Antibody response to lipopolysaccharides and recombinant proteins of Shiga toxin (STX)-producing Escherichia coli (STEC) in children with haemolytic uraemic syndrome in Poland.

Typical haemolytic uraemic syndrome (STEC-HUS), caused by Shiga toxin (Stx)-producing Escherichia coli (STEC), is a serious, life-threating disease that mainly affects children. Bacteriological and genetic tests are commonly used in the routine laboratory diagnosis of STEC-HUS; however, serological methods have emerged as useful and reliable diagnostic tools, especially when bacterial isolation fails. In this study, we present the results of the serological investigation of 72 paediatric patients suspected for HUS, hospitalized during 2011-2019 at the Department of Pediatrics and Nephrology of Children's Hospitals in Poland. During the routine laboratory investigation STEC strains were isolated only from nine stool samples. However, serological investigations confirmed 45 cases of STEC infections in children with HUS. In this study, 22 (48·9%) paediatric patients were infected by E. coli serotype O26, 11 (24·4%) by serotype O145, 9 (20·0%) by serotype O157, and 3 (6·7%) by E. coli serotype O111. In the majority of these patients, in addition to a high level of IgA, IgG and IgM antibodies to lipopolysaccharide of particular E. coli serotypes, antibodies to recombinant proteins Tir, Stx2b and intimin were detected. Our results confirm that serological tests are useful in the diagnosis of STEC-HUS. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that serological analysis greatly complements bacterial isolation and helps in the diagnosis and confirmation of Shiga toxin (verotoxin)-producing Escherichia coli (STEC) infections. Serological tests can be performed to qualify the patient for the typical haemolytic uraemic syndrome (STEC-HUS). In Poland, STEC-HUS in children is mostly caused by the E. coli serotype O26, which indicates that there is an increasing number of non-O157 STEC infections associated with human diseases in Europe.

Incidence of pertussis in patients of general practitioners in Poland.

We estimated the incidence of pertussis in patients consulting general practitioners (GPs). Between July 2009 and April 2011, we conducted a prospective cohort study of patients attending 78 general practices (158 863 persons overall). We included patients aged ≥ 3 years, with cough lasting 2-15 weeks, who gave informed consent. GPs interviewed eligible patients, collected a blood specimen, and a nasopharyngeal swab. At follow-up 30-60 days after the initial visit, physicians collected a second blood specimen and conducted patient interview. Cases were confirmed by specific IgA and/or IgG antibody titre exceeding significantly the general population background level or detection of bacterial DNA by real-time PCR. During the study period, 3864 patients with prolonged cough consulted the participating GPs, of those 1852 met the inclusion criteria, 1232 were recruited, and 288 were confirmed as pertussis cases (4% by PCR, 96% by serology). The adjusted incidence rate was 201.1/100 000 person-years [95% confidence interval (CI) 133.9-302.0], ranging from 456.5 (95% CI 239.3-870.8) in the 15-19 years group to 94.0 (95% CI 33.4-264.5) in the 25-29 years group. The reporting ratio was 61, ranging from 4 in those aged 3-5 years, to 167 in those aged 65-69 years. The study confirmed high incidence of pertussis in all age groups in the general population, in particular in adults, not appropriately documented by the existing surveillance system.

Comparison of seven commercial enzyme-linked immunosorbent assays for the detection of anti-diphtheria toxin antibodies.

