Novel combinatorial therapy of oncolytic adenovirus AdV5/3-D24-ICOSL-CD40L with anti PD-1 exhibits enhanced anti-cancer efficacy through promotion of intratumoral T-cell infiltration and modulation of tumour microenvironment in mesothelioma mouse model.

Introduction: Malignant mesothelioma is a rare and aggressive form of cancer. Despite improvements in cancer treatment, there are still no curative treatment modalities for advanced stage of the malignancy. The aim of this study was to evaluate the anti-tumor efficacy of a novel combinatorial therapy combining AdV5/3-D24-ICOSL-CD40L, an oncolytic vector, with an anti-PD-1 monoclonal antibody. Methods: The efficacy of the vector was confirmed in vitro in three mesothelioma cell lines - H226, Mero-82, and MSTO-211H, and subsequently the antineoplastic properties in combination with anti-PD-1 was evaluated in xenograft H226 mesothelioma BALB/c and humanized NSG mouse models. Results and discussion: Anticancer efficacy was attributed to reduced tumour volume and increased infiltration of tumour infiltrating lymphocytes, including activated cytotoxic T-cells (GrB+CD8+). Additionally, a correlation between tumour volume and activated CD8+ tumour infiltrating lymphocytes was observed. These findings were confirmed by transcriptomic analysis carried out on resected human tumour tissue, which also revealed upregulation of CD83 and CRTAM, as well as several chemokines (CXCL3, CXCL9, CXCL11) in the tumour microenvironment. Furthermore, according to observations, the combinatorial therapy had the strongest effect on reducing mesothelin and MUC16 levels. Gene set enrichment analysis suggested that the combinatorial therapy induced changes to the expression of genes belonging to the "adaptive immune response" gene ontology category. Combinatorial therapy with oncolytic adenovirus with checkpoint inhibitors may improve anticancer efficacy and survival by targeted cancer cell destruction and triggering of immunogenic cell death. Obtained results support further assessment of the AdV5/3-D24-ICOSL-CD40L in combination with checkpoint inhibitors as a novel therapeutic perspective for mesothelioma treatment.

Comparative study between virus neutralisation testing and other serological methods detecting anti-SARS-CoV-2 antibodies in Europe, 2021.

One consequence of the ongoing coronavirus disease pandemic was the rapid development of both in-house and commercial serological assays detecting anti-SARS-CoV-2 antibodies, in an effort to reliably detect acute and past SARS-CoV-2 infections. It is crucial to evaluate the quality of these serological tests and consequently the sero-epidemiological studies that are performed with the respective tests. Here, we describe the set-up and results of a comparative study, in which a laboratory contracted by the European Centre for Disease Prevention and Control offered a centralised service to EU/EEA Member and pre-accession Member States to test representative serum specimens with known serological results, with the gold standard technique (virus neutralisation tests) to determine the presence of neutralising antibodies. Laboratories from 12 European countries shared 719 serum specimens with the contractor laboratory. We found that in-house serological tests detecting neutralising antibodies showed the highest percent agreement, both positive and negative, with the virus neutralisation test results. Despite extensive differences in virus neutralisation protocols neutralisation titres showed a strong correlation. From the commercial assays, the best positive percent agreement was found for SARS-CoV-2 IgG (sCOVG) (Siemens - Atellica IM Analyzer). Despite lower positive percent agreement of LIAISON SARS-CoV-2 TrimericS IgG kit (Diasorin Inc.), the obtained results showed relatively good correlation with neutralisation titres. The set-up of this study allowed for high comparability between laboratories and enabled laboratories that do not have the capacity or capability to perform VNTs themselves. Given the variety of in-house protocols detecting SARS-CoV-2 specific neutralising antibodies, including the virus strain, it could be of interest to select reference isolates for SARS-CoV-2 diagnostic to be made available for interested EU Member States and pre-accession countries.

Estimated Incidence of Symptomatic Lyme Borreliosis Cases in Lublin, Poland in 2021.

