[The occurrence of infections caused by Francisella tularensis in humans in Poland and laboratory diagnosis of tularemia].

Tularemia is a serious infectious zoonotic disease, caused by Gram-negative bacterium Francisella tularensis. Natural reservoir of infection are small mammals such a mice, voles, squirrels and rabbits. Transmission to humans occurs through contact with infected animals or contaminated environments, or through arthropod vectors. Because of its extreme infectivity it is a dangerous biological agent to human health. Tularemia has a broad geographical distribution, however is mainly in the northern hemisphere, in areas with cooler climates particularly in North America, Europe, Russia and Japan. Most of the cases among European countries have been reported in the Scandinavian region. The prevalence rate of tularemia in Poland is small, although in recent years stable increase has been observed. According to official epidemiological data during the years 2010-2016 only 61 cases of tularemia were reported in Poland. A laboratory diagnosis of tularemia is based on serological investigation, classical microbiology and molecular biology. The aim of this study was to evaluate the prevalence of infections caused by Francisella tularensis in humans in Poland and present characteristics of laboratory diagnosis of tularemia.

[Enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Salmonella Typhi lipopolysaccharide O and capsular polysaccharide Vi antigens in persons from outbreak of typhoid fever]. / Zastosowanie odczynu immunoenzymatycznego ELISA do poszukiwania przeciwcial dla lipopolisacharydowych antygenów somatycznych O oraz otoczkowego antygenu Vi paleczek Salmonella Typhi u osób z epidemicznego ogniska duru brzusznego.

INTRODUCTION: The laboratory diagnosis of typhoid fever is dependent upon either isolation of S. Typhi from a clinical sample or the detection of raised titers of serum antibodies in the Widal test or the passive hemagglutination assay (PHA). In this study we evaluated the usefulness of ELISA for detection of antibodies to S. Typhi lipopolysaccharide O and capsular polysaccharide Vi antigens in the sera of persons from outbreak of typhoid fever. MATERIAL AND METHODS: Fifteen serum samples from patients with laboratory confirmed typhoid fever and 140 sera from persons suspected for contact with typhoid fever patients from outbreak in 1974/75 in Poland were tested by ELISA. Additionally, as the control group, we tested 115 sera from blood donors for the presence of S. Typhi anti-LPS and anti-Vi antibodies. RESULTS: Anti-LPS and anti-Vi antibodies were detected in 80% and 53.3% of sera obtained from patients with laboratory confirmed typhoid fever, respectively. The high percentages of positive results in ELISA were also noted in the group of persons suspected for contact with typhoid fever patients (51.4% and 45%) but not in the group of blood donors (7.8% and 6.1%, respectively). CONCLUSIONS: The ELISA could be a useful tool for the serological diagnosis of typhoid fever in patients who have clinical symptoms but are culture negative, especially during massive outbreaks of typhoid fever.

[Seroprevalence of Mycoplasma pneumoniae in Poland in 2008-2013]. / Wystepowanie zakazen Mycoplasma pneumoniae w Polsce w latach 2008-2013 na podstawie wyników badan serologicznych.

INTRODUCTION: Mycoplasma pneumoniae is a common causative agent of tracheobronchitis and atypical pneumonia, mainly in children and adolescents. The infections are often seen as epidemics occurring in autumn-winter seasons at intervals of 4-7 years. Epidemiological studies showed that M. pneumoniae is responsible for 30% to 40% of all cases of bacterial respiratory infections in Poland. The aim of the study was estimate the seroprevalence of M. pneumoniae in Poland in 2008-2013 in comparing to results obtained in other European countries. MATERIAL AND METHODS: The results of diagnostic serological tests (ELISA) in particular immunoglobulin classes for infection with M. pneumoniae performed in 16.825 persons were retrospectively analyzed. The patients were mostly children at the preschool and school age with clinical symptoms of respiratory tract infection. The data were obtained from Bacteriology Department of National Institute of Public Health-National Institute of Hygiene in Warsaw and from 13 Sanitary and Epidemiological Stations through the country which send quarterly or monthly reports. RESULTS: The serological results showed that in autumn-winter seasons of 2011-2012 the "early antibodies" (IgA and/or IgM) for M. pneumoniae were twice more often diagnosed in sera of patients with respiratory tract infection than in analogous seasons of 2008-2010. The antibodies were detected in 34% and 42% of patients, respectively in third quarter of 2011 and 2012. CONCLUSIONS: Epidemic increase of M. pneumoniae infections in Poland in autumn-winter seasons of 2011-2012 was mainly observed due to diagnosis of the IgA and/or IgM antibodies in serological tests.