Determination of immune status of patients to diphtheria toxin is based mainly on the results of commercially available ELISA kits. The aim of the present study was to compare the results obtained by ELISAs from seven different manufacturers: Mikrogen, Immunolab, Sekisui Virotech, NovaTec, Virion\Serion, IBL International and Euroimmun. All assays were performed according to the manufacturers' instructions. The concentrations of the anti-diphtheria toxin antibodies in 72 serum samples were calculated on the basis of curves constructed from standards supplied by manufacturers and the new reference material-International Standard for Diphtheria Antitoxin (10/262). The repeatability and reproducibility of all the ELISA kits tested were good. Number of sera with concentrations of the anti-diphtheria toxin antibodies below the WHO-recommended level of protection (0.1 IU/ml) were dependent on the ELISA used: Mikrogen, 20/72 samples (27.7%); Immunolab, 11/72 samples (15.3%); Sekisui Virotech, 0/72 samples (0%); NovaTec 18/72 samples (25.0%); Serion 12/72 samples (16.7%); IBL International, 7/72 samples (9.7 %); and Euroimmun, 17/72 samples (23.6%). However, the results obtained in particular ELISAs, with the exception of Sekisui Virotech, were much more consistent when the concentrations of the anti-diphtheria toxin antibodies in 72 sera measured by using curves constructed from the International Standard 10/262. The data obtained clearly demonstrated that manufacturer-dependent differences between anti-diphtheria IgG ELISA kits exist. The differences in recommendations accepted by the individual manufacturers together with differences shown in our studies in sensitivity greatly affect the clinical interpretation of results.

Seasonality of Yersinia enterocolitica bioserotype 1B/O:8 infections in Poland.

Both serological and bacteriological investigations revealed a cyclic, seasonal pattern of Yersinia enterocolitica 1B/O8 infections in Poland during the years 2008­2011. A large increase in incidence was observed in the second quarter and a decrease in the third quarter of each year. Such seasonal changes were not seen in the case of infections caused by the other enteropathogenic Yersinia bioserotypes.

[Use of recombinant P1 protein of Mycoplasma pneumoniae for the serodiagnosis of mycoplasmosis]. / Zastosowanie metody inzynierii genetycznej do uzyskania bialka P1 Mycoplasma pneumoniae oraz ocena jego przydatnosci w serodiagnostyce mykoplazmozy u ludzi.

INTRODUCTION: Mycoplasma pneumoniae is a common etiologic agent of community-acquired respiratory infection. In serological diagnosis of M. pneumoniae infections tests have been described based on the purified P1 protein, which is the most important virulence factor of this pathogen, as antigen. The aim of his study was to express and purify a recombinant protein P1 M. pneumoniae and next evaluate this protein as high specific antigen in serodiagnosis of mycoplasmosis. METHODS: Protein P1 M pneumoniae was expressing in E. coli BL21 (DE3) using the pET-30 Ek/LIC expression vector. Based on published literature, we decided to express C-terminal region [P1-C1] encompassing amino acid residues 1160 to 1521. Purification was accomplished by immobilized metal (Ni2+) affinity column chromatography (His-trap). Serum samples collected from 221 patients with mycoplasmosis, positive in complement fixation test (CFT), 87 patients with other then mycoplasmosis bacterial infections and 80 blood donors were screened for anti-P1 recombinant protein IgA, IgG and IgM antibodies by using the home-made ELISA. RESULTS: SDS-PAGE and Coomassie brilliant blue staining confirmed a high purity of the recombinant P1 protein preparation with an expected molecular mass of 39,7 kDa. The specificity of the recombinant protein was confirmed by western blot analysis using serum samples from rabbits immunized by M pneumoniae. The results of ELISA revealed that more then 70.0% of patients with mycoplasmosis confirmed by CFT, had antibodies to recombinant P1 protein in diagnostically significant level (x + 2SD). The antibodies were found only sporadically in sera obtained from patients with other then mycoplasmosis bacterial infections and clinically healthy persons. A comparison of results obtained in home-made ELISA with results of commercial western blot (Virotech) showed similar, ranged from 84.2% to 97.4%, compatible of results. The IgM antibodies to recombinant P1 protein were found in 87.2% sera obtained in acute phase of disease, in 80.0% sera obtained 2-4 weeks after onset of clinical symptoms and only in 43.8% sera obtained in chronic mycoplasmosis. CONCLUSIONS: The present study confirmed the earlier observations of the high usefulness of recombinant P1 protein for reliable serologic diagnosis of M. pneumoniae infection.

The presence of the ail gene in clinical strains of Yersinia enterocolitica isolated from stools in Poland and characteristics of gene variant.

