Characteristics and assessment of the usefulness of serological tests in the diagnostic of infections caused by coronavirus SARS-CoV-2 on the basis of available manufacturer's data and literature review.

Recognized in 2019 in Wuhan, China, the new SARS-CoV-2 coronavirus is responsible for the occurrence of a global pandemic disease called COVID-19. So far, confirmation of infection is based on the detection of virus RNA in a sample taken from a person meeting the suspected case definition. However, in the laboratory diagnosis of SARS-CoV-2 infections, in addition to genetic tests, serological methods can also be used to detect specific antibodies of the IgM, IgG and IgA class produced after contact with antigens or to detect viral antigen. Currently, a number of rapid immunochromatographic, chemiluminescent and ELISA immunoassay tests developed by different manufacturers for the diagnosis of COVID-19 are available on the market. Despite this fact, so far there is no WHO or ECDC recommendations or even reliable research regarding the usefulness of serological investigations in the laboratory diagnosis of infections caused by SARS-CoV-2.

Usefulness of in-house obtained recombinant proteins Yop of Yersinia enterocolitica as highly specific antigens in ELISA and recom-dot performed in the serodiagnosis of yersiniosis.

INTRODUCTION: Proper analysis of the human immune response is crucial in the laboratory diagnosis of many bacterial infections-The current serological diagnosis of yersiniosis often is carried out using ELISA with native antigens. However, recombinant proteins increase the specificity of the serological assays, particularly in patients with chronic, non- specific infections. The aim of the present study was to evaluate the usefulness of in-house obtained recombinant proteins Yop of Yersinia enterocolitica as highly specific antigens in ELISA and recom-dot performed in the serodiagnosis of yersiniosis. MATERIALS AND METHODS: Recombinant YopD, YopB, YopE and V-Ag proteins of Y enterocolitica were expressing in E. coli BL21 (DE3) using the pET-30 Ek/LIC expression vector (Novagen). Purification was accomplished by immobilized metal (Ni2) affinity column chromatography (His-trap). The proteins were used as antigens in standard ELISA and recom-dot assay, which was performed on nitrocellulose strips. The study population, used for characterization of the humoral immune response to the recombinant proteins, consisted of 74 patients suspected for Y enterocolitica infection and 41 clinically healthy blood donors. Some of the results obtained by ELISA and recom-dot were compared with results obtained by commercial western-blot Yersinia (Virotech). RESULTS: In the group of patients suspected for yersiniosis in clinical investigation the most positive results were obtained in ELISA with the recombinant protein YopD (IgA respectively 25 (42.4%), IgG 41 (69.5%), IgM 24 (40.7%). The percentage ofpositive results in the group of blood donors did not exceed 10.0% in IgG and 5.0% in IgA/IgM classes of immunoglobulin. The results obtained in the recom-dot assay showed that among 74 tested serum samples obtained from individuals suspected of yersiniosis the most common IgA, IgG and IgM antibodies were found for recombinant protein YopD (respectively IgG in 60.8%, IgA in 37.8% and IgM in 33.8% of serum samples). IgG antibodies to the protein V-Ag were observed in 32.4%, protein YopB in 27.0% and for the protein YopE in 18.9% serum samples. Immunoglobulin A, and M for the recombinant proteins were found much less frequently than IgG antibodies (respectively 12.2% and 10.8% for V protein-Ag in 10.8% and 14.9% protein and_YopB 2,7% and 10.8% for the protein YopE). Further studies showed that results obtained in recom-dot assay with recombinant protein YopD were comparable with the results of commercial western-blot Yersinia. CONCLUSIONS: The study showed that in-house obtained recombinant proteins can be used as highly specific antigens in ELISA and recom-dot performed in the serodiagnosis of yersiniosis. The most useful was the protein YopD.

Characterisation of Yersinia Secretion Apparatus--Pathogenicity Island (Ysa-PI) of Yersinia enterocolitica 1B/O8 in Poland: an Idle Ysa is a Specific Hallmark of the Epidemic Sensu Stricto Strain.