[Serodiagnosis of clinical infections caused by Mycoplasma pneumoniae in Poland in 1970-2010]. / Serologiczna diagnostyka objawowych zakazen wywolywanych w Polsce w latach 1970-2010 przez Mycoplasma pneumoniae.

The aim of the study was evaluation of the reliability of serodiagnosis of mycoplasmosis in Poland after replaced of classical assays, as complement fixation test (CFT) and immunoelectroprecipitation test (IEPT), by the ELISA method. The data were obtained from National Public Health Institute in Warsaw (NPHI), which receives quarterly reports of serologically confirmed infection from Sanitary and Epidemiological Stations through the country. Previously, from the 1970 to 1999 the serodiagnosis in Poland was performed only by uniform CFT using the same M. pneumoniae FH antigen prepared at the Mycoplasma Laboratory of NPHI. The first data of M. pneumoniae serological investigation performed by commercial ELISA, mainly Virotech, which gradually replaced the CFT, were obtained in 2000. In the studied period 1970-2010 a total of over 300 thousand patients with respiratory tract infections (85% were children below 18 years old) were tested. During these studies five epidemics of mycoplasmosis were noted in Poland before 2000. However, after introduction of ELISA for serodiagnosis this characteristic distinct difference in epidemic and endemic occurrence of M pneumoniae infections have not been seen. In our opinion it may be caused by changes in the epidemiological pattern of M. pneumoniae infections in Poland as well as, paradoxical, by the decrease of sensitivity and specificity of serological investigation performed by ELISA since 2000. First of all, for the economical reasons 98% of the patients were tested by ELISA only once, secondly, the mycoplasmosis in many laboratories was confirmed only on the basis of the presence of IgG antibodies. The results of our analysis showed also usefulness in the serodiagnosis of mycoplasmosis, mainly during the first 2 weeks of the disease, of IEPT, which detect only specific IgM antibodies to M pneumoniae antigens.

[Application of enzyme linked immunosorbent assay (ELISA) for diagnosis of antibodies to lipopolysaccharide of Klebsiella rhinoscleromatis in patients with rhinoscleroma]. / Ocena przydatnosci immunoenzymatycznego odczynu ELISA do wykrywania przeciwcial dla lipopolisacharydu paleczek Klebsiella rhinoscleromatis u chorych na twardziel.

The ELISA were performed on polystyrene microtiter plates (Nunc, MaxiSorp) coated with LPS (2a antigen) at the final concentration of 10 microg/ml. The antigen was extracted from Klebsiella rhinoscleromatis Rh32 by the trichloroacetic acid and separated by ethanol (Boivin method). The antibodies against the LPS were detected by ELISA in serum samples collected from 65 patients suspected in clinical investigation for rhinoscleroma in Poland from 1970 to 2009. Additionally, the specificity of the antigen was tested using serum sample of immunized rabbit and 30 sera of patients from control group, with high level of antibodies to different bacterial pathogens. All serum samples were diluted 1:100. The concentrations of IgA, IgG and IgM antibodies were expressed as optical density (OD) measured at the wavelength of 450 nm. The cut-off limit of serum antibodies was set at mean antibody OD determined in the sera of 30 blood donors exceeded by three standard deviations. The presence of IgA and IgG antibodies were detected by ELISA in 33 (50,8%) and IgM in 28 (43,1%) of patients. Most of the serum samples (75%) with high level of specific antibodies were obtained from patients before 1980. On the other hand antibodies to K. rhinoscleromatis were detected only in 2 (6,7%) patients from the control group and none of blood donors. In conclusion, our home-made ELISA, based on purified LPS of K. rhinoscleromatis showed high specificity and sensitivity in the diagnosis of antibodies to K. rhinoscleromatis in comparison to the complement fixation test. The presence of high level of specific IgA, IgG and IgM antibodies in the sera obtained in different stages of disease may showed that during the rhinoscleroma is permanent stimulation of antibody production.

