Backbone Torsion Angle Determination Using Proton Detected Magic-Angle Spinning Nuclear Magnetic Resonance.

Protein torsion angles define the backbone secondary structure of proteins. Magic-angle spinning (MAS) NMR methods using carbon detection have been developed to measure torsion angles by determining the relative orientation between two anisotropic interactions─dipolar coupling or chemical shift anisotropy. Here we report a new proton-detection based method to determine the backbone torsion angle by recoupling NH and CH dipolar couplings within the HCANH pulse sequence, for protonated or partly deuterated samples. We demonstrate the efficiency and precision of the method with microcrystalline chicken α spectrin SH3 protein and the influenza A matrix 2 (M2) membrane protein, using 55 or 90 kHz MAS. For M2, pseudo-4D data detect a turn between transmembrane and amphipathic helices.

Targeting Viral Surface Proteins through Structure-Based Design.

The emergence of novel viral infections of zoonotic origin and mutations of existing human pathogenic viruses represent a serious concern for public health. It warrants the establishment of better interventions and protective therapies to combat the virus and prevent its spread. Surface glycoproteins catalyzing the fusion of viral particles and host cells have proven to be an excellent target for antivirals as well as vaccines. This review focuses on recent advances for computational structure-based design of antivirals and vaccines targeting viral fusion machinery to control seasonal and emerging respiratory viruses.

Cytomegalovirus Infection in Postrenal Transplant Recipients: 18 Years' Experience From a Tertiary Referral Center.

BACKGROUND: Cytomegalovirus (CMV) reactivation or infection is one of the most important infectious complications in transplant recipient leading to significant morbidity and mortality. Its early detection and prompt treatment is imperative to improve transplant outcome. The present study estimated the frequency of CMV in renal transplant recipients (RTR). Various aspects of pp65Ag assay and quantitative real-time polymerase chain reaction (qRT-PCR) were evaluated in relation to the recent guidelines for CMV detection and treatment. METHODS: Retrospectively, data of clinically suspected cases of CMV (1610 out of total 2681 renal transplants) were analyzed along with a comparison of pp65Ag assay and qRT-PCR. RESULTS: The overall incidence of CMV syndrome was 14.25%; however, the incidence of CMV viremia in the clinically suspected group was 23.73%. The proportion of positive cases with pp65Ag assay and qRT-PCR were 13.6% (95% CI; 7.9-22.3) and 19.3% (95% CI; 12.4-28.8) with a substantial agreement (Cohen's kappa = 0.632) between the 2 techniques. CMV positive recipients were treated with ganciclovir until their viral count was negative or up to 3 weeks, followed by 3 months of prophylaxis with valganciclovir. No graft failure or mortality was reported secondary to CMV infection until 3 to 5 years of follow-up. RESULTS: CMV infection is quite prevalent in RTR, and early detection and immediate treatment or prophylaxis is of utmost importance. qRT-PCR is the gold standard and preferred over other methods; however pp65Ag assay still holds its importance in low-economic countries and populations where CMV infection is more prevalent and financial constraints are a major limitation.

Molecular characterization of Epstein-Barr virus variants detected in the oral cavity of adolescents in Cali, Colombia / [Caracterización molecular de las variantes del virus de Epstein-Barr detectadas en la cavidad oral de adolescentes de Cali, Colombia].

Introduction: The Epstein-Barr virus (EBV) is an ubiquitous and oncogenic virus associated with the development of diseases such as infectious mononucleosis, Burkitt's lymphoma, nasopharyngeal carcinoma, and other neoplasms. Currently, two types are recognized: EBV-1 and EBV-2, which have genetic differences with their EBNA nuclear antigens. Likewise, due to the high degree of heterogeneity and variability found in the LMP1 protein of the virus, variants associated with pathogenesis or specific geographic regions have been described. Objective: To identify and characterize molecularly EBV variants detected in the oral cavity of 84 adolescents in Cali, Colombia. Materials and methods: Conventional PCR amplification, purification, and sequencing of the gen EBNA3C were carried out to typify the virus and the C-ter domain of the LMP1 protein to identify variants. We also conducted a phylogenetic and nucleotide variant analysis of the obtained sequences versus pathogenic or geographic variants reported in GenBank-NCBI. Results: The predominant viral subtype was EBV-1 (79%); 72.6% was grouped with the pathogenic variant Raji, derived from B lymphocytes of a patient with Burkitt>s lymphoma, 13.7% was related to a variant of Mediterranean origin, and 13.7% was not grouped with any of the reference variants. Conclusions: This is the first time that variants of LMP1-EBV have been identified in Cali, Colombia. Additional studies are necessary to characterize the unidentified variant and to determine if it is pathogenic or if it is just an isolate present in the city of Cali.