Yersinia secretion apparatus (Ysa), the chromosomal type three secretion system (T3SS) is considered to contribute to virulence of high-pathogenicity Yersina enterocolitica biovar 1B. DNA-sequence of Ysa pathogenicity island was determined for clinical isolate DM0110 of Y enterocolitica 1B/08 with origin in Poland. We found a premature stop-codon in the regulatory gene ysrR (mutation at position 269). Altered ysrR was detected in all tested 78 isolates of Y enterocolitica 1B/O8 collected from clinical samples in Poland from 2004 to 2013. Since aberrations in YsrR are considered to inactivate Ysa, our findings may suggest Ysa is not indispensable for Y enterocolitica 1B/O8 to infect humans.

Consistency of Bordetella pertussis vaccine seed strains and potency of whole-cell pertussis vaccine still in use in Poland.

In Poland, where the wP vaccine has been used since 1960, pertussis rates increased in the mid-1990s. In 2012, the rate of pertussis recognised by surveillance was unexpectedly found to be two-fold higher than in the previous decade. Quality measures on potency and vaccine working seeds were introduced, to confirm the possible impact of manufacturing inconsistency or potency lowering on the observed increase in pertussis. Shewhart charts on potency values for lots released between 2001 and 2013 did not reveal any significant fluctuations. Working seeds of three vaccine strains used within last decade for wP manufacturing belong to the PFGE group III and were highly related. According to PFGE and SDS-PAGE data, all vaccine strains were found consistent according profiling on the genomic and protein levels. According to the sequencing data, they harboured ptxA2, ptxC1, prn1, fim2-1, fim3-1, tcfA2, ptxP1 and were assigned as MLST-2 type. Other factors apart from vaccine manufacturing inconsistency might be responsible for the increase in pertussis noted in 2012 in Poland.

[Selected mechanisms of pathogenicity of Campylobacter jejuni]. / Wybrane mechanizmy chorobotwórczosci paleczek Campylobacter jejuni.

Campylobacter jejuni is one of the most common causes of bacterial diarrhea worldwide. Various virulence factors of C. jejuni contribute to survival and participate in the induction of infection in the human body. The virulence mechanisms such as motility, toxin production or invasive properties allow the bacteria to survive in the human body. The article presents the current knowledge on selected virulence mechanisms of C. jejuni.

[Frequency of antibodies to the recombinant protein P39 of C. jejuni in patients with gastrointestinal disorders and reactive arthritis in Poland]. / Czestosc wystepowania przeciwcial dla rekombinowanego bialka P39 paleczek C. jejuni u wybranych osób z zaburzeniami zoladkowo-jelitowymi oraz z reaktywnym zapaleniem stawów w Polsce.

INTRODUCTION: Campylobacterjejuni is has been found to be the leading cause of bacterial gastroenteritis worldwide. Clinical manifestation on enterocolitis caused by C. jejuni are diarrhea, fever, abdominal pain and in some patients, fecal blood. After C. jejuni infection, squeals may occur such as reactive arthritis. The aim of the study was to investigate the frequency of antibodies to the recombinant protein P39 sticks C. jejuni in patients with gastrointestinal disorders and reactive arthritis in Poland. MATERIAL AND METHODS: Serum samples collected from 46 patients with bacteriology confir- med infection caused by Campylobacter jejuni, 472 sera from patients with gastrointestinal disorders, 97 serum samples obtained from patients with reactive arthritis and 84 sera from healthy adults and children. Sera were screened for anti-P39 C. jejuni recombinant protein IgA, IgG andIgM antibodies by using the home-made ELISA. Protein P39 C. jejuni was expressing in E. coli BL21 (DE3) using the pET-30 Ek/LIC expression vector. Purification was accomplished by immobilized metal (Ni2+) affinity column chromatography (His Bind Resign, Novagen). RESULTS: SDS-PAGE and Coomassie brilliant blue staining confirmed a high purity of the recombinant P39 protein preparation with an expected molecular mass of 39 kDa. The results of ELISA with the P39 recombinant protein revealed that IgA antibodies in diagnostically significant level (x + 2SD) were found in 18.8%, IgM in 14.8% and IgG in 7.8% of sera obtained from patients with of gastrointestinal disorders. On the other hand, antibodies to recombinant P39 protein in sera obtained from patients with reactive arthritis were found in more than twice the percentage than in patients with gastrointestinal disorders (IgA in 34.0%, IgG in 26.8% and IgM in 19.6%). CONCLUSIONS: In conclusion, based on the data obtained, C. jejuni may be important factor in triggering the gastrointestinal disorder and reactive arthritis in humans in Poland.