Usefulness of PCR melting profile method for genotyping analysis of Klebsiella oxytoca isolates from patients of a single hospital unit.

The development of rapid and simple typing methods is required in order to identify possible sources of human exposure to opportunistic pathogens. Klebsiella spp. belongs to a group of bacteria that are opportunistic pathogens responsible for an increasing number of multiresistant infections in hospitals. Recently, we showed the high genetic diversity of K. oxytoca using a large collection of strains isolated from the patients of several hospitals in Poland over a 50-year period. Our results showed that the internal transcribed spacer polymerase chain reaction method (ITS-PCR) is useful for the phylogenetic delineation of genetic groups in K. oxytoca and the high discriminatory power of the PCR melting profiles (PCR MP) method can be useful for epidemiological studies of K. oxytoca. In the present study the usefulness of PCR MP was tested on two sets of strains isolated from a single unit over a short period of time. The results revealed that PCR MP has a high discriminatory power and can be useful for epidemiological studies of closely related strains of K. oxytoca isolated from a single unit over a short period of time to identify the source, reservoirs and the tract of infection spread. The advantage of PCR MP for the above application was shown by using the procedure at increasing denaturation temperature during PCR to confirm genotyping results. Considering this feature and the high discriminatory power of PCR MP, as shown in this report for determination of the genetic similarities of consecutive K. oxytoca strains, we propose that PCR MP is one of the best techniques for short-term epidemiology analysis.

[Evaluation of the effectiveness of serodiagnosis of Mycoplasma pneumoniae infections by ELISA in Poland in 2006-2008]. / Ocena efektywnosci diagnostyki testem ELISA zakazen wywolywanych przez Mycoplasma pneumoniae w Polsce, w latach 2006-2008.

This study analyses the results of over 15 thousand patients tested by ELISA against the presence of antibodies to Mycoplasma pneumoniae in Poland in 2006-2008. Most of the tested patients were hospital-treated children with lower respiratory tract infections or pneumonia. The positive results of ELISA were obtained in 4.513 (29.6%) patients. Among them IgG and IgM antibodies were diagnosed in 24.0%, antibodies only class IgG in 44.0% and only IgM in 32.0% of cases. The results of our study showed the oversensitivity of serodiagnosis of mycoplasmosis based only on the presence in the tested sera of IgG antibodies.

Multiplex-PCR assay for identification of Klebsiella pneumoniae isolates carrying the cps loci for K1 and K2 capsule biosynthesis.

Multiplex-PCR assay for identification of Klebsiella pneumoniae isolates carrying gene clusters for biosynthesis of capsular polysaccharide (CPS) types K1 and K2 was developed. Genes wzc and orf10 of the cps cluster were applied as K1 and K2 specific markers respectively. The assay specificity was confirmed using 147 isolates of Klebsiella spp. including 77 K-antigen reference strains. The multiplex-PCR assay was found simple and cost-effective tool for identification of K. pneumoniae clinical isolates of K1 and K2 geno-serotypes.

[Single strand conformation polymorphism of selected genes of the Klebsiella pneumoniae cluster waa for core lipopolysaccharide biosynthesis]. / Zróznicowanie profili jednoniciowego DNA (SSCP) wybranych genow locus waa zwiazanych z wytwarzaniem lipopolisacharydu u paleczek Klebsiella pneumoniae.

We aimed to determine single strand conformation polymorphism (SSCP) of selected waa cluster genes (waaA, waaE, waaL, waaQ and waaZ) involved in core lipopolysaccharide (LPS) synthesis in reference and epidemic strains of Klebsiella pneumoniae. Number of 24 reference strains belonging to serogroups O1, O2a, O2a2e, O2a2e2h, O2a2f2g, O3, O4, O5, O7, O8, and O12 was tested together with 13 epidemic strains from 5 outbreaks and 6 casual isolates using PCR and Multitemperature-SSCP. Based on PCR-SSCP results, from 4 to 8 patterns (genotypes) were distinguished for each analysed gene. Predomination of single genotype ranging from 28% to 76% for waaL and waaE respectivelly was observed in tested strains. The average predomination for other genes was about 36%. Although no correlation was observed between genotypes and serogroup of tested strains, it is notheworthy that epidemiologically linked isolates belonged to the same genotype. Therefore reported here heterogeneity of tested genes may be potentially useful for K. pneumoniae strains subtyping by SSCP or DNA sequencing.