Introducción. El virus de Epstein-Barr (EBV) es un virus ubicuo y oncogénico, asociado con el desarrollo de enfermedades como la mononucleosis infecciosa, el linfoma de Burkitt, el carcinoma nasofaríngeo y otras neoplasias. Actualmente, se reconocen dos subtipos: EBV-1 y EBV- 2, que tienen diferencias genéticas con sus antígenos nucleares (Epstein-Barr Nuclear Antigens, EBNA). Debido a la gran heterogeneidad y variabilidad encontradas en la proteína LMP1 del virus, se han descrito variantes asociadas con ciertas enfermedades o con regiones geográficas específicas. Objetivo. Identificar y caracterizar molecularmente las variantes del EBV detectadas en la cavidad oral de 84 adolescentes de Cali, Colombia. Materiales y métodos. Se hizo la amplificación por reacción en cadena de la polimerasa (Polymerase Chain Reaction, PCR) convencional, así como la purificación y la secuenciación del gen EBNA3C se realizó para subtipificar el virus y del dominio C-ter de la proteína LMP1 para identificar variantes. Además, se llevó a cabo un análisis filogenético y de variantes nucleotídicas de las secuencias obtenidas comparadas con variantes patogénicas y geográficas reportadas en el GenBank (National Center for Biotechnology Information, NCBI). Resultados. El subtipo viral predominante fue el EBV-1 (79 %); el 72,6 % se agrupó con la variante patogénica Raji, derivada de linfocitos B de un paciente con linfoma de Burkitt; el 13,7 % se relacionó con una variante de origen geográfico del Mediterráneo y otro 13,7 % no se agrupó con ninguna de las variantes de referencia. Conclusiones. Este es el primer estudio que reporta variantes del gen LMP1-EBV en Cali, Colombia. Se requieren nuevos estudios para caracterizar la variante sin identificar y determinar si es patogénica o si es una variante geográfica presente exclusivamente en la ciudad.

Encapsulating Quantum Dots within HIV-1 Virions through Site-Specific Decoration of the Matrix Protein Enables Single Virus Tracking in Live Primary Macrophages.

Labeling and imaging with quantum dots (QDs) provides powerful tools to visualize viral infection in living cells. Encapsulating QDs within virions represents a novel strategy for virus labeling. Here, we developed infectious HIV-1 virions encapsulating QDs through site-specific decoration of the viral matrix protein (MA) and used them to visualize early infection events in human primary macrophages by single-particle imaging. The MA protein was fused to a biotin acceptor peptide (BAP) tag, biotinylated, complexed with streptavidin-conjugated QDs in live cells, and incorporated into virions during virus assembly. The QD-encapsulated virions were tracked during infection of macrophages at a single particle level. The dynamic dissociation of MA and Vpr was also tracked in real time, and the results demonstrated that MA has multiple dynamic behaviors and functions during virus entry. More importantly, we tracked the dynamic interplay of QD-encapsulated virions with cellular mitochondria in live primary macrophages. We also found that HIV-1 can induce fission of mitochondria during the early phases of infection. In summary, we have constructed a type of QD-encapsulated virus particle and used this technology to further our understanding of the early events of HIV-1 infection.

The interplay between local immune response and Epstein-Barr virus-infected tonsillar cells could lead to viral infection control.

