Maria Kozioł-Montewka

The phenotypic and genetic biofilm formation characteristics of coagulase-negative staphylococci isolates in children with otitis media

OBJECTIVE Medical biofilms are involved in a number of chronic infections including otitis media with effusion and chronic rhinosinusitis, which are common pediatric infectious diseases. The purpose of the study was to analyze the phenotypic and genotypic indicators of biofilm formation of coagulase negative staphylococci isolates in children with otitis media with effusion, and in children with chronic rhinosinusitis as a comparison group by using three different detection methods. METHODS Forty nine children aged from 2 to 6 years old, diagnosed with otitis media with effusion were enrolled to the study. The comparative group consisted of twenty three strains of coagulase-negative staphylococci from the strains collection isolated from nose swabs from children 3 to 7 years old suffering from rhinosinusitis for longer than 12 weeks. Cultured strains were tested for biofilm formation ability with three tests: Congo red agar, tissue culture plate methods and detection of ica operon. RESULTS Out of 97 ear effusion specimens, obtained from 49 children suffering from OME, 38 were found positive in conventional culture resulting in isolation of 50 different bacterial species. Nested-PCR method confirmed bacterial presence in 95 (97.9%) cases. Among 50 different bacterial species isolated, 30 (30.9%) CNS and 20 (20.6%) other than CNS species. Detection of slime producing phenotype of CNS was performed with CRA plate test. Among OME isolates, 11 (36.7%) were CRA plate test positive. In case of isolates from CRS, 8 (34.8%) strains revealed black coloration on CRA. Using TCP method, strong adherence to microtiter plate was observed in two Staphylococcus epidermidis strains from OME and two S. epidermidis from CRS. By using the ica operon test, the genotypic ability to form biofilm was identified in 7 (23.3%) S. epidermidis strains cultured from ears effusion and in 3 (13%) strains from nose swabs. CONCLUSIONS CNS strains revealed genotypic and phenotypic features responsible for the ability to form the biofilm in vivo. The presence of ica genes and phenotypic ability to form a biofilm by CNS strains emphasizes the pathogenic character of these strains in some cases of otitis media with effusion.

Identification of latent tuberculosis infection in rheumatic patients under consideration for treatment with anti-TNF-α agents

Introduction Immunosuppressive therapy with anti-tumour necrosis factor-α (TNF-α) agents in rheumatic patients modulates the immune system and may increase the risk of reactivating infections that are normally maintained in a latent state, such as tuberculosis. The purpose of this study was to analyse the value of QuantiFERON TB Gold In-Tube (QFT IT) and tuberculin skin test (TST) in BCG vaccinated patients with rheumatoid arthritis and ankylosing spondylitis who were qualified to receive TNF-α blockers. Material and methods Ninety patients with rheumatoid arthritis and ankylosing spondylitis were included in the study. The control group consisted of 20 healthy participants. Chest X-ray, TST and QFT IT were carried out in all persons. Results In rheumatic patients positive results of QFT IT and TST tests were identified in 15 cases (16.7%) whereas negative results of both tests were detected in 56 cases (62.2%). In the group of examined patients, 11 (12.2%) had QFT IT-/TST+ test results. In patients with QFT IT+/TST– status one active tuberculosis case was detected. In the control group QFT IT positive results were found in 4 cases (20%) and TST positive in 11 cases (55%). Treatment with TNF-α blockers was introduced in 26 rheumatology patients with the following test status: 3 with QFT IT+/TST+; 20 with QFT IT-/TST-; 3 with QFT IT-/TST+. Conclusions In the BCG vaccinated population the QFT IT assay may potentially improve the identification and selection for therapy for latent TB infection before treatment with anti-TNF agents.

