Detection of Polioviruses in Sewage Using Cell Culture and Molecular Methods.

The work presented here demonstrates the utility of a two-step algorithm for environmental poliovirus surveillance based on: preselection of sewage samples tested for the presence of enteroviral genetic material-RT-PCR assay and detection of infectious viruses by cell culture technique (L20B for polioviruses and RD for polio and other non-polio enteroviruses). RD and L20B cell lines were tested to determine their sensitivity for isolation of viruses from environmental samples (sewage). Finally, we wanted to determine if sewage concentration affects the results obtained for RT-PCR and cell cultures.

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.

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.

Seroprevalence of measles-specific antibodies in the group predominantly affected by measles in 2006-2009 in Poland.

INTRODUCTION: Irrespective of the high vaccination coverage against measles, sporadic measles outbreaks still occur in Poland. In 2006-2009, a slight increase in the number of measles cases was observed. Of these cases, people born in 1976-198 9 were predominantly affected. AIM: The aim of the study was to evaluate the immunity to measles in the aforesaid age group. MATERIAL AND METHODS: The serum samples were selected from the serum bank in which material collected from the general population living in 5 provinces in Poland is stored. These samples were collected from patients hospitalized due to emergencies in 2010-2011. The antibody titre against measles was determined in each serum sample by ELISA test (Genzyme Virotech). Linear regression models using log-transformed antibody titres were used to compare the values. RESULTS: The serum samples collected from 483 persons, including 111 females and 372 males were tested. All patients had antibody titres exceeding 0.5 IU/ml. The antibody titre was statistically significantly associated with the vaccination coverage in each age group in particular province. CONCLUSIONS: The results of this study suggest good immunity to measles in the general population in Poland. The disparities between randomly selected provinces demonstrated a relation with the coverage rates as well as the differences in measles incidence which is observed recently between these provinces.

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.

RSV respiratory infection in children under 5 y.o.--dynamics of the immune response Th1/Th2 and IgE.

INTRODUCTION: The imbalance of Th1/Th2 cytokine concentrations and increased level of IgE might be useful tool for prediction of severity of RSV infection among young children and possibility of sequels. The ratio of cytokines Th1/Th2 varied during the disease. THE AIM of our studies was the assessment of immunological response by dynamics of Th1 and Th2 cytokines and IgE in RSV infections. MATERIAL AND METHODS: Study was done on sera collected from 36 young children hospitalized because of RSV infection and from 16 children with other respiratory tract infection (HMPV, EV, HPIV1-3). Assaying of the serum levels of cytokine Th1 (IL-2, IFN-g, TNF), Th2 (IL-4, IL-6, IL-10) and concentration of IgE has been done. Paired sera (48 patients) were collected in the interval 4-14 days. Reference group consist of 18 children (< 6 months of life) hospitalised because other than respiratory diseases with negative results for viruses tested by PCR. RESULTS: Among children with respiratory infection the Th1/Th2 ratio was shifted towards Th2, level of IgE increased in comparison to the reference group. Changes in concentration of IL-6, IFN-g, IL-10 were related to RSV infection, mainly bronchitis and bronchiolitis, while the dynamic of TNF concentration was independent on aetiological agent. It was found that the risk factors (prematurity, artificial nutrition) correlated with RSV bronchitis and the levels of cytokines and IgE. Increased level of IL-6 and IL-10 were shown in prematures, and increased concentration of IgE--among artificial nourished children. The time of serum collection affected the level of cytokines and IgE and the effect was depended on the aetiological agent. In RSV infections was observed significant decrease with time of IL-6, IL-10 and IFN-gamma but not IgE (still significantly higher than in the reference group). While the significant decrease of IgE was determined only in other than RSV infections. CONCLUSIONS: Determined level of cytokines and IgE varied depending on the time of serum collection. Observed dynamics in paired sera and IgE might have prognostic value in disease and sequels of RSV infections. Prevention RSV infection in premature infants should be done in any possible way. Breastfeeding is one of the critical elements of prevention of RSV infection.

Sequence based typing and pre-absorption test in retrospective analysis of a pseudo-outbreak of Legionella infections differentiates true cases of legionellosis.