Epstein Barr virus (EBV) gains access to the host through tonsillar crypts. Our aim was to characterize microenvironment composition around EBV+ cells in tonsils from pediatric carriers, to disclose its role on viral pathogenesis. LMP1 expression, assessed by immunohistochemistry (IHC), was used to discriminate EBV + and - zones in 41 tonsil biopsies. Three regions were defined: Subepithelial (SE), interfollicular (IF) and germinal center (GC). CD8, GrB, CD68, IL10, Foxp3, PD1, CD56 and CD4 markers were evaluated by IHC; positive cells/100 total cells were counted. CD8+, GrB+, CD68+ and IL10+ cells were prevalent in EBV+ zones at the SE region (p < 0.0001, p = 0.03, p = 0.002 and p = 0.002 respectively, Wilcoxon test). CD4+ and CD68+ cell count were higher in EBV + GC (p = 0.01 and p = 0.0002 respectively, Wilcoxon test). Increment of CD8, GrB and CD68 at the SE region could indicate a specific response that may be due to local homing at viral entry, which could be counterbalanced by IL10, an immunosuppressive cytokine. Additionally, it could be hypothesized that CD4 augment at the GC may be involved in the EBV-induced B-cell growth control at this region, in which macrophages could also participate.

Modified Anoikis Assay That Functionally Segregates Epstein-Barr Virus LMP1 Strains into Two Groups.

Nasopharyngeal carcinoma (NPC) is a metastatic Epstein-Barr virus (EBV)-associated cancer that expresses the viral oncogenic protein, latent membrane protein 1 (LMP1). During epithelial metastasis, detached cells must overcome anoikis-induced cell death and gain the ability to reattach and restore growth potential. Anoikis assays have revealed cell survival mechanisms during suspension, but few studies have tracked the fate of cells surviving anoikis-inducing conditions. In this study, a modified anoikis assay was used to examine if the expression of LMP1 confers the recovery of epithelial cells from anoikis. Cells expressing LMP1 mutants and strains were evaluated for distinguishing properties in survival during suspension, reattachment, and outgrowth potential. Expression of LMP1 promoted the outgrowth of the NPC cell line HK1 following anoikis induction that was not attributed to enhanced cell survival in suspension or reattachment. The mechanism of LMP1-induced outgrowth required Akt signaling and the conserved PXQXT motif on LMP1, which activates Akt. Deletion of any of the three LMP1 C-terminal activation regions (CTAR) abrogated anoikis recovery, suggesting that additional LMP1-regulated signaling pathways are likely involved. Of the seven LMP1 strains, only B958, China1, and Med+ promoted HK1 outgrowth from anoikis. This distinguishing biological property segregates LMP1 strains into two categories (anoikis recovering and nonrecovering) and suggests that LMP1 strain-specific sequences may be important in determining metastatic outgrowth potential in NPC tumors.IMPORTANCE LMP1 is one of the most divergent sequences in the EBV genome and phylogenetically segregates into seven LMP1 strains. The LMP1 strains differ in geographical distribution and NPC tumor prevalence, but the molecular basis for this potential selection is not clear. While there are signaling motifs conserved in all LMP1 sequences from circulating EBV isolates, phylogenetic studies of NPC also suggest that there may be sequence selection for tumor-associated LMP1 strains and polymorphisms. The present study describes a modified anoikis assay that can distinguish LMP1 strains into two groups by biological properties. The pleiotropic LMP1 signaling properties and sequence diversity may offer a unique opportunity to illuminate the complex mechanisms of metastasis. Although the host genomic landscape is variable between NPC tumors, the present functional-mapping studies on LMP1 support the notion that viral proteins could serve as molecular tool kits and potentially reveal sequence-associated risk factors in NPC metastatic progression.

Immunomodulatory properties of quercetin-3-O-α-L-rhamnopyranoside from Rapanea melanophloeos against influenza a virus.