MPO and cytokines in the serum of cancer patients in the context of Candida colonization and infection

This study investigated the immunological factors, such as neutrophils number, the level of myeloperoxidase and IL-12, IL-10, TNF-α, IFN-γ, that additionally might correlate with increased susceptibility to Candida infections in cancer patients. A total of 105 cancer patients were evaluated. Patients were examined twice for Candida colonization and presence of Candida antigen and DNA in bloodstream. Serum concentrations of MPO and selected cytokines were quantified by ELISA. The values for myeloperoxidase were decreased in Candida-colonized as well as deep-infected cancer patients groups, compared to healthy persons. In the group of patients suspected of deep candidiasis, we observed significantly elevated level of IFN-γ compared to control. In the group of Candida-colonized patients, the concentrations of IL-12, TNF- α and IFN-γ were significantly heightened when compared to control. MPO deficiency seems to be one of the important risk factor for deep candidiasis independently of the neutrophil count. The disturbances in cytokines levels in cancer patients group can be connected with underlying cancer disease, its treatment as well as Candida infection. The decreased level of TNF-α, in particular may be connected with Candida invasion.

Prevalence of Legionella pneumophila in water distribution systems in hospitals and public buildings of the Lublin region of eastern Poland.

OBJECTIVE The aim of this study was to determine the prevalence of L. pneumophila in water supply systems, hospitals and public buildings in the Lublin region of eastern Poland. MATERIAL AND METHODS The study was carried out in 26 different objects in the Lublin region. The number of Legionella bacteria in water samples was determined by the membrane filtration method and/or by surface inoculation in accordance with the standards. RESULTS The study showed the presence of L. pneumophila in 166 hot water samples (74.77%). In 34.33% (n=57) of water samples the count of tested bacteria exceeded the acceptable level of >100 CFU/100 ml. Of the samples where an acceptable level of bacteria was exceeded, 49 samples had an average level of L. pneumophila (100-1,000 CFU/100 ml), and the level in 8 samples was high (>1,000 CFU/100 ml). CONCLUSIONS The water samples collected form the hot water supply system of hospitals and public buildings showed exceeded counts of L. pneumophila, indicating the risk of infection. The constant monitoring of water distribution systems is an important element of the control of infections caused by these organisms.

The role of programmed death ligand 1 pathway in persistent biomaterial-associated infections

Staphylococcus epidermidis is commonly involved in biomaterial-associated infections. Bacterial small colony variants (SCV) seem to be well adapted to persist intracellularly in professional phagocytes evading the host immune response. We studied the expression of PD-L1/L2 on macrophages infected with clinical isolates of S. epidermidis SCV and their parent wild type (WT) strains. The cytokine pattern which is triggered by the examined strains was also analysed. In the study, we infected macrophages with S. epidermidis WT and SCV strains. Persistence and release from macrophages were monitored via lysostaphin protection assays. Moreover, the effect of IFN-γ pre-treatment on bacterial internalisation was investigated. Expression of PD-L1/L2 molecules was analysed with the use of FACS. Inflammatory reaction was measured by IL-10, TNF-α ELISAs, and transcriptional induction of TNF-α. Our study revealed that clinical SCV isolates were able to persist and survive in macrophages for at least 3 days with a low cytotoxic effect and a reduced proinflammatory response as compared to WT strains. Bacteria upregulated PD-L1/L2 expression on macrophages as compared to non-stimulated cells. The results demonstrated that the ability of S. epidermidis SCVs to induce elevated levels of anti-inflammatory cytokine, IL-10, and reduced transcriptional induction of TNF-α, together with expression of PD-L1 on macrophages and the ability to persist intracellularly without damaging the host cell could be the key factor contributing to chronicity of SCV infections.