Lyme borreliosis (LB), the most common tick-borne disease in Europe, is endemic to Poland. Despite public health surveillance with mandatory reporting of LB cases by physicians and laboratories, many symptomatic LB cases are not included in surveillance in Poland. We estimated the extent of the under-ascertainment of symptomatic LB cases via surveillance in the Polish province of Lublin to better understand Poland's LB burden. The number of incident symptomatic LB cases in Lublin in 2010 was estimated from two seroprevalence studies conducted among adults in Lublin, as well as estimates of the proportion of asymptomatic LB cases and the duration of LB antibody persistence. The estimated number of incident symptomatic LB cases was compared to the number of surveillance-reported cases in Lublin to derive an under-ascertainment multiplier. This multiplier was applied to the number of surveillance-reported cases in 2021 to estimate the number and population-based incidence of symptomatic LB cases in Lublin in 2021. We estimate that there are 5.9 symptomatic LB cases for every surveillance-reported LB case in Lublin. Adjusting for under-ascertainment, the estimated number of symptomatic LB cases in Lublin in 2021 was 6204 (population-based incidence: 467.6/100,000). After adjustment for under-ascertainment, the incidence of symptomatic LB in Lublin, Poland, is high.

Tick-borne encephalitis epidemiology and surveillance in Poland, and comparison with selected European countries before and during the COVID-19 pandemic, 2008 to 2020.

BackgroundTick-borne encephalitis (TBE) is the most common viral central nervous system (CNS) infection in Poland. Previous research suggests that its incidence was underestimated in the pre-pandemic period. The COVID-19 pandemic caused a considerable burden on surveillance systems, which could further impact reporting.AimWe aimed to assess the completeness of reporting of TBE in the years 2008 to 2020 and explore the potential impact of the COVID-19 pandemic on reporting to the epidemiological surveillance system, compared with hospitalisations for TBEV and other viral neuro-infections.MethodsWe compared the Polish epidemiology of TBE and other viral infections of the CNS from national surveillance reports with data on hospitalisations from 2008 to 2020 and data from selected European countries.ResultsBetween 2008 and 2020, 3,016 TBE cases were reported to surveillance compared with 3,620 hospitalisations. There was an increasing trend in hospitalisations, while surveillance data demonstrated the opposite, with the largest discrepancy observed in the first pandemic year (354 hospitalisations vs 159 cases reported to surveillance). Serological testing for TBE was used more in the known endemic region of north-eastern Poland and less in non-endemic areas. Other European countries reported higher TBE case numbers and an increase during the COVID-19 pandemic, whereas Poland observed an opposite trend.ConclusionThe sensitivity of TBE surveillance in Poland requires improvement. There are considerable regional differences. Regions that test for TBE intensively report most cases. Policymakers should be made aware of the value of quality epidemiological data for planning prophylactic measures in risk areas.

In vitro immune evaluation of adenoviral vector-based platform for infectious diseases.

New prophylactic vaccine platforms are imperative to combat respiratory infections. The efficacy of T and B memory cell-mediated protection, generated through the adenoviral vector, was tested to assess the effectiveness of the new adenoviral-based platforms for infectious diseases. A combination of adenovirus AdV1 (adjuvant), armed with costimulatory ligands (ICOSL and CD40L), and rRBD (antigen: recombinant nonglycosylated spike protein rRBD) was used to promote the differentiation of T and B lymphocytes. Adenovirus AdV2 (adjuvant), without ligands, in combination with rRBD, served as a control. In vitro T-cell responses to the AdV1+rRBD combination revealed that CD8+ platform-specific T-cells increased (37.2 ± 0.7% vs. 23.1 ± 2.1%), and T-cells acted against SARS-CoV-2 via CD8+TEMRA (50.0 ± 1.3% vs. 36.0 ± 3.2%). Memory B cells were induced after treatment with either AdV1+rRBD (84.1 ± 0.8% vs. 82.3 ± 0.4%) or rRBD (94.6 ± 0.3% vs. 82.3 ± 0.4%). Class-switching from IgM and IgD to isotype IgG following induction with rRBD+Ab was observed. RNA-seq profiling identified gene expression patterns related to T helper cell differentiation that protect against pathogens. The analysis determined signaling pathways controlling the induction of protective immunity, including the MAPK cascade, adipocytokine, cAMP, TNF, and Toll-like receptor signaling pathway. The AdV1+rRBD formulation induced IL-6, IL-8, and TNF. RNA-seq of the VERO E6 cell line showed differences in the apoptosis gene expression stimulated with the platforms vs. mock. In conclusion, AdV1+rRBD effectively generates T and B memory cell-mediated protection, presenting promising results in producing CD8+ platform-specific T cells and isotype-switched IgG memory B cells. The platform induces protective immunity by controlling the Th1, Th2, and Th17 cell differentiation gene expression patterns. Further studies are required to confirm its effectiveness.