[Molecular characterisation of the haemolytic isolates of Bacillus anthracis originated in Poland]. / Molekularna charakterystyka hemolizujacych szczepów bacillus anthracis izolowanych w Polsce.

UNLABELLED: Bacillus anthracis is generally considered non-haemolytic, when cultured on the solid media. However, strains capable to lyse sheep erythrocytes have been reported. Anthrolysin O, an orthologue of cereolysin was proposed as a putative haemolysin of B. anthracis. AIM: to determine whether anthrolysin O, haemolytic enterotoxin HBL and the pleiotropic regulator PlcR that activates antrholysin O production are associated with a haemolytic activity of B. anthracis strains isolated in Poland. MATERIAL: in total 8 B. anthracis strains - the fully virulent BL1 and seven the pXO2 lacking strains including: a vaccine strain Sterne 34F2 together with three haemolytic and three non-haemolytic strains isolated from different samples of the same animal died from anthrax in Poland. METHODS: The haemolytic activity was detected using Columbia agar plates supplemented with 5% of sheep blood. Anthrolvsin O, cereolysin and gene hblA encoding the key subunit of the HBL were detected by PCR. In addition, the plcR gene fragment containing the B. anthracis specific non-sense mutation was analysed by the DNA sequencing. Ten marker loci based MLVA genotyping was performed to distinguish tested strains. RESULTS: The alo gene encoding anthrolysin O was detected in both the haemolytic and non-haemolytic strains while hblA was absent. The B. anthracis specific plcR non-sense mutation was detected in both the groups of tested strains, suggesting that the haemolysis in tested strains may rather be conferred by the PlcR-independent factors. Moreover, haemolytic and non-haemolytic strains were indistinguishable by the MLVA. Obtained results may argue the haemolytic and non-haemolytic strains are isogenic and most probably a single mutational event is responsible for the haemolytic phenotype induction.

[Single strand conformation polymorphism (SSCP) of selected loci of the cps region for capsule synthesis in epidemic and casually isolated strains of Klebsiella pneumoniae in Poland]. / Polimorfizm profili jednoniciowego DNA (SSCP) rejonu locus cps wspólnego paleczkom Klebsiella pneumoniae u szczepów epidemicznych i przypadkowo izolowanych.

The SSCP of ORFs 1, 2, 3, 15 from the cps region for capsule biosynthesis was determined for 56 epidemic isolates, 4 reference strains of K. pneumoniae including A5054 and B5055, and 12 strains isolated casually from stool samples. From 6 up to 14 different SSCP-profiles were observed for tested loci, which combined together distinguished 31 SSCP-genotypes. Epidemic strains could be diversified into 15 genotypes whereas 11 genotypes were detected for 12 casual isolates. Strains from the same outbreak belonged to a single genotype. Strains from different outbreaks represented separate genotypes, however majority of them was located in the same main branch of a dendrogram based on the cluster analysis of the SSCP-profiles diversity. Obtained results may suggest high genetic diversity of tested loci. The SSCP genotyping of multiple cps loci was found as potentially useful tool for tracing epidemic strains during an outbreak.

[Occurrence of selected loci of the cps region for capsule K1 or K2 synthesis in epidemic strains of Klebsiella pneumoniae isolated from infants in Poland]. / Wystepowanie wybranych loci regionu cps zwiazanych z wytwarzaniem otoczki typu K1 i K2 przez szczepy Klebsiella pneumoniae izolowane w Polsce od niemowlat w ogniskach zakazen szpitalnych.

Number of 60 epidemic strains of K. pneumoniae and 15 isolated occasionally from stool samples were tested for presence of 4 and 11 selected loci of the cps cluster of K1 and K2. Following open reading frames (ORFs): 1, 2, 3, 4, 7, 9, 10, 14, 15 (gnd) of cps K2 strain Chedid and genes magA, gmd, wzc, wca of cps K1 strain DTS were searched by PCR in tested and reference strains O1:K1 A5054 and O1:K2 B5055. The ORFs 1 to 3 and ORF15 were detected in both the reference and epidemic strains as well as in the greatest majority of the occasional isolates. Thus, such ORFs were found K. pneumoniae cps common domains, while tested ORFs 4 to 14 were observed in strain B5055 and 11 epidemic isolates from patients of the same hospital ward. Only exception was a single strain O3 occasionally isolated from faeces. The tested genes of cps K1 were detected only in strain A5054 and in two O3 occasional isolates from faeces. Interestingly, these genes as well ORFs 4 to 14 were detected together in the appropriate reference and tested strains with only two exceptions. Therefore, the cps sector occupied by ORFs 4 to 14 was found as group-specific domain. The occurrence ratio of cps K2 group-specific loci among epidemic strains from infants was 18%, while the K1 group-specific loci were absent.