BACKGROUND: Influenza infection is a major public health threat. The role of influenza A virus-induced inflammatory response in severe cases of this disease is widely recognized. Drug resistance and side effects of chemical treatments have been observed, resulting in increased interest in alternative use of herbal medications for prophylaxis against this infection. The South African medicinal plant, Rapanea melanophloeos (RM) (L.) Mez of the family Myrsinaceae was selected owing to its traditional use for the treatment of several diseases such as respiratory ailments and also previous preliminary studies of anti-influenza activity of its methanolic extract. The aim of this study was to investigate the immunomodulatory properties of a glycoside flavone isolated from RM against influenza A virus. METHODS: The non-cytotoxic concentration of the quercetin-3-O-α-L-rhamnopyranoside (Q3R) was determined by MTT assay and tested for activity against influenza A virus (IAV) in simultaneous, pre-penetration and post-penetration combination treatments over 1 h incubation on MDCK cells. The virus titer and viral load targeting NP and M2 viral genes were determined using HA and qPCR, respectively. TNF-α and IL-27 as pro- and anti-inflammatory cytokines were measured at RNA and protein levels by qPCR and ELISA, respectively. RESULTS: Quercetin-3-O-α-L-rhamnopyranoside at 150 µg/ml decreased the viral titer by 6 logs (p < 0.01) in the simultaneous procedure. The NP and M2 genes copy numbers as viral target genes, calculated based on the Ct values and standard formula, significantly decreased in simultaneous treatment (p < 0.01). The expression of cytokines was also considerably affected by the compound treatment. CONCLUSIONS: This is the first report of quercetin-3-O-α-L-rhamnopyranoside from RM and its immunomodulatory properties against influenza A virus. Further research will focus on detecting the specific mechanism of virus-host interactions.

Expression of human cytomegalovirus components in the brain tissues of patients with Rasmussen's encephalitis.

Rasmussen's encephalitis (RE) is a rare and severe progressive epileptic syndrome with unknown etiology. Infection by viruses, including human cytomegalovirus (HCMV), has been speculated to be a potential trigger for RE. However, no viral antigens have been detected in the brains of patients with RE; thus, a possible clinical linkage between viral infections and RE has not been firmly established. In this study, we evaluated the expression of HCMV pp65 antigen in brain sections from 26 patients with RE and 20 non-RE patients by immunohistochemistry and in situ hybridization, and assessed the associations between HCMV infection and clinical parameters. Elevated expression of HCMV pp65 protein and DNA was observed in 88.5% (23/26) and 69.2% (18/26) of RE cases, respectively. In the non-RE group, HCMV pp65 antigen was detected only in two cases (10%), both of which were negative for DNA staining. Additionally, the intensity of HCMV pp65 staining was correlated with a shorter duration of the prodromal stage, younger age of seizure onset, and more severe unilateral cortical atrophy. Elevated expression of HCMV pp65 was observed in RE brain tissue and was correlated with the clinical features of RE disease. In summary, our results suggested that HCMV infection may be involved in the occurrence and progression of RE disease. Thus, further studies are needed to determine whether early treatment with anti-HCMV antibodies could modulate the course of RE.

Epstein-Barr Virus Lytic Reactivation Activates B Cells Polyclonally and Induces Activation-Induced Cytidine Deaminase Expression: A Mechanism Underlying Autoimmunity and Its Contribution to Graves' Disease.

Graves' disease is an autoimmune disease that results in and is the most common cause of hyperthyroidism, and the reactivation of persisting Epstein-Barr virus (EBV) in B lymphocytes induces the differentiation of host B cells into plasma cells. We previously reported that some EBV-infected B cells had thyrotropin receptor antibodies (TRAbs) as surface immunoglobulins (Igs), and EBV reactivation induced these TRAb+EBV+ cells to produce TRAbs. EBV reactivation induces Ig production from host B cells. The purpose of the present study was to examine total Ig productions from B cell culture fluids and to detect activation-induced cytidine deaminase (AID), nuclear factor kappa B (NF-κB), and EBV latent membrane protein (LMP) 1 in culture B cells during EBV reactivation induction and then we discussed the mechanisms of EBV reactivation-induced Ig production in relation to autoimmunity. We showed that the EBV reactivation induces the production of every isotype of Ig and suggested that the Ig production was catalyzed by AID through LMP1 and NF-κB. The results that the amount of IgM was significantly larger compared with IgG suggested the polyclonal B cell activation due to LMP1. We proposed the pathway of EBV reactivation induced Ig production; B cells newly infected with EBV are activated by polyclonal B cell activation and produce Igs through plasma cell differentiation induced by EBV reactivation. LMP1-induced AID enabled B cells to undergo class-switch recombination to produce every isotype of Ig. According to this mechanism, EBV rescues autoreactive B cells to produce autoantibodies, which contribute to the development and exacerbation of autoimmune diseases.