UNLABELLED: The aim of this study was elimination of false positive results obtained by the Chlamylege kit. Two serological kits (IgM ELISA L. pneumophila sgs1-7; ImmuView(TM) L. pneumophila sg1/sg3) and pre-absorption tests (with L. pneumophila sg1 and sg3 reference strains antigens) were used. 153 sera (79 patients) were examined. The high correlations were found between the results by both tests. Positive results by ELISA (sgs1-7) were found in 19/79 patients; by ImmuView(TM) (sg1+sg3) in 16/63. In 8 patients, the dynamics of the IgM in pairs of sera was high (ratio ≥2). In 5/8 of those patients seroconversion was determined. Selected pre-absorbed sera (15 pairs) were tested simultaneously by the same tests. In 8/15 pairs of sera, the reduction of IgM levels in pre-absorbed sera was higher than 10. The reduction of IgM differed in sg1 and sg3 tests. The probability of infections due to L. pneumophila sg3 (7 patients) and L. pneumophila sg1 (5 patients) was based on the results of pre-absorption tests. The correlation between ELISA and ImmuView(TM) tests of pre-absorbed sera was statistically significant (Po=0.0389). Moreover, genotyping of L. pneumophila (SBT) directly in the sera of selected 15 patients (high IgM reduction) was carried out. Completed 7 alleles profile (ST36) was determined in one patient. However, a second patient had the same profile of 5 alleles, and similar reactions in pre-absorption tests. At least 4 sources of infections were suggested on the base of genotyping and pre-absorption results. CONCLUSIONS: Positive results obtained by molecular techniques (eg.PCR) in the diagnosis of Legionella infections should be supplemented by other tests for confirmation of legionellosis. The sequence based typing carried out directly in clinical specimens seems to be a promising method.

Infections caused by RSV among children and adults during two epidemic seasons.

Respiratory Syncytial Virus (RSV) is one of the most common causes of lower respiratory tract infections in young children, immunocompromised patients (children and adults), patients with chronic respiratory diseases and elderly people. Reinfections occur throughout the life, but the severity of disease decreased with subsequent infection. The aim of this study was to analyze the frequency of RSV infections in two selected subpopulations: young children (below 5 y.) and adults with chronic respiratory diseases (25-87 y.). Nasopharyngeal swabs (334) collected from October 2008 to March 2010 were examined. The presence of RSV genome was determined by RT-PCR and the presence of RSV antigen by quick immunochromatographic test. Positive results of RT-PCR were found in 45.2% of all swabs: 48.6% samples in 2008; 41.5% in 2009; 50.8% in 2010. The highest frequency of RSV-positive samples was in fall-winter months, but differences in RSV epidemic seasons were found. In the first season (2008-2009) an increased number of RSV infections was observed from November 2008, but in the second season--from January 2010. Generally, the frequency of RSV-positive RT-PCR among children was 53%, among adults 25%. The highest difference was observed in the first three-month period of 2010. RT-PCR positive samples were found in 68.5% of children and 5.9% of adults. However, the RSV antigen was found in 44.4% of samples collected from adults in this period. Our results indicate that the contribution of RSV infections during epidemic season of respiratory tract infections in Poland was really high among children and adults.

Detection of hMPV antigen by EIA in clinical specimens.

UNLABELLED: Human Metapneumovirus (hMPV) is one of the latest discovered viruses. It has been classified to Paramyxoviridae family. It is the second viral etiological agent, after RSV, which causes respiratory tract infections (RTI) in children, especially children below 5 years old. It is estimated that 5-25% of RTI in children is due to hMPV. In adults hMPV reinfections are bounded to upper respiratory tract infections. The aim of the study was to establish usefulness of ELISA test in detecting hMPV antigen and to analyze hMPV infection in connection to clinical diagnosis. MATERIAL/METHODS: 273 nasopharyngeal swabs from children (189 swabs) and adults (84 swabs) with respiratory tract infections collected from 2008 to 2010 were examined. Due to similarity of hMPV and RSV viruses and overlapping of their epidemic season rapid immunochromatographic test for RSV antigen detection was also performed in case of 120 samples, hMPV antigen was detected in 24.5% of all swabs (n = 67): in 0.0% probes in 2008, 29.0% in 2009 and 36.8% in first quarter of 2010. The highest rate ofhMPV infection was detected from summer of 2009 till the end of March 2010 (VIII-IX 2009 - 62.5%, X-XII 2009 - 44.1% and I-III 2010 -36.8%). We analyzed respiratory tract diseases reported in patients with hMPV infection. Infection due to hMPV was found in 26.5% of children and 24.0% of adults with recognized pneumonia, respectively in 28.4 and 17.6% of patients with bronchitis. Bronchiolitis was diagnosed in two children with hMPV. RSV and hMPV coinfections were confirmed in 15 out of 120 examined probes. Cross reaction pattern was excluded thanks to ELISA hMPV antigen test which was performed with suspension of RSV and thanks to statistical analysis. Coinfections were confirmed in 8% of pneumonia, 11% of bronchitis and 24.2% of the rest concomitant diagnoses. CONCLUSIONS: We found hMPV infection as the significant agent ofpneumonia not only in children but also in adults. ELISA hMPV antigen test can be used in diagnosis of etiological agent of respiratory infections in children and adults and in coinfections as well.