[Occurrence of selected genes of the Klebsiella pneumoniae clusters waa and wb for lipolysaccharide synthesis in reference and epidemic strains]. / Wystepowanie wybranych genów loci waa i wb zwiazanych z wytwarzaniem lipopolisacharydu u referencyjnych i izolowanych z materialu klinicznego szczepow paleczek Klebsiella pneumoniae.

The goal of presented study was to determine by PCR differences in existence or homology level of selected genes involved in K. pneumoniae lipopolysaccharide (LPS) synthesis and application of obtained results for genotyping. Number of 26 reference strains of K. pneumoniae belonging to serogroups O1, O2a, O2a2e, O2a2e2h, O2a2f2g, O3, O4, O5, O7, O8, and O12 was tested together with 13 epidemic strains from 5 outbreaks and 6 casual isolates for the existence of 7 (waaA, waaE, waaL, waaQ, waaZ, waaX and uge) and 4 (wbdA, wbdC, manB, wbbO) genes of the waa and wb clusters for LPS biosynthesis. Based on PCR results, 10 and 11 genotypes were distinguished in tested strains for genes from waa and wb clusters respectively. Derived dendrograms were topologically dissimilar, however observed correlation between clonal groups and O-group was marginal for both compared clusters. Since we aimed to develop genotyping method for K. pneumoniae, genes from clusters waa and wb were used together to enhance the distinguishing capacity. Twenty-one genotypes were distinguished in 45 tested strains (DI=0,46) when 11 genes were applied for typing. Although no apparent correlation between genotype and serogroup was observed, epidemic isolates from 5 outbreaks were diversified into 5 genotypes, whereas strains from the same outbreak were indistinguishable. Described here genotyping method is determinative and was found time and cost effective. This method may be applied in every clinical laboratory equipped in an ordinary PCR apparatus.

[The characteristic of Staphylococcus aureus strains isolated from food's samples]. / Charakterystyka szczepów Staphylococcus aureus izolowanych z próbek zywnosci.

The study was carried out of 22 isolates of S. aureus isolated from 7 different incriminated food's samples from foodborne-disease outbreaks. The possibility of these isolates to producing of enterotoxins by commercial test SET-RPLA (Oxoid) was tested. The genotyping of these isolates was done by pulse-filed gel electrophoresis, acc. to Pfaller in own modification. On the basis of the DNA restriction patterns of the 22 isolates--5 strains were singled out, one of these strains--strain V (isolat nr 7) was not relationship to others. It was found that this strain V was one enterotoxin produced. Additionaly, all tested strains, in spite of the strain nr V, were isolated from the 2 or 3 samples of different kinds of foods. In the present study it has been shown too, that several similar colonies should be isolated for farther studies to assess microbiological contamination of the food products properly.

[Virulotypes of Bacillus anthracis strains isolated in Poland]. / Wirulotypy szczepów Bacillus anthracis izolowanych na terenie Polski.

Bacillus anthracis--the causative agent of anthrax--possesses several virulence genes located in the chromosome as well as in two B. anthracis virulence plasmids: pXO1 and pXO2. In the presented study, we determined occurrence of six virulence markers located in the virulence plasmids (capA, capB, capC, pagA, lef and cya) for capsule and toxin production together with virulence-associated gene gerXA and chromosomal gene sap, which are responsible for germination and S-layer biosynthesis respectively. Fourteen strains of B. anthracis isolated in Poland and belonging to five different MLVA genotypes were analyzed by PCR for presence of the aforementioned genes. Two virulotypes were found in tested strains. The only variation was absence of capA, capB and capC due to a lack of pXO2.

Intriguing diversity of Bacillus anthracis in eastern Poland--the molecular echoes of the past outbreaks.