Epstein-Barr virus and its association with Fascin expression in colorectal cancers in the Syrian population: A tissue microarray study.

Colorectal cancer (CRC) is the third most common malignancy in both men and women worldwide. Colorectal carcinogenesis is a complex, multistep process involving environmental and lifestyle features as well as sequential genetic changes in addition to bacterial and viral infections. Viral infection has a proven role in the incidence of approximately 20% of human cancers including gastric malignancies. Accordingly, Epstein-Barr virus (EBV) has been recently shown to be present in human gastric cancers, which could play an important role in the initiation and progression of these cancers. Therefore, this work explores the prevalence of EBV in 102 CRC tissues from the Syrian population using polymerase chain reaction (PCR) and tissue microarray (TMA) analysis. We found that EBV is present in 37 (36.27%) of CRC samples. Additionally, the expression of LMP1 onco-protein of EBV was found to be correlated with Fascin expression/overexpression in the majority of CRC tissue samples, which are intermediate/high grade invasive carcinomas. Our data indicate that EBV is present in CRC and its presence is associated with more aggressive cancer phenotype. Consequently, future investigations are needed to expose the role of EBV in CRC initiation and progression.

Clinical Usefulness of Monitoring Cytomegalovirus-Specific Immunity by Quantiferon-CMV in Pediatric Allogeneic Hematopoietic Stem Cell Transplantation Recipients.

Cytomegalovirus (CMV) is a well-established cause of morbidity and mortality in pediatric recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT). CD8⁺ T-cells are important for controlling CMV infection. We conducted a prospective pilot study to investigate the clinical utility of measuring the CMV-specific T-cell immune response using the QuantiFERON-CMV assay (QF-CMV) in pediatric allo-HSCT recipients. Overall, 16 of 25 (64%) patients developed CMV infection. QF-CMV was evaluated in these 16 patients during the early and late phases of the first CMV infection post allo-HSCT. Whereas the initial QF-CMV results during the early phase of CMV infection did not correlate with the course of the corresponding infection, the QF-CMV results post resolution of the first CMV infection correlated with the recurrence of CMV infection until 12 months post allo-HSCT; no recurrent infections occurred in the four QF-CMV-positive patients, while recurrent infections manifested in five of eight QF-CMV-negative (62.5%) and all three QF-CMV-indeterminate patients (P=0.019). In spite of the small number of patients examined, this study supports the potential application of monitoring CMV-specific T-cell immunity using the QF-CMV assay to predict the recurrence of CMV infection in pediatric allo-HSCT recipients.

Molecular Detection of Avian Influenza Virus from Sediment Samples in Waterfowl Habitats on the Delmarva Peninsula, United States.

Avian influenza viruses (AIV) affect many species of birds including waterfowl and may persist in sediment in aquatic habitats. Sediment samples were collected from two areas representative of prime migration and overwintering waterfowl habitat in Dorchester County, Maryland in the fall and winter of 2013-2014. Samples were screened for the presence of AIV via reverse transcriptase-quantitative PCR targeting the matrix gene. Although 13.6% of sediment samples were positive for the AIV matrix gene across all collection dates and locations, differences in detection were noted with location and collection season. Percentage of AIV-positive sediment samples recovered corresponded to trends in waterfowl abundance at collection sites both temporally and spatially. These findings provide further support for the assertion that the presence of AIV in the aquatic environment is likely affected by the total number, site-specific density, and array of waterfowl species.

Label-free luminescent detection of LMP1 gene deletion using an intermolecular G-quadruplex-based switch-on probe.