[Serum immunoglobulin IgG subclass distribution of antibody responses to Francisella tularensis in patients with tularemia]. / Wystepowanie przeciwcial dla paleczek Francisella tularensis w poszczególnych podklasach IgG u osób z tularemia.

INTRODUCTION: The present study was aimed at determining the IgG subclass distribution against F. tularensis in patients with tularemia. METHODS: The total number of 56 serum samples obtained from patients with serologically confirmed tularemia were tested by in-house ELISA with bacterial sonicate as the antigen for the presence of IgG1, IgG2, IgG3 and IgG4 antibodies to F. tularensis. Based on the results of determining the level of antibodies in the sera of 30 blood donors, the cut-off limit of serum antibodies for each subclass was set at arithmetic mean plus three standard deviations. RESULTS: Antibodies of subclass IgG1 to F. tularensis were diagnosed in 41 (73.2%), IgG2 in 52 (92.9%) and IgG3 in 13 (23.2%) serum samples. The arithmetic mean of OD450 of antibodies IgG2 was over three-times higher than antibodies IgG1 and IgG3 measured in all of tested serum samples. The concentration of IgG4 was below the detection level. CONCLUSION: In conclusion, IgG2 antibodies to F. tularensis are predominating IgG subclass in tularemia. This study showed also that subclasses of IgG1 and IgG3 but not IgG4 antibodies to F. tularensis are produced during natural infection in humans.

[Electrophoretic and immunological analysis of native proteins secreted in vitro under conditions inducing Ysa (Yersinia secretion apparatus) by clinical isolates of Yersinia enterocolitica 1B/O8 in Poland]. / Elektroforetyczna i immunologiczna analiza natywnych bialek wydzielniczych wytwarzanych in vitro w warunkach indukcji Ysa (Yersinia secretion apparatus) przez izolaty Yersinia enterocolitica 1B/O8 wyosobnione od ludzi w Polsce.

INTRODUCTION: The high pathogenicity Yersinia enterocolitica 1B/O8 produce variety of virulence factors including chromosomal T3SS known as Ysa-Ysp system that is considered to act at the early stage of infection. The aim of the study was to examine the ability to produce Ysa-Ysp proteins in vitro by human clinical isolates of the epidemic Y. enterocolitica 1B/O8 strains in native conditions and immunological characterization of expressed proteins. METHODS: Seven Y. enterocolitica 1B/O8 isolates with known epidemiological link and the reference high pathogenicity strain WA-314 and six strains from the Institute Pasteur (France) were examined for production of Ysa-Ysp proteins according with procedure described by Matsumoto and Young (Mol. Microbiol., 2006, 59:689-76). All the isolates and strains were characterized by SDS-PAGE to determined Ysa-Ysp proteins profile. The immunological characterization was performed by using western-immunobloting method using sera from two immunized rabbits and from two patients with bacteriology confirmed Y. enterocolitica 1B/O8 infection. RESULTS: The reference strain WA-314 yielded typical Ysa-Ysp proteins profile. In contrast all the tested Y. enterocolitica 1B/O8 human isolates yielded the same SDS--PAGE profile that was apparently distinct from profile of Ysa-Ysp proteins of reference strain WA-314. CONCLUSIONS: The Y. enterocolitica 1B/O8 isolates of the epidemic strain circulating in Poland were found to be unable to produce Ysa-Ysp proteins in vitro under conditions sufficient to stimulate expression of the Ysa-Ysp proteins in the reference strain WA-314 and strains from the Institute Pasteur (France). Our results may suggest that the ability to produce Ysa--Ysp proteins in concentrations sufficient to induce production of specific antibodies is not indispensible for Y. enterocolitica 1B/O8 infection in humans. The western-immunoblotting analysis of human serum samples showed that the antibodies were not induced by Ysa and Ysp proteins during infection caused by the epidemic strain of Y. enterocolitica 1B/O8 circulating in Poland. Similar, negative result was found with serum of a rabbit immunized intravenously by the reference strain WA-314. The project was funded by the National Science Centre in Cracov, Poland, grant N N401 076039.