The non-American variant of ail gene was found independently of the virulence-associated plasmid pYV in all 152 tested clinical Y. enterocolitica O:3 strains isolated from humans in Poland. Data obtained by sequencing exhibited high genetic stability of the ail gene present in the tested isolates obtained from different regions of the country. The PCR-RFLP technique was sufficient for fast identification and characterization of the ail gene variant present in the tested strains.

[Use of PCR methods for detection of selected genes of Mycoplasma pneumoniae]. / Zastosowanie metody PCR do wykrywania wybranych genów Mycoplasma pneumoniae.

The aim of this study was standardization of PCR for the detection of gene encoding the P1 protein, 16S rRNA and elongation factor Tu of M. pneumoniae. A total of 13 strains of M. pneumoniae, 28 strains of other mycoplasmas and 14 strains of different bacteria causing respiratory tract infections were tested. In all of tested M. pneumoniae strains the presence of the sought genes was confirmed. The specificity of DNA was confirmed by the restriction endonuclease analysis with enzymes Hind III, Alu I and Hha I. With none of primers specific for the M. pneumoniae genes amplification of DNA from other bacteria was noted. The PCR method with the selected primers allowed to detect from 10(2) to 10(4) cfu M. pneumoniae/ml suspended in broth. The obtained results indicate that the PCR method can be used for detection of M. pneumoniae genes. A very good sensitivity and specificity predestine++ PCR as a potential quick diagnostic method for identification of M. pneumoniae in clinical specimens.

[Evaluation of the usefulness for the latex agglutination test for serodiagnosis of Mycoplasma pneumoniae infections]. / Ocena przydatnosci lateksowego testu aglutynacyjnego do serologicznej diagnostyki zakazen wywolywanych przez Mycoplasma pneumoniae.

The usefulness of latex agglutination test prepared in our laboratory for the diagnosis of M. pneumoniae infections was assessed. A total of 628 serum samples obtained from patients with respiratory tract infections were tested by complement fixation test and by latex test, from among them 274 serum samples were additionally tested by ELISA--Ig A/--IgG/--IgM and by immunoelectroprecipitation test. The highest sensitivity and specificity was displayed by the latex test in relation to ELISA when determining mycoplasmal antibodies of IgM class (respectively 82.1% and 89.6%) and to the complement fixation test (81.0% and 89.0%). Positive latex test results in our investigations were associated only with the presence of IgM antibodies and were not dependent on the IgA and IgG antibody classes. The latex agglutination test may be used in routine serodiagnosis of mycoplasmosis under condition that the results obtained in this test will be confirmed by the complement fixation test or ELISA.

Susceptibility of Polish clinical strains of Yersinia enterocolitica serotype O3 to antibiotics.

A total of 199 clinical strains of Yersinia enterocolitica serotype O3, biotype 4 were tested for their susceptibility to antibiotics (158 strains carried the virulence plasmid pYV and 41 strains did not). A total of 114 isolates were tested by a standard disk diffusion method for 21 antibiotics. Almost all strains tested were resistant to ampicillin and cefazolin and susceptible to amoxycillin/clavulanate, cefaclor, cefamandole, cefuroxime, cefotaxime, ceftriaxone, aztreonam, imipenem, gentamicin, amikacin, netilmicin, tetracycline, doxycycline, chloramphenicol, ciprofloxacin, sulphamethoxazole, trimethoprim, co-trimoxazole and furazolidone. In addition, minimal inhibitory concentrations of 15 antibiotics were determined by the agar dilution method for all 199 strains (158 carrying plasmid pYV and 41 strains that did not). Third-generation cephalosporins such as cefotaxime and ceftriaxone and a fluoroquinolone (ciprofloxacin) were the most active antimicrobial agents tested followed by aztreonam, imipenem, trimethoprim, tetracycline, gentamicin, chloramphenicol, amoxycillin/clavulanate, cefaclor, cefuroxime, amikacin, furazolidone and sulphamethoxazole. The present study demonstrated a high susceptibility of clinical strains of Y. enterocolitica to most of the tested antibiotics. In general there was no significant difference between susceptibility to antibacterial agents of strains with or without plasmid pYV.