We have synthesized a series of luminescent iridium(III) complexes and investigated their ability to act as luminescent split G-quadruplex probes. After screening, the iridium(III) complex 1 [Ir(2-phenylquinoline)2(3,4,7,8-tetramethyl-1,10-phenanthroline)]PF6 was validated as a highly-selective G-quadruplex probe and was utilized to construct a label-free intermolecular G-quadruplex-based assay for the selective and sensitive detection of LMP1 gene deletion. This "mix-and-detect" assay is simple and selective, and could detect down to 10 nM of the target gene in aqueous solution with a linear range from 10 to 500 nM. We also investigated the performance of our split G-quadruplex-based sensing platform for LMP1 gene deletion in the presence of cellular debris, demonstrating the robustness of this sensing system in biological samples. Comparative assays were also performed using either organic dyes or labeled oligonucleotides as signal-transducing agents.

Circulating cytomegalic cells in a patient with advanced HIV presenting with cytomegalovirus rhinosinusitis.

We report a patient with newly diagnosed late-stage HIV presenting with fever, rhinosinusitis and cognitive impairment. Analysis of cerebrospinal fluid and nasal turbinate tissue confirmed cytomegalovirus (CMV) infection. CMV pp65 antigen assay conducted on peripheral blood leukocytes revealed large CMV infected mononuclear cells (diameter ∼ 50 µm) with an unusual cytoplasmic pattern of pp65 staining. These large cells were also seen in buffy coat Wright's stained smears; their size, morphology and pp65 antigen uptake pattern were consistent with CMV infected cytomegalic endothelial cells. While CMV sinus infections are occasionally encountered in AIDS patients, this is the first report to document CMV infection of the nasal mucosa with secondary sinusitis and chronic suppurative otitis media. Furthermore, the detection of circulating cytomegalic cells is described--a rare finding with pathological and clinical significance in immunocompromised patients with CMV disease.

Epstein-Barr virus-positive pleural effusion: Clinical features, cytomorphologic characteristics, and flow cytometric immunophenotyping.

OBJECTIVES: The etiology of pleural effusions (PEs) varies, and a percentage of PEs remains unexplained despite an intensive workup. One previous study documented a high prevalence of Epstein-Barr virus (EBV)-DNA in unselected and unexplained PEs. Our aim is to characterize the clinical and cytomorphologic features of EBV-associated PEs, which have not been described. METHODS: A database search was performed for PEs with EBV-DNA identified in the fluid by real-time quantitative polymerase chain reaction. The corresponding fluid cytology and chemistry were reviewed, and the patients' demographic data and clinical features were recorded. RESULTS: A total of 20 cases of EBV-DNA-positive PE were found. All patients had a history of lung transplantation. Most of the PE EBV loads were relatively low. Cytologically, polymorphous lymphocytosis was present in more than 70% of PEs. Scattered lymphocytic mitosis and apoptosis were seen in some cases. Mesothelial cells varied in number, and some cases showed reactive atypia. The lymphocytes were predominantly T cells with the CD4/CD8 ratio varying from 10:1 to 3:20. CONCLUSIONS: EBV infection/reactivation can account for certain proportions of "idiopathic" PEs. Polymorphous lymphocytosis is the most common cytologic feature, but atypical features (in both lymphocytes and mesothelial cells) can be seen.

Human cytomegalovirus tegument protein pp65 is detected in all intra- and extra-axial brain tumours independent of the tumour type or grade.

Human cytomegalovirus (HCMV) has been indicated being a significant oncomodulator. Recent reports have suggested that an antiviral treatment alters the outcome of a glioblastoma. We analysed the performance of commercial HCMV-antibodies applying the immunohistochemical (IHC) methods on brain sample obtained from a subject with a verified HCMV infection, on samples obtained from 14 control subjects, and on a tissue microarray block containing cores of various brain tumours. Based on these trials, we selected the best performing antibody and analysed a cohort of 417 extra- and intra-axial brain tumours such as gliomas, medulloblastomas, primary diffuse large B-cell lymphomas, and meningiomas. HCMV protein pp65 immunoreactivity was observed in all types of tumours analysed, and the IHC expression did not depend on the patient's age, gender, tumour type, or grade. The labelling pattern observed in the tumours differed from the labelling pattern observed in the tissue with an active HCMV infection. The HCMV protein was expressed in up to 90% of all the tumours investigated. Our results are in accordance with previous reports regarding the HCMV protein expression in glioblastomas and medulloblastomas. In addition, the HCMV protein expression was seen in primary brain lymphomas, low-grade gliomas, and in meningiomas. Our results indicate that the HCMV protein pp65 expression is common in intra- and extra-axial brain tumours. Thus, the assessment of the HCMV expression in tumours of various origins and pathologically altered tissue in conditions such as inflammation, infection, and even degeneration should certainly be facilitated.