Empirical validation of Polish guidelines for the management of acute streptococcal pharyngitis in children

OBJECTIVE Group A Streptococcus (GAS) pharyngitis is currently the only commonly occurring form of acute pharyngitis for which antibiotic therapy is definitely indicated. Polish guidelines advocate the use of modified Centor score (MCS) to assess the probability of GAS pharyngitis. They advise performing throat culture or rapid antigen detection test (RADT) in children with score 2-3 in MCS and treating with antibiotic only those in whom GAS was detected. Negative RADT results should be confirmed by culture. In children with score 4, the guidelines allow to introduce empiric antibiotic therapy. Phenoxymethyl penicillin is recommended as a drug of choice to treat GAS pharyngitis. The aim of our study was to evaluate the accuracy of strategy recommended by Polish guidelines in identifying those children with acute pharyngitis who require antibiotic treatment. Hence, diagnostic values of score 4 in MCS and RADT were assessed using throat culture as a reference standard. Phenoxymethyl penicillin efficacy in GAS eradication and prevention of post-streptococcal complications were estimated as well. METHODS Ninety children between 2 and 15 years of age with acute pharyngitis symptoms suggesting GAS etiology (MCS ≥ 2), participated in our study. At the initial visit MCS was evaluated and two throat swabs were collected to perform RADT and culture. In children with GAS pharyngitis treated with penicillin, microbiological cure was assessed by performing two control throat cultures. Next, children were under observation for 3 months. RESULTS Positive predictive value of score 4 in MCS turned out to be 48.05% (95% CI: 36.5-59.7%). RADT sensitivity, specificity and diagnostic accuracy proved to be 100%, 96%, and 98%, respectively. GAS eradication rate in children treated with penicillin turned out to be 92.5%. No post-streptococcal sequelae occurred in any child in 3-month observation. CONCLUSIONS Empiric antibiotic therapy in children with score 4 in MCS will result in significant overtreatment of those with nonstreptococcal pharyngitis. New generation RADT diagnostic value in GAS detection proved to be equivalent to that of culture, which obviates the need of backup culture in children with negative RADT results. Phenoxymethyl penicillin revealed high eradication efficacy and proved to prevent post-streptococcal sequelae in children with acute GAS pharyngitis.

Assessment of cytokine release after in vitro stimulation of whole blood with legionella pneumophila in immunocompromised patients.

This study we examined ex vivo potential of the immune response after stimulation of whole blood with L. pneumophila SG 1, SG 2-14 and L. pneumophila standard strain ATCC 33152 in immunocompromised patients, such as: hemodialysis patients and patients after renal transplantation. The levels of TNF-α and IFN-γ in supernatants were measured with the use of commercial ELISA kits. The synthesis of TNF-α and IFN-γ after stimulation with L. pneumophila were analyzed in two aspects: differentiated stimulatory activity in relation to SG 1, SG 2-14 and ATCC 33152 L. pneumophila and differentiated response of the hemodialysis patients and patients after renal transplantation in relation to the control group. The positive and negative results of anti-L. pneumophila antibodies of two groups of our patients were found for the analysis of the stimulatory activity of L.pneumophila as a primary or secondary response. In patients with immunosuppression the response in the secretion of cytokines (TNF-α and IFN-γ) was reduced after stimulation of L. pneumophila SG 1 but in varying degrees after stimulation of L. pneumophila SG 2-14, which indicates that the risk of the infection is varied.

Evaluation of High-Mobility Group Box 1 Protein Concentration in Serum of Patients with M. tuberculosis Infection

Infection with Mycobacterium tuberculosis is accompanied by an intense inflammatory response. Recently, a new mediator of inflammation, HMGB1 protein has been identified that contributes to acute lung injury. However, its role in the systemic inflammatory response in tuberculosis has not been thoroughly investigated. We investigated the systemic levels of HMGB1 and TNF-α in patients with active and latent lung tuberculosis as a prognostic marker of disease activity. The study was performed to 70 patients with confirmed Mycobacterium tuberculosis infection and other than tuberculosis lung diseases and in 20 healthy persons. Serum HMGB1 and TNF-α concentrations were measured by ELISA. The highest concentration of HMGB1 was detected in the bloodstream of people with Mtb infection (latent and active). Its concentration increased significantly in sera of patients with active tuberculosis (47.5 ng/ml), compared to patients with other lung diseases (36.87 ng/ml). TNF-α had significantly higher concentration in a patients group compared to healthy controls, with the highest concentration in the LTBI group of patients (0.136 ng/ml). We observed a strong positive correlation between TNF-α and HMGB1 concentrations in patients with tuberculosis infections. We conclude that HMGB1 is secreted during active and latent tuberculosis in the highest amounts compared to other lung diseases.