From Immunosuppression to Immunomodulation - Turning Cold Tumours into Hot.

Cancer cells employ various mechanisms to evade and suppress anti-cancer immune responses generating a "cold" immunosuppressive tumour microenvironment. Oncolytic viruses are a promising tool to convert tumour immunosuppression to immunomodulation and improve the efficacy of cancer treatment. Emerging preclinical and clinical findings confirm that oncolytic viruses act in a multimodal scheme, triggering lyses, immunogenic cell death and finally inducing anti-cancer immune responses. In this paper, we tested the local administration of a novel oncolytic adenovirus AdV-D24-ICOSL-CD40L expressing co-stimulatory molecules ICOSL and CD40L to induce the production of tumour infiltrating lymphocytes to the site of injection. Subsequently, in immunocompetent mouse models, we studied possible correlation between tumour infiltrates and anti-cancer efficacy. Described results showed that the delivery of oncolytic viruses encoding immunomodulatory transgenes in combination with anti-PD1 resulted in synergistic inhibition of both melanoma and mesothelioma tumours. Importantly anti-cancer effect positively correlated with cytotoxic CD8+ tumour-infiltrating lymphocytes exerting a central role in the tumour volume control thus generating beneficial outcomes that will undoubtedly provide new insights into possible future treatment strategies to combat cancer. Altogether our findings highlight the importance of oncolytic vectors able to modulate anti-cancer immune responses that can correlate with efficacy in solid malignancies.

Novel Insights Into Mesothelioma Therapy: Emerging Avenues and Future Prospects.

Malignant mesothelioma is a rare and aggressive cancer that develops in the thin layer surrounding the mesothelium and is mainly caused by asbestos exposure. Despite improvements in patient prognosis with conventional cancer treatments, such as surgery, chemotherapy, and radiotherapy, there are still no curative treatment modalities for advanced disease. In recent years, new therapeutic avenues have been explored. Improved understanding of the mechanisms underlying the dynamic tumor interaction with the immune system has led to the development of immunotherapeutic approaches. Numerous recent clinical trials have shown a desire to develop more effective treatments that can be used to fight against the disease. Immune checkpoint inhibitors, oncolytic adenoviruses, and their combination represent a promising strategy that can be used to synergistically overcome immunosuppression in the mesothelioma tumor microenvironment. This review provides a synthesized overview of the current state of knowledge on new therapeutic options for mesothelioma with a focus on the results of clinical trials conducted in the field.

Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model.

Malignant melanoma, an aggressive form of skin cancer, has a low five-year survival rate in patients with advanced disease. Immunotherapy represents a promising approach to improve survival rates among patients at advanced stage. Herein, the aim of the study was to design and produce, by using engineering tools, a novel oncolytic adenovirus AdV-D24- inducible co-stimulator ligand (ICOSL)-CD40L expressing potent co-stimulatory molecules enhancing clinical efficacy through the modulation of anti-cancer immune responses. Firstly, we demonstrated the vector's identity and genetic stability by restriction enzyme assay and sequencing, then, by performing in vitro and in vivo pre-clinical studies we explored the anti-cancer efficacy of the virus alone or in combination with anti PD-1 inhibitor in human melanoma cell lines, i.e., MUG Mel-1 and MUG Mel-2, and in immunocompetent C57BL/6 melanoma B16V mouse model. We showed that both monotherapy and combination approaches exhibit enhanced anti-cancer ability and immunogenic cell death in in vitro settings. Furthermore, AdV-D24-ICOSL-CD40L combined with anti PD-1 revealed a fall in tumor volume and 100% survival in in vivo context, thus suggesting enhanced efficacy and survival via complementary anti-cancer properties of those agents in melanoma therapy. Collectively, the novel oncolytic vector AdV-D24-ICOSL-CD40L alone or in combination with anticancer drugs, such as check point inhibitors, may open novel therapeutic perspectives for the treatment of melanoma.