Universal biosensor for detection of influenza virus.

Influenza is a contagious disease caught by humans caused by viruses belonging to the family Orthomyxoviridae. Each year, the influenza virus infects millions of people and kills hundreds of thousands of them. Traditional diagnostic methods, such as virus propagation and isolation, antigen capture immunoassays and molecular methods are not sufficient for the detection of the influenza virus. Development of a valid diagnostic assay for quick detection (in less than an hour) of the virus, with high sensitivity, is a challenge for researchers all over the world. Here we present a new, universal immunosensor for detection of the influenza A virus. By using electrochemical impedance spectroscopy (EIS) and direct attachment of antibodies to the gold electrode the assay allows detection of the pathogen with sensitivity similar to molecular methods in relatively short time. Application of universal anti-M1 antibodies allows detection of all serotypes of influenza A virus. The simple design of the sensor facilitates miniaturization of the device and its implementation for routine diagnostics during first contact with the patient, before applying a proper treatment.

Quantification of cytomegalovirus viral load.

Cytomegalovirus (CMV), a member of the Herpesviridae family, is worldwide distributed. After the primary infection, CMV induces a latent infection with possible reactivation(s). It is responsible for severe to life-threatening diseases in immunocompromised patients and in foetuses and newborns of infected mothers. For monitoring CMV load, classical techniques based on rapid culture or pp65 antigenemia are progressively replaced by quantitative nuclear acid tests (QNAT), easier to implement and standardize. A large variety of QNAT are available from laboratory-developed assays to fully-automated commercial tests. The indications of CMV quantification include CMV infection during pregnancy and in newborns, and viral surveillance of grafted and non-grafted immunocompromised patients, patients with bowel inflammatory diseases and those hospitalised in intensive care unit. A close cooperation between virologists and clinicians is essential for optimizing the benefit of CMV DNA monitoring.

Development of an Epstein-Barr virus-associated lymphoproliferative disorder in a patient treated with azacitidine for chronic myelomonocytic leukaemia.

Some chemotherapeutic agents can cause iatrogenic lymphoproliferative disorders. In analogy to what has been observed with other nucleoside analogues such as cladribine and fludarabine, we document the first case of an Epstein-Barr virus-positive, iatrogenic immunodeficiency-associated, lymphoproliferative disease, formally resembling polymorphic post-transplant lymphoproliferative disease in a patient treated with azacitidine (Vidaza) for chronic myelomonocytic leukaemia (CMML). A 78-year-old female patient was diagnosed with CMML in January 2012, and treatment with azacitidine was initiated, which lasted for five cycles from February until June 2012. The patient was hospitalized in June 2012 under the suspicion of pneumonia. Transformation of the CMML was suspected at that time too. During hospitalization, a generalized enlargement of the lymph nodes and the spleen was noticed. The patient rapidly deteriorated and finally died of respiratory insufficiency. At autopsy, an Epstein-Barr virus-associated lymphoproliferative disorder, resembling polymorphic post-transplant lymphoproliferative disease with involvement of the lymph nodes, the spleen and the lung and causing necrotizing pneumonia, was diagnosed. Diagnostic criteria for diffuse large B-cell lymphoma or infectious mononucleosis-like lymphoproliferative disease were not met. This is the first documented case of an azacitidine-associated lymphoproliferative disease, raising awareness for possible not yet known side effects of this drug, which should be kept in mind by oncologists and pathologists.