The diversity of sulfate-reducing bacteria in the seven bioreactors.

Anaerobic technology has a wide scope of application in different areas such as manufacturing, food industry, and agriculture. Nowadays, it is mainly used to produce electrical and thermal energy from crop processing, solid waste treatment or wastewater treatment. More intensively, trend nowadays is usage of this technology biodegradable and biomass waste processing and biomethane or hydrogen production. In this paper, the diversities of sulfate-reducing bacteria (SRB) under different imputed raw material to the bioreactors were characterized. These diversities at the beginning of sampling and after cultivation were compared. Desulfovibrio, Desulfobulbus, and Desulfomicrobium genus as dominant among sulfate reducers in the bioreactors were detected. The Desulfobulbus species were dominant among other SRB genera before cultivation, but these bacteria were detected only in three out of the seven bioreactors after cultivation dominant.

Evaluation of ibuprofen toxicity for zebrafish (Danio rerio) targeting on selected biomarkers of oxidative stress.

OBJECTIVES: The aim of the study was to investigate the effects of subchronic exposure of zebrafish to ibuprofen, using selected oxidative stress parameters as a target. DESIGN: Toxicity tests were performed on Danio rerio according to OECD No. 203 and No. 215. In the growth test, fish were exposed to subletal concentrations of ibuprofen (0.0001, 0.05, 1, 8, and 25 mg.L-1) for 28 days. For the assessment of free radical defense in fish, the catalytic activities of glutathione reductase (GR), glutathione S-transferase (GST), glutathione peroxidase (GPx), and catalase (CAT), as well as the concentration of malondialdehyde (MDA) were measured. RESULTS: Ibuprofen did not affect the activity of glutathione reductase and catalase. A significant (p<0.01) increase in the activity of glutathione peroxidase was found, which was proved dose-dependent (10.58 nmol NADPH per min per mg protein in the control and 20.53, 26.36, 26.89, and 45.87 nmol NADPH per min per mg protein in the ibuprofen concentrations of 0.5, 1, 8, and 25 mg.L-1. An increased (p<0.05) activity of glutathione S-transferase in the highest concentration was found compared to control. Malondialdehyde levels were found significantly (p<0.01) decreased from control in the concentrations of 0.0001 and 8 mg.L-1, but no dose-dependence was found. CONCLUSION: The results suggest that ibuprofen causes the increase in the activity of some antioxidative and biotransformation enzymes in zebrafish (GPx and GST). We also found a significant decrease in lipid peroxidation in the concentrations of 0.0001 and 8 mg.L-1 compared to control.

Gene polymorphisms involved in manifestation of leucopenia, digestive intolerance, and pancreatitis in azathioprine-treated patients.

BACKGROUND: Approximately 10-28 % of patients experience adverse drug reactions related to treatment with thiopurines. The most serious reaction is myelosuppression, typically manifested as leucopenia, which occurs in approximately 2-5 % of patients. Other adverse drug reactions that often accompany thiopurine therapy are pancreatitis, hepatotoxicity, allergic reactions, digestive intolerance, arthralgia, febrile conditions, and rash. OBJECTIVE: The objective of this study was to assess the relationship between variant alleles of thiopurine S-methyltransferase (SNPs 238G > C, 460G > A and 719A > G), inosine triphosphate diphosphatase (SNPs 94C > A and IVS2 + 21A > C), and xanthine dehydrogenase (837C > T) and the occurrence of adverse drug reactions to azathioprine therapy. METHODS: Genotype was determined for 188 Caucasians diagnosed with inflammatory bowel disease treated with a standard dose of azathioprine (1.4-2.0 mg/kg/day). Allelic variants were determined by PCR-REA and real-time PCR methods. Results were statistically evaluated by use of Fisher's test and by odds ratio calculation. RESULTS: Variant genotype thiopurine S-methyltransferase predisposes to development of leucopenia (P = 0.003, OR = 5, CI 95 %, 1.8058-13.8444). Although not statistically significant, we observed a trend that suggested correlation between the occurrence of digestive intolerance and the variant genotype inosine triphosphate diphosphatase (P = 0.1102; OR 15.63, CI 95 %, 1.162-210.1094), and between the occurrence of pancreatitis and the variant allele xanthine dehydrogenase 837T (P = 0.1124; OR 12,1, CI 95 %, 1.15-126.37). CONCLUSION: The variant genotype thiopurine S-methyltransferase has been associated with the occurrence of leucopenia. The involvement of polymorphisms in inosine triphosphate diphosphatase and xanthine dehydrogenase genes in the development of digestive intolerance and pancreatitis will require further verification.

Increased occurrence of Treponema spp. and double-species infections in patients with Alzheimer's disease.

Although the link between microbial infections and Alzheimer's disease (AD) has been demonstrated in multiple studies, the involvement of pathogens in the development of AD remains unclear. Here, we investigated the frequency of the 10 most commonly cited viral (HSV-1