[Demographic characteristics of reported cases of legionellosis in years 2005-2009 in Poland in comparison to EWGLI data]. / Charakterystyka demograficzna zachorowan na legioneloze w Polsce w latach 2005-2009 w swietle danych gromadzonych przez European Working Group for Legionella Infections (EWGLI).

In Poland, all forms of legionellosis are obligatory reported: Legionella pneumonia and extrapulmonary legionellosis (such as Pontiac fever). Total 210 cases caused by Legionella sp. were reported in years 2003-2010. In our study analysis of age, sex, place of residence of 163 patients with legionellosis reported in 2005-2009 were done. The majority (74.2%) cases were reported in Region Mazowieckie. Age and sex structure of patients in Poland in years 2005-2009 differ from the data of EWGLI. In Poland, legionellosis was much more frequently reported in children - 46.6% of the total reported, than in other countries. It might be a result of collaboration with the paediatricians during research conducted by our team in 2005-2007. Reported cases of legionellosis were also observed in 10 children aged < or = 2 years. Totally, legionellosis was observed more frequently among girls than in boys (M: F = 1:1,71), and in males (M: F = 1,64:1). It was also found that in smaller towns (< 100 thousand inhabitants) legionellosis was much more frequently diagnosed in girls (Po = 0.0152, 71.1% of ill children) and men (65.8% of adults). In larger cities (> 100 thousand.) differences in incidence between girls and boys and men and women were lower. To sum up: the number of reported cases of legionellosis depends largely on the availability of laboratory tests and the knowledge of physicians. Extension of diagnostic testing in the direction of Legionnaires' disease patients belonging to other than the high risk group increased number of detected cases, including those not so severe.

[Immunity against tick borne encephalitis virus (TBE) in population of forest workers in Bialowieza]. / Odpornosc dla wirusa kleszczowego zapalenia mózgu (kzm) w populacji pracowników lesnych z terenu Bialowiezy.

In Poland, 200 cases of tick borne encephalitis are annually registered. It is about half of all registered cases of viral encephalitis. Immunity acquired after vaccination with TBE inactivated vaccine persists defined time dependent on number of dose of vaccine and individual features of vaccinated person. The aim of this paper was to determine the frequency of IgG antibodies against TBE virus among forestry workers from Bialowieza National Park with regard to information about vaccination against TBE virus. In the group of 59 persons vaccinated against TBE virus, 54 (91.5%) had IgG antibodies against TBE virus. In the group of 32 persons (non-vaccinated or for whom the information about vaccination was not given) 26 (81.3%) were seropositive. Relationships between VE value (determining level of antibodies), age and period passed from the last dose of vaccination were analysed. Taken to account the most disadvantageous circumstances (age = 60, regression intercept minus two standard deviation) antibodies level of VE = 11 (lower limit of positive result) remains for 2,72 years from last dose of vaccine against TBE virus. This result confirm recommendation about necessity to use of complementary dose within 3-5 years, at the same time it should be considered recommendation of necessity ofrevaccination persons 60 years old or above as frequently as every 3 years.

Tick-borne encephalitis outbreak in Hungary due to consumption of raw goat milk.

