show active EBV infection, which may progress to virus-associated lymphoproliferative disorders. Many clinical researches show a strong role for viral load measurement in predicting and monitoring EBV-associated diseases, especially in immunosuppressed patients. The aim of this work was to design and to optimize novel real-time PCR assay for detection and quantification of Epstein-Barr virus DNA. MATERIALS AND METHODS: In described experiment TaqMan chemistry-based primers and probes were designed to specific EBV sequence of BALF5 viral gene. To test laboratory utility of the designed method, 80 sera samples, positive for EBV DNA in routine investigations, were also analyzed and 1st International WHO WBV Standard was applied for recalculation of the results to international units. RESULTS: Developed real-time PCR assay gave positive result only in the samples containing genetic material of EBV. Mean viral load of the 80 clinical samples tested was 2,838 and 3,241 log10 copies/ml for analyzed and reference method, respectively. Correction with EBV Standard led to equalization of these results (3,229 and 3,244 log10 international units/ml respectively). CONCLUSIONS: The obtained data indicate that this TaqMan-based qPCR assay is accurate, rapid and reliable method for the diagnosis and monitoring of EBV DNAemia in clinical samples, coming from immunosuppressed individuals.

[Novel multiplex real-time PCR assay for detection and differentiation of herpes simplex virus type 1 and 2 DNA]. / Wykorzystanie nowej techniki multiplex real-time PCR do wykrywania i róznicowania DNA wirusów opryszczki typu 1 i 2.

INTRODUCTION: Herpes simplex viruses type 1 and 2 are the cause of world spread multiple infections with different course and severity. The aim of this work was to design and to optimize multiplex real-time PCR assay for simultaneous detection of HSV-1 and HSV-2. The second aim of the project was to check if the designed method is laboratory useful analyzing different clinical specimens. MATERIALS AND METHODS: In this experiment primers and probes were designed to specific viral sequences: for HSV-1 to the gene of viral DNA polymerase; for HSV-2 to the UL5 sequence. For performing qPCR assay TaqMan chemistry was used. Reference strains HSV-I McIntyre and HSV-2 MS were used as a positive control. To test laboratory utility of the designed method 58 different clinical specimens were analyzed. RESULTS: Developed multiplex real-time PCR gave positive result only in the samples containing genetic material of HSV-1/2. Of the 58 clinical samples tested, 27 proved to be positive for HSV-1 and 17 for HSV-2. The 7 samples showed the presence of both types of DNA herpes simplex virus, and 7 others were found for both HSV-1 and HSV-2 negative. CONCLUSIONS: Obtained results show that the designed method is highly specific and can possibly be used to simultaneously detect and differentiate HHV-1/2. Both high specificity and very short time of analysis have great importance in diagnosing immunocompromised patients, which ought to be diagnosed quickly and effectively in order to provide appropriate treatment.

[Usefulness of wide-range microbiological diagnostics proceedings in case of simultaneous infection with four herpesviruses after allogeneic haematopoietic stem cell transplantation--a case report]. / Przydatnosc wielokierunkowej diagnostyki mikrobiologicznej na przykladzie jednoczesnego zakazenia czterema herpeswirusami jako powiklania po allogenicznym przeszczepieniu komórek krwiotwórczych--opis przypadku.

INTRODUCTION: Infections caused with a variety of bacteria, fungi and viruses are still responsible for high level of mortality and morbidity in immunosupressed individuals. A case of fatal post-transplant reactivation with four herpesviruses in 49-year-old immunocompromised male with MDS-RAEB2, subjected to allogeneic haematopoietic stem cell transplantation was described. METHODS: Full microbiological examination of was performed in different types of clinical samples (whole blood, stool). Sera specimens were tested for the presence of different viral DNA using the real-time PCR assays. RESULTS AND CONCLUSIONS: DNA of HSV-1, VZV, HHV-6 and EBV in serum samples was detected using molecular biology techniques. Viral level of HSV-1 and VZV was constantly increasing despite routine applied oral acyclovir therapy. These findings underline the value of real-time PCR technique used in current therapeutic procedures and for monitoring of antiviral therapy with nucleoside analogs. We found that real-time PCR is a useful tool in detection and monitoring of disseminated herpesviral infection, especially for the detection of low-copy viraemia in clinical specimens.