The SARS-CoV-2 ORF10 is not essential in vitro or in vivo in humans.

SARS-CoV-2 genome annotation revealed the presence of 10 open reading frames (ORFs), of which the last one (ORF10) is positioned downstream of the N gene. It is a hypothetical gene, which was speculated to encode a 38 aa protein. This hypothetical protein does not share sequence similarity with any other known protein and cannot be associated with a function. While the role of this ORF10 was proposed, there is growing evidence showing that the ORF10 is not a coding region. Here, we identified SARS-CoV-2 variants in which the ORF10 gene was prematurely terminated. The disease was not attenuated, and the transmissibility between humans was maintained. Also, in vitro, the strains replicated similarly to the related viruses with the intact ORF10. Altogether, based on clinical observation and laboratory analyses, it appears that the ORF10 protein is not essential in humans. This observation further proves that the ORF10 should not be treated as the protein-coding gene, and the genome annotations should be amended.

From Conventional Therapies to Immunotherapy: Melanoma Treatment in Review.

In this review, we discuss the use of oncolytic viruses and checkpoint inhibitors in cancer immunotherapy in melanoma, with a particular focus on combinatory therapies. Oncolytic viruses are promising and novel anti-cancer agents, currently under investigation in many clinical trials both as monotherapy and in combination with other therapeutics. They have shown the ability to exhibit synergistic anticancer activity with checkpoint inhibitors, chemotherapy, radiotherapy. A coupling between oncolytic viruses and checkpoint inhibitors is a well-accepted strategy for future cancer therapies. However, eradicating advanced cancers and tailoring the immune response for complete tumor clearance is an ongoing problem. Despite current advances in cancer research, monotherapy has shown limited efficacy against solid tumors. Therefore, current improvements in virus targeting, genetic modification, enhanced immunogenicity, improved oncolytic properties and combination strategies have a potential to widen the applications of immuno-oncology (IO) in cancer treatment. Here, we summarize the strategy of combinatory therapy with an oncolytic vector to combat melanoma and highlight the need to optimize current practices and improve clinical outcomes.

Prospects of Replication-Deficient Adenovirus Based Vaccine Development against SARS-CoV-2.

The current appearance of the new SARS coronavirus 2 (SARS-CoV-2) and it quickly spreading across the world poses a global health emergency. The serious outbreak position is affecting people worldwide and requires rapid measures to be taken by healthcare systems and governments. Vaccinations represent the most effective strategy to prevent the epidemic of the virus and to further reduce morbidity and mortality with long-lasting effects. Nevertheless, currently there are no licensed vaccines for the novel coronaviruses. Researchers and clinicians from all over the world are advancing the development of a vaccine against novel human SARS-CoV-2 using various approaches. Herein, we aim to present and discuss the progress and prospects in the field of vaccine research towards SARS-CoV-2 using adenovirus (AdV) replication deficient-based strategies, with a comprehension that may support research and combat this recent world health emergency.

Molecular diagnosis of COVID-19 ­ present experiences / Molekularna diagnostyka COVID-19 ­ dotychczasowe doswiadczenia.

Severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2), a new highly emerging and pathogenic for human RNA virus, is responsible for the present COVID-19 pandemic. Molecular diagnostic methods, including real-time reverse transcription-PCR (RT-PCR) assay are the recommended methods for the identification and laboratory confirmation of COVID-19 cases. RT-PCR allows for detection the RNA of the virus in clinical specimens from patients suspected of COVID-19 with high specificity and sensitivity. Testing is still crucial for rapid detection of infected persons, implementation of appropriate measures to suppress further virus transmission and mitigate its impact. In response to demand of a molecular diagnostic test for SARS-CoV-2, within a first few months ongoing pandemic many commercial kits has become available on the market. However, these tests have varied in number and type of molecular targets, time of reaction as well as quality. In this study we compared different commercial tests for the detection of SARS-CoV-2 in clinical samples sending to Laboratory of Department of Virology, NIPH-NIH.

Problem of rubella in Poland after compensatory outbreak in 2013 / Problem rózyczki w Polsce po epidemii wyrównawczej w 2013 roku

The attempt to estimate the real number of rubella cases in the years 2015-2016 in Poland was presented in this paper. The relations between number of reported cases of measles in 2006-2015y., the number of laboratory-confirmed cases of rubella among cases suspected of measles and the proportion of rubella among patients suspected of measles in the last 10 years as well as the results of serological examination in samples from 74 patients suspected of rubella collected in the first half of 2016 year were analysed. The sera from patients suspected of rubella were collected in cooperation with the State Sanitary Inspection. The analysis of data collected during the study-cases of suspected measles (2006-2016) and rubella infections (in 2016y.) indicated high over-registration of rubella in recent years in Poland, which is associated with a very low rate of laboratory confirmed cases.

Problem of immunoglobulin M co-detection in serological response to bacterial and viral respiratory pathogens among children suspected of legionellosis.

The objective of this research was an analysis of the serological response to respiratory bacterial and viral pathogens, in 156 children admitted to hospital in Warsaw with a suspicion of legionellosis. Levels of immunoglobulin (Ig) M to Bordetella pertussis, Mycoplasma pneumoniae, respiratory syncytial virus (RSV), adenoviruses, human parainfluenza virus (HPIV) t. 1-4 and influenza t. A + B viruses were determined retrospectively by ELISAs. In the prospective examinations (only Legionella pneumophila sg1), a positive level of IgM was found in 35 patients, but in 59 children together with retrospective tests. There were positive results for B. pertussis (21 children), followed by HPIV (10 children), M. pneumoniae (5 patients), RSV (4 persons), adenoviruses (3 children), and influenza A + B virus (3 persons). Positive results for > 1 agent were found in 16 children. The most often co-detected IgM were to L. pneumophila sg1 and B. pertussis (9 children) and L. pneumophila sg1 and M. pneumoniae (5 patients). The distribution of IgM to L. pneumophila sg1, B. pertussis and HPIV among children ≤ 4 years differed significantly from IgM specific to other pathogens. A high number of HPIV infections, mainly single, was found among infants. Positive results of IgM to L. pneumophila sg1 were mainly found in children aged 4-5 years. and the oldest children (over 10 years.). However, among the oldest children, anti-L. pneumophila sg1 antibodies were often found together with IgM to B. pertussis. Infections due to more than 2 pathogens were only observed among patients with pneumonia, especially due to L. pneumophila sg1 and/or B. pertussis. Conversely, co-detection of IgM ELISA for L. pneumophila and M. pneumoniae were mainly detected among patients hospitalized without pneumonia.

Cross-reactions in IgM ELISA tests to Legionella pneumophila sg1 and Bordetella pertussis among children suspected of legionellosis; potential impact of vaccination against pertussis?