[Evaluation of the usefulness of commercial enzyme-linked immunosorbent assays for diagnosis of Mycoplasma pneumoniae infections]. / Ocena przydatnosci komercyjnych testów immunoenzymatycznych i testu opracowanego we wlasnym zakresie do diagnostyki zakazen wywolywanych przez Mycoplasma pneumoniae.

The usefulness of the ELISA distributed by BioChem ImmunoSystems, Medial Polska, Biomedica/Virotech and prepared in our laboratory (ELISA FH-K) for diagnosis of the M. pneumoniae infections was estimated. Eighty six serum samples obtained from 86 patients with respiratory tract infections were simultaneously tested by ELISA-IgM/-IgG and by complement fixation test which was accepted as a reference test. The highest sensitivity in relation to the CFT was displayed by the ELISA BioChem ImmunoSystems and Medial Polska (100%), slightly lower sensitivity by the ELISA Biomedica/Virotech--96.5% and ELISA FH-K--90.9% when determining mycoplasmal antibodies of IgM. The lowest sensitivity was displayed by the ELISA Biomedica/Virotech when determining antibodies of the IgG class (54.9%). The specificity of ELISA in relation to the CFT was generally higher when detecting mycoplasmal antibodies of the IgM class then of IgG class. The study demonstrated that all 4 ELISA may be used in routine serodiagnosis of M. pneumoniae infection. For the improve of sensitivity of ELISA it's recommended to measure simultaneously the level of mycoplasmal antibodies of IgM and IgG.

[Susceptibility to selected antibiotics of Yersinia enterocolitica 03 strains, carrying and not carrying plasmid pYV]. / Wrazliwosc na wybrane antybiotyki paleczek Yersinia enterocolitica grupy O3 posiadajacych i nie posiadajacych plazmidu pYV.

A total of 199 clinical strains of Yersinia enterocolitica serotype O3, biotype 4 were tested for their susceptibility to antibiotics (158 strains carried the virulence plasmid pYV and 41 strains did not). 114 isolates were tested by standard disk diffusion method for 21 antibiotics. Almost all tested strains were resistant to ampicillin and cefazolin and susceptible to amoxycillin/clavulanate, cefaclor, cefamandole, cefuroxime, cefotaxime, ceftriaxone, aztreonam, imipenem, gentamicin, amikacin, netilmicin, tetracycline, doxycycline, chloramphenicol, ciprofloxacin, sulphamethoxazole, co-trimoxazole, trimethoprim and furazolidone. In addition minimal inhibitory concentrations (MICs) of 15 antibiotics were determined by agar dilution method for all 199 strains (158 plasmid positive and 41 strains plasmid negative). Third-generation cephalosporins such as cefotaxime and ceftriaxone and a fluoroquinolone (ciprofloxacin) were the most active antimicrobial agents, tested followed by aztreonam, imipenem, trimethoprim, tetracycline, gentamicin, chloramphenicol, amoxycillin/clavulanate, cefaclor, cefuroxime, amikacin, furazolidone and sulphamethoxazole. The present study demonstrated a high susceptibility of clinical strains of Y. enterocolitica to most of the tested antibiotics. In general, there was no significant difference between susceptibility of virulence plasmid pYV positive and virulence plasmid negative strains to antibacterial agents.

Occurrence of serologically verified Mycoplasma pneumoniae infections in Poland in 1970-1995.

This study analyses the numbers of serologically verified Mycoplasma pneumoniae infections in Poland in 1970-1995. The investigations were performed using the complement fixation test (CFT) with sonicated antigen in National Institute of Hygiene in Warsaw and since 1985 in 33 laboratories throughout the country. The result was accepted as positive when antibody titre was 60 or higher, or at least a four-fold increase in titre occurred during the illness. During these studies five epidemics of mycoplasmosis were noted in Poland. They occurred regularly every 5 years during the autumn-winter season in 1970/1971, 1975/1976, 1980/1981 and 1985/1986. The last epidemic, which started in 1991 and culminated in 1992-1993, seems to have inaugurated a change from epidemic to endemic occurrence of M. pneumoniae infection in Poland. At the peak of the epidemic, depending on the region of country, in 20-38% of patients with respiratory tract infection serological confirmation of mycoplasmosis was obtained.