The multiple locus VNTRs analysis (MLVA) revealed the presence of five genotypes in a group of 10 Bacillus anthracis isolates from epidemiologically unrelated cases of bovine-anthrax in eastern Poland. Eight tested isolates possessed the pagA and capB genes indicating the presence of both virulence plasmids, while two isolates revealed only pagA and lacked pXO2. The MLVA and DNA sequence analysis indicated that seven tested isolates represent four novel genotypes. Five tested strains revealed a unique 144 bp vrrB2 variant as well as 220 bp variant of vrrB1, implying the relatedness to the lineage B2. Consequently, we propose establishing of novel B2 strains sub-lineage. Multiple anthrax outbreaks, which took place in Poland several decades ago were proposed as a cause of intriguing diversity of B. anthracis observed in this study.

[Out-of-laboratory control screening of growth mediums used by sanitary service laboratories for isolation of pathogenic enteric bacteria from fecal specimens]. / Zewnatrzlaboratoryjna ocena jakosci wybiórczo-róznicujacych podlozy do wykrywania paleczek Shigella i Salmonella w próbkach kalu.

The aim of the study was to test the selective-differential plating mediums used for isolation of Salmonella and Shigella for routine stool specimens examination for epidemiological and sanitary purpose. Three plates of any such medium used in the laboratories in 37 Sanitary Service Stations in Poland were obtained. The specimens of Mac Conkey Lactose Bile Salt Agar and SS Agar were obtained from all laboratories, Hektoen Enteric medium from 8 and EosinMethylene Blue Agar from only one laboratory. The desiccated substrates of these mediums originated from 11 manufactures. The mediums were inoculated by "drops" method. The five control strains of selected taxons were chosen from National Institute of Hygiene strains collection. The quality of growth was evaluated by comparison with the growth on two control mediums: the general outlook of bacterial colonies, size and number of cfu/ml was taken under consideration. It was found that the results were satisfying for Hektoen medium, Levine and all but two Mac Conkey's medium specimens. The results of growth on SS medium were much worse: only on 10 specimens out of 39 checked supported properly the growth of all the five control strains. On 18 specimens the growth appeared after 48 hours of incubation and the size of colonies was too small to be isolated. On 11 there was no growth on 1, 2 or 3 control strains. The need of systematic extra-laboratory control of plating mediums used for examination stool specimen was shown.

[Yersiniosis--unappreciated infectious disease]. / Jersinioza--niedoceniana choroba zakazna.

Yersiniosis is an acute or chronic zoonosis caused by rods belonging to species Y. enterocolitica and Y. pseudotuberculosis. The natural reservoirs of these rods are domestic and wild living animals. Pathogens are transmitted to host by contaminated food, water, and soil. The clinical manifestations of yersiniosis are variable from mild diarrhoeas to serious pathological organic lesions. The diagnosis of yersiniosis is often possible only on the results of the bacteriological or serological examinations because of lack of typical clinical manifestations. The antibiotic treatment is necessary in case of yersiniosis involving enterocolitis, septicemia, and organic lesions.

[Evaluation of commercial usefulness for microparticle agglutination Serodia-Myco II test for serodiagnosis of Mycoplasma pneumonia infections]. / Ocena przydatnosci komercyjnego testu aglutynacyjnego Serodia-Myco II do serologicznej diagnostyki zakazen wywolywanych przez Mycoplasma pneumoniae.

The usefulness of Serodia-Myco II agglutination test (Fujirebio, Japan) for diagnosis of the M. pneumoniae infections was evaluated. A total of 66 serum samples obtained from patients with respiratory tract infections were tested by Serodia-Myco II test, complement fixation (CF) test, ELISA-IgG/-IgM, and by latex agglutination (LA) test prepared in our laboratory. Using CF test and ELISA as the reference tests, Serodia-Myco II test gave too many false positive results. This test in relation to CF test, ELISA-IgM, ELISA-IgG, and LA test showed a very high sensitivity, virtually 100%, with a low specificity, below 50%. It seems that oversensitivity of the Serodia-Myco II test is caused by too low cut off (40) value recommended by the manufacturer. The Serodia-Myco II test may be used in routine serodiagnosis of mycoplasmosis under condition that cut off value will be raised to 160 and the positive results of this test will be confirmed by the CF test or ELISA.