A tick-borne encephalitis outbreak involving 25 patients of 154 exposed persons occurred in Hungary in August 2007. None of the patients had a history of tick-bite, however all of them drank unpasteurized raw goat milk from the same farm. The aim of this study was to identify the goats on the farm which could have spread the infection through their milk. Blood samples were taken from 75 goats on the farm and were examined by various serological methods, namely indirect immunofluorescent assay, hemagglutination inhibition, microneutralization and an ELISA adapted to testing material from goats, to determine antibody levels in the serum. The four methods have proved different levels of specificity. The least specific was the indirect immunofluorescent assay, which showed a low titre in all sera. Comparison of the results of the other three methods indicates that two sera were positive for anti-TBEV IgG and one for anti-TBEV IgM. The goat with the IgM positive serum sample could have been a source of the infected milk. It has been concluded that serological results for goats by the different methods should be compared before final diagnosis because the specificity of methods in use can differ significantly.

[Detection of TBEV RNA in ticks as a tool for valuation of endemic area wide and sensitivity of TBE surveillance]. / Obecnosc RNA wirusa kleszczowego zapalenia mózgu w kleszczach Ixodes ricinus jako narzedzie oceny zasiegu obszarów endemicznych i czulosci nadzoru nad zachorowaniami na KZM.

In this study we present the nested RT-PCR strategy designed for detection of TBEV RNA in ticks Ixodes ricinus. The presented nested RT-PCR method using 2 different primer pairs specific primers for NS5 gene provides specific TBEV cDNA detectable by electroforesis in agarose gel. Of the 177 polls of ticks investigated, TBEV RNA was detected in 14, which accounts for 7.9% of all pools. We confront the PCR results of tested ticks to routine surveillance data. The obtained results showed that the TBEV RNA is detectable in ticks collected in areas in Poland, which are defined as an non-endemic. The nested RT-PCR method can be used as a tool of epidemiological surveillance as well as for screening of occurrence of circulating TBEV.

[Hantavirus specific IgG antibodies in population of zoologists and forest workers]. / Wystepowanie przeciwcial klasy IgG swoistych dla hantawirusów w populacji pracowników lesnych i zoologów.

Serological diagnostic tests were introduced for detection of immunoresponse against to etiological agents of current infectious diseases. Interpretation criteria for those tests should eliminate unspecific reactions, cross-reactivity with related viruses as well as traces amounts of immunological response against to the past infections. In the case of past hantaviruses infections for obtaining an adequate seroepidemiological data revalidation of this tests are required. In present studies Puumala IgG results obtained in sera from healthy population had been analysed by statistical method and interpretation criteria were recalculated. Panels of 86 sera from forest workers and 47 zoologists working with small mammals were evaluated for hantavirus specific IgG (Hantavirus Puumala ELISA test). Puumala specific antibodies were detected in 7 zoologist's sera and in 5 sera gave equivocal results. All sera collected from forest's workers were negative. Statistical analysis based on negative results in forest workers group suggests that the cut off of the ELISA test suitable for diagnosis of suspected nephropathia epidemica cases is too restrictive for seroepidemiological research of hantavirus in healthy population.

[The cross and unspecific reactions in serological examination for antibodies against hantavirus Puumala]. / Problem wystepowania reakcji krzyzowych i nieswoistych w badaniach odpowiedzi humoralnej dla hantawirusa Puumala.

A serological survey of 78 zoologist capturing small wild rodents in their environment, 9 patients with suspected hantavirus infections and 21 patients with acute renal dysfunction for antibodies to hantaviruses was conducted in Poland. Survey was done by the indirect ELISA with Puumala and Hantaan virus antigens. Out of the 78 mammalogists 15 were seropositive for hantavirus Puumala IgG without history of clinical illness. Analysis of relation between reactive zoologist's sera IgG with antigens of Puumala and Hantaan viruses suggests that these persons had contact with Puumala/Tula viruses rather than with Dobrava/Saaremaa complex. Analysis of results of IgG and IgM presence by ELISA test have confirmed correct interpretation criteria proposed by manufacturer for serological diagnosis of suspected hantavirus infection. Both cross and unspecific reactions in the some sera have been observed. Low number of patients with suspected hantavirus infection suggests the existence of underestimation in registration of data collected in Poland and existence of non-diagnosed infections with hantaviring.

[Occurrence of human hantavirus infections in Poland]. / Ocena problemu wystepowania zakazen ludzi hantawirusami na terenie Polski, ze szczególnym uwzglednieniem wirusa Puumala.