The objective of this study was preliminary evaluation of IgM cross-reaction in sera collected from children hospitalized because of suspected legionellosis. Sera with positive IgM results to L. pneumophila sgs1-7, B. pertussis or with simultaneous detection of IgM antibodies to L. pneumophila sgs1-7 and B. pertussis, or IgM to L. pneumophila sgs1-7 and M. pneumoniae in routine tests, were selected. In total, an adapted pre-absorption test was used for the serological confirmation of legionellosis in the sera of 19 children suspected of legionellosis, and also in 3 adult persons with confirmed Legionnaires' disease. Sera were pre-absorbed with antigens of L. pneumophila sg1, B. pertussis or both, and tested by ELISA tests. The reduction of IgM antibody level by pre-absorption with antigen/antigens was determined. Reduction of anti-Lpsgs1-7 IgM by pre-absorption with L.pneumophila sg1 antigen ranged from 1.5 to 80, and reduction of anti-Bp IgM by pre-absorption with B. pertussis ranged from 2.0 to 23.8. Reduction by both antigens varied depending on the age of the patients: among children <4 yrs.old, the reduction of anti-B. pertussis IgM by both antigens was higher than for B. pertussis antigen alone. Based on the high difference (≥ 2 times) between reduction by L.pneumophila sg1 and by B. pertussis antigen, legionellosis was confirmed in 8/19 children. The majority of them also indicated IgM positive/borderline results for B. pertussis or M.pneumoniae in routine ELISA tests. As a preliminary, we posed a hypothesis of a potential impact of an anti-pertussis vaccination on the results obtained in anti-L. pneumophila ELISA IgM tests among young children.

Selected aspects of filoviruses in the view of Ebola virus disease epidemic.

Ebola virus disease (EVD) is a zoonosis of high virulence in humans. Current epidemic in West Africa is the largest EVD epidemic reported so far, exceeding the number of cases notified and geographical regions affected. This article discusses selected aspects of Ebola virus biology and ecology which are of significance for the processes of primary infection in humans and the spread of epidemic in population. A special attention was drawn to the issues essential for the diagnosis of infection and safety of testing.

Patients' age and the dynamics of IgM for L. pneumophila sg1.

MATERIAL AND METHODS: The results of IgM L. pneumophila sg1 test in 304 adults and 270 children performed at NIPH-NIH in 2004-2007 were analyzed to determine the effects of patients' age and the interval between collected sera on the results and the interpretation. RESULTS: Significant difference in the level of IgM, depending on the age of the patients (P0 = 0.0084) was found. Positive results (in total 20.4% of patients) were the most frequently observed in patients aged 19-29 years (42.5%), and the least--in patients 60 y.o. and < 2 y.o. (7%). Average and median levels of IgM in these two groups (+60 y.o. and < 2 y.o.) were similar and significantly different from the results in the other groups. From 44 adults and 33 children > or = 2 sera were collected. There was a significant difference in the interval between collecting the first and second serum sample in adults (mainly 3-5 weeks) and children (mainly 2-4 weeks). Significant increase of IgM levels was observed in children when the interval between 1 and 2 sample didn't exceed 4 weeks, while in adults this change was also observed at > 5 weeks (25% of patients). No significant differences in the analysis of the IgM ratio in children (1.25-14) and adults (1.5-26) was found, but longer persistence of IgM in adults than in children was observed. CONCLUSIONS: Demonstrated trend of faster decline in the level of IgM among children than in adults indicated that in suspected case of legionellosis in children, the serum sample should be taken up to 4-5 weeks after the onset, and at intervals of 1-2 weeks maximum.

Non-influenza viruses in acute respiratory infections among young children. High prevalence of HMPV during the H1N1V.2009 pandemic in Poland.

UNLABELLED: In Poland the majority of hospitalized cases of pneumonia (annually more than 70000) were reported without determination of an aetiological agent (J18 of ICD-10), also because diagnosis of viral ARTI is limited to identification of influenza viruses or sometimes RSV. MATERIAL AND METHODS: For determination the contribution of non-influenza viruses in ARTI among children, 381 nasopharyngeal swabs from hospitalized in period X.2008-IV.2011y. children (aged 1 day - 5 y.o.) were tested for RSV, HMPV, HEV/HRV, HPIV 1-3, HAdV, HBoV. RESULTS: At least one viral agent was detected in 72.7% of patients. The most predominant was RSV infection (49%), followed by HEV/HRV (15.5%); HMPV (8.7%), Adenoviruses (7.4%), HPIVt.1-3 (5.8%) and HBoV (5.5%). Seven periods based on the median of examined children/month were determined: 3 with increased number of ARTI. RSV infections, diagnosed in all periods, were predominate in five periods, mainly in LRTI cases. In the 3th period - HMPV was predominant, in the 5th - HEV/HRV. It was found that clinical manifestation of HMPV infections varied depending on the period. CONCLUSIONS: Relatively high prevalence of HBoV or HMPV cases of ARTI, especially different clinical picture in some periods (ARTI without pneumonia or bronchiolitis), indicated necessary of more detailed molecular and epidemiological studies. Also our results indicate the need for improved diagnostic capabilities of virological tests in acute upper and lower respiratory tract infections in children.