[Serum immunoglobulin IgG subclass distribution of antibody responses to pertussis toxin and filamentous hemagglutinin of Bordetella pertussis in patients with whooping cough]. / Wystepowanie przeciwcial dla toksyny krztuscowej i wlókienkowej hemaglutyniny Bordetella pertussis w poszczególnych podklasach IgG w przebiegu krztusca.

INTRODUCTION: The present study was aimed at determining the IgG subclass distribution against pertussis toxin (PT) and filamentous hemagglutinin (FHA) of Bordetella pertussis in patients with whooping cough. METHODS: The total number of 222 serum samples obtained from patients suspected in clinical investigation for pertussis were tested separately by in-house ELISA for the presence of IgG antibodies to pertussis toxin and filamentous hemagglutinin. The percentage distribution of specific anti-PT and anti-FHA IgG subclass response was calculated only on the basis of group of sera confirmed in the present study as positive for total IgG antibodies (183 sera to PT antigen and 129 to FHA antigen). Paired serum specimens were obtained from 36 patients. Based on the results of determining the level of antibodies in the sera of 40 blood donors, the cut-off limit of serum antibodies for each subclass was set at arithmetic mean plus two standard deviations. RESULTS: Antibodies of IgG1 to pertussis toxin and filamentous hemagglutinin were diagnosed in 151 (82.5%) and 99 (76.7%), IgG2 in 72 (39.0%) and 50 (38.8%), IgG3 in 17 (9.3%) and 43 (33.3%), IgG4 in 55 (30.1%) and 53 (41.1%) serum samples, respectively. There were no significant differences in percentage of sera with IgG1, IgG2 and IgG3 in relation to age of the patients. However, the frequency of occurrence of IgG4 antibodies was highest in the group of the youngest children to the age of 6 years old (61.8% for PT and 68.0% for FHA), and decrease with age, reaching the minimum in the group of patients above 40 years old (13.2% and 4.2% for PT and FHA, respectively). We also found significantly higher frequency of IgG4 to PT and FHA antigens in men than in women. Statistically significant, essential changes in the pattern of IgG subclass during the course of infection were not found. CONCLUSIONS: In conclusion, this study showed that all four subclasses of IgG antibodies to pertussis toxin and filamentous hemagglutinin are produced during whooping cough.

[Characteristics of Clostridium tetani and laboratory diagnosis of tetanus]. / Charakterystyka Clostridium tetani i laboratoryjna diagnostyka tezca.

The causative agent of tetanus is the obligate anaerobic bacterium--Clostridium tetani. These bacteria form endospores that are able to survive long periods of exposure to air and other adverse environmental conditions. Infection generally occurs through wound contamination. We can distinguish several forms of tetanus: generalized, local and neonatal. Diagnosis of tetanus is based primarily on the patient's clinical symptoms (muscle cramps, painful back muscle spasms, generalized contractions of the arcuate curvature of the body) as well as on microbiological diagnosis. This article is a brief review of C. tetani and diagnosis of infections caused by these organisms in humans.

Development and evaluation of a latex agglutination test for the rapid serodiagnosis of tularemia.

A latex agglutination test (LAT) was developed for a rapid detection of antibodies against Francisella tularensis. The assay is performed by mixing serum with antigen-coated latex beads and read within 5 min. Developed LAT has been proved to be a specific, sensitive, fast, easy-to-perform and cost-efficient tool for the routine diagnosis of tularemia.