A spread of hantavirus infections causing hemorrhagic fever with renal syndrome is one of the rising epidemiological problems in the world. There is evidence that this type of viruses exists in almost all European countries. It is known that in Poland all the species of wild rodents hosting European hantaviruses are present. However, the data on hantavirus are very limited and information on incidence and prevalence of hantavirus infections among humans and animals in our country is lacking. Up to now, only one study in Poland has dealt with hantavirus infections in humans. Therefore the aim of the presented study was to show whether those infections are present in Poland and how prevalent they are. Two risk groups of hantavirus infection were investigated: group I--people with renal disorders (hospitalized patients with proteinuria and people suspected to hantavirus infection; n=30 people) and group II--healthy people employed at the universities, scientific institutions who due to their profession have contact with small rodents as well as students who captured those rodents when practicing or preparing their master theses (n=76) and forest workers (n=86). No one from the group of patients with renal disorders was positive for hantavirus Puumala IgG. Moreover, none of the forest workers had evidence of hantavirus infection. The hantavirus IgG were found only in the group of mammologists--19% persons were positive. Mammologists are an occupational subpopulation who remain in direct contact with host and/or with host excretions (virus is present in urine, feces, saliva of infected animals) and seem to be the most sensitive indicator of hantaviruses presence in environment. These results prove occurrence of hantavirus infections in Poland, however it seems that they are not very prevalent.

[Measles elimination programme on the world and Poland]. / Podstawy programu eliminacji odry na swiecie i w Polsce.

Measles is still one of the leading causes of children mortality, despite of availability of a cheap, effective and safe vaccine for more than 40 years. Effective global eradication of smallpox and the success of polio eradication have provided an incentive to achieve the measles eradication all over the world. Elimination is achieved when no endemic measles cases has been observed in given area. This study analyzes epidemiological situation of measles, measles vaccination and laboratory diagnosis. In this report we describe the role of Global Measles Laboratory Network and integrated measles/rubella surveillance. The National Laboratory in Poland is based at the Department of Virology in National Institute of Hygiene, Warsaw. Following the implementation of 2-dose measles vaccine schedule the epidemiologic situation of measles has improved. In 2006 results of genotyping indicate that recent outbreaks were caused by local strains of the virus (D4 and D5).

[Effect of cytomegalovirus strain selection on stimulation of cytokine panel Th1/Th2]. / Wplyw doboru szczepów wirusa cytomegalii na stymulacje cytokin panelu Th1/Th2.

CMV infection can undergoing as primary or secondary infection, which can be represented by reactivation of endogenous infection or by superinfection with another viral strain. Taking for consideration high prevalence of seropositive persons it may be concluded that superinfections occur quite often. For the other hand it is known, that differences among CMV strains are deep and regard as well viral structure as its biological properties. The aim of this study was investigation of immunological profile in range of cytokines production after stimulation with different CMV strains. The study was conducted with leukocytes obtained from CMV seropositive person cultivated and stimulated in vitro with AD169, Towne and Davis. After 24, 48, 72 and 96 hours incubation concentrations of IL-2, IL-4, IL-5, IL-10, INF-gamma and TNF-alpha in supernatants was measured by flow cytometry with Cytometric Bead Array technique. Obtained results allow to conclude that difference in level of stimulated cytokines and their panel is dependent on viral strain used. The experiment also allows to optimisation of stimulation conditions.

Differences in stimulation of CD45+ cells from mouse spleen population enriched in dendritic cells depending on the virulence of herpes simplex virus type 1 strains.

The interaction of CD28 with one of the B7 molecules (CD80 and CD86) on professional antigen-presenting cells (APC) is generally considered to be the most important co-stimulatory signal for T cell activation. Several lines of evidence suggest that dendritic cells (DC), the most potent antigen presenting cells known, play a role in the immunological control of herpes simplex virus (HSV) infections. The fact that CD86 is strongly up-regulated together with other co-stimulatory molecules during DC maturation suggests that it plays an important role in induction of immune response. To determine the effect of virulence on up-regulation of CD86, we stimulated population of spleen cells enriched in dendritic cells by HSV-1 strains characterised by different pathogenicity. We analysed cells, which express CD45 molecule. HSV-1 ts, earlier described as less virulent for mice, stimulated an increased expression of co-stimulatory molecule CD86 than wild strain did.