Intestinal Colonization with Oxalobacter formigenes and its Relation to Urinary Oxalate Excretion in Pediatric Patients with Idiopathic Calcium Urolithiasis

BACKGROUND AND AIM Oxalobacter formigenes is an intestinal bacterium that utilizes oxalate as the only source of energy. It has been suggested that the lack of colonization with this organism may be a risk factor for calcium oxalate urolithiasis. Because this problem was not investigated in pediatric stone formers, we decided to assess it in our patients. METHODS The presence of O. formigenes in stool samples of 76 children and adolescents (aged 4.1-18 years) with idiopathic calcium urolithiasis (36 with chemically confirmed calcium oxalate stones and 40 children with a strong clinical suspicion of this type of urolithiasis) was assessed using PCR method. Simultaneously, urinary oxalate excretion was measured in this group. Fifty healthy, age- and sex-matched subjects served as controls. RESULTS O. formigenes was found in 21/76 patients (27.6%). In controls, frequency of colonization was similar (26%). The median 24h urinary oxalate excretion in patients colonized with O. formigenes was significantly lower in comparison with non-colonized patients, 0.319 (range 0.141-0.546) and 0.437 (range 0.198-0.967) mmol/1.73 m(2)/24h, respectively. CONCLUSIONS Higher urinary oxalate excretion in children with calcium urolithiasis may be a result of the absence of O. formigenes. The reasons for similarly low intestinal colonization with this bacterium in normal subjects and stone formers remain speculative. Thus, further studies are necessary to clarify this issue.

Exposure to ticks and seroprevalence of Borrelia burgdorferi among a healthy young population living in the area of southern Podlasie, Poland

OBJECTIVES The objective of the study was assessment of risk of infection with Borrelia burgdorferi in the area of southern Podlasie in Poland, near the border with Belarus, by analysis of post-exposure procedure, and evaluation of asymptomatic infection in adolescents bitten by a tick, confirmed by serologic tests. MATERIAL AND METHODS The study was conducted among 128 healthy individuals aged 16-20 who declared being bitten by a tick. The level of IgM and IgG class antibodies was determined using the immunoenzymatic test (Borrelia 14 kD + OspC IgM ELISA and Borrelia IgG + VlsE ELISA, DRG Diagnostics). Positive and doubtful results were confirmed using the Western blot method (EUROLINE-WB, EUROIMMUN). RESULTS In the study group, the largest number of respondents (59.4%) declared tick bite in the region of the lower extremities, most often in the knee pit. Among the methods for removing the tick the largest number of respondents indicated removing it with the use of tweezers, with a simple, swift steady movement (29.7%), and pulling it out with the fingers (22.7%). In the ELISA test, a positive or doubtful result in at least one class was observed in 25.0% of respondents (n=32/128): in IgM class - 23.4% (n=30/128), and in IgG class - 4.7% (n=6/128). After verification with the Western blot test, infection was confirmed in 5.5% of respondents (n=7/128): in IgM class - 1.6% (n=2/128), in IgG class - 3.9% (n=5/128). In IgM class antibodies, the Western blot test confirmed positive or doubtful results of the ELISA test in 6.7%, while in IgG class antibodies in 83.3%. CONCLUSION Evaluation of the actual infection with Borrelia spp. using serologic tests is difficult due to a certain non-specificity of the ELISA test, especially in IgM class antibodies, and difficulties with performance of a wide scope of specific Western blot tests. The variety of methods of tick removal declared by adolescents suggests that a wider education of society concerning appropriate methods of removing the tick should become an especially important element of prophylactic actions in the area of borreliosis.