[Electrophoretic and immunologic comparative analysis of Mycoplasma pneumoniae and Mycoplasma genitalium proteins]. / Elektroforetyczna i immunologiczna analiza porównawcza komórkowych bialek Mycoplasma pneumoniae i Mycoplasma genitalium.

The Mycoplasma pneumoniae FH strain routinely used in our laboratory for over 25 years as antigen in serological tests, 2 reference M. pneumoniae strains from ATCC (29342 and M129) and 3 isolates of M. pneumoniae obtained in 1995 from pneumonia patients were compared by SDS-PAGE, complement fixation test (CFT) and by Western-immunoblotting against human and rabbit serum samples with high level of mycoplasmal antibodies. On SDS-PAGE all M. pneumoniae strains showed the same number of 23 polypeptides on the gel with identical molecular weights. The same strains on immunoblotting against human and rabbit serum samples showed six bands: 170, 89, 75, 55, 38 and 33 kDa with the strongest antibody staining in 170-(P1 protein) and 89-kDa bands. Because of its known antigenic relationships Mycoplasma genitalium was used for comparison. The pattern of M. genitalium proteins on SDS-PAGE was similar to pattern of M. pneumoniae but distinguishable. On immunoblotting six proteins of M. genitalium (135, 127, 110, 95, 75 and 45 kDa) reacted with human and rabbits immunoglobulins for M. pneumoniae antigens. Furthermore in complement fixation test both antigens, prepared from M. pneumoniae and M. genitalium, reacted as well with human and rabbit immunoglobulins for M. pneumoniae and with rabbit immunoglobulins for M. genitalium. These cross-reactions observed in serological techniques could give false positive results in routine diagnosis of M. pneumoniae infections. In such situations showing on immunoblott of presence in tested serum sample of antibodies to 170- and 89 kDa proteins could confirm M. pneumoniae infection.

Epidemiology of Mycoplasma pneumoniae infections in Poland : 28 years of surveillance in Warsaw 1970-1997.

Mycoplasma pneumoniae is a common cause of lower respiratory tract disease in humans, particularly among older children, adolescents, and young adults. Infections are endemic in cities and epidemic increases are observed at intervals of 4 to 7 years. M. p

[Evaluation of the reliability of serological diagnostic tests for Mycoplasma pneumoniae infection carried out by laboratories in sanitary epidemiological stations (WSSE)]. / Ocena wiarygodnosci wyników serologicznych badan diagnostycznych w kierunku zakazen Mycoplasma pneumoniae wykonywanych w laboratoriach WSSE.

In the action undertaken for evaluating of the reliability of serological tests for M. pneumoniae infection 33 laboratories of the Sanitary Epidemiological Stations participated. Every laboratory had to determine twice at an interval of 2-4 weeks the level of mycoplasma antibodies by the complement fixation test in serum samples divided into 4 groups: sera not containing these antibodies-titre < 5, or containing them in titres of 60, 120 and 320. The correct results of the determinations were obtained in 27 laboratories (81.8%) for samples without M. pneumoniae antibodies, and in 22 (66.6%) and 14 (42.4%) for samples with titres of 60 and 120, and 320 respectively. Only 4 laboratories (12.1%) obtained correct results of these determinations for every sample and in both testing series. In these series considered separately correct results were obtained in 9 (27.3%) and 8 (24.2%) laboratories. Faulty results in all samples in both testing series were reported from 2 (6.1%) laboratories. In the individual series all false results were obtained in 4 (12.1%) and 3 (9.1%) laboratories. The study showed that for raising of the quality and reliability level of serological investigations for M. pneumoniae infection a permanent practice should be periodic training of laboratory workers and frequently repeated interlaboratory controls of the reliability of test results.