Imported cases of dengue in Poland and their diagnosis.

Infections with dengue virus are transmitted by mosquitoes. In tropical areas, it is mainly spread by Aedes aegypti while in countries with lower temperatures by Aedes albopictus. Since 2010, autochthonous cases of dengue are also reported in Europe. There are 4 serotypes of dengue virus (DENV). No correlation between clinical presentation of disease and virus type, however, were determined. Nevertheless, reinfection with different type of DENV may lead to a serious, life-threatening condition. An estimated 100 million persons are infected with dengue virus per year. Of them, approximately a half (mainly children) develop the symptoms of dengue fever (DF), dengue haemorrhagic fever (DHF) or dengue shock syndrome (DSS). Fatality is high in case of severe dengue. Dengue is a serious condition provided there is a presence of IgG antibodies directed against antigens of particular DENV serotypes, associated with primary infection caused by different serotype or transferred from infected mother to her child. For adequate dengue laboratory diagnosis, it is required to apply a set of various diagnostic methods. Within the family Flaviviridae, cross-reactivity is reported, which may lead to the occurrence of false-positive results. In Poland, differential diagnosis with different Flavivirus species is of special importance as it is an endemic area for tick-borne encephalitis (TBE). Thus, data regarding history of patient's immunization against TBE or yellow fever should be also taken into consideration as important in interpretation of results of serological examination.

Persistent colonization of 2 hospital water supplies by L. pneumophila strains through 7 years--sequence-based typing and serotyping as useful tools for a complex risk analysis.

Contamination with Legionella spp. of hot water system (HWS) in hospitals is a considerable problem and elimination of bacteria poses difficulties. Obligatory control of Legionella spp. in hospital HWS was implemented in Poland in 2008y. After that, Legionella spp. has been isolated repeatedly from HWS of the majority of hospitals. The aim of our study was to confirm the permanent colonization with Legionella spp. of 2 hospital HWSs based on the antigenic (serogroup/subgroups) and genetic properties (SBT, rtxA) of L.pneumophila strains isolated in 2004-2011. The dynamic of L.pneumophila population was also examined due to methods of disinfections applied during 7 years. Totally, 134 environmental samples were collected from two hospitals in 2004-2011 (118 from HWSs). During the study disinfection by chlorine dioxide was implemented in both hospitals, while thermal shock was added in the hospital A. Isolated L.pneumophila were serogrouped (105 strains) using Dresden MAb Panel, genotyped by sequence based typing (53) and by harboring of rtxA gene (58 isolates). Legionella spp. were still presented in both systems after 7 years. Exactly the same strains (ST1, ST87, ST114, ST992) were found in the hospital B. While changes of L.pneumophila population were observed in the hospital A: strains still occurred after 7 years (ST835 Sg6, ST114 Sg6); modified antigenic properties (ST835 - Sg12 vs. Sg6); eliminated or maybe not detected (ST81, ST838, ST959). Moreover, the majority of examined strains ST1 (Sg1, OLDA) harboured rtxA gene (hospital B). Our results and data in the EWGLI SBT base indicated higher risk of Legionella infection in the hospital B than A--because of heavy colonization with L.pneumophila ST1. The risk assessment of Legionella infection based only on technical parameters, extent of colonization/contamination level may be not completed. It should be supplemented with the additional examination: serotyping, genotyping and virulence testing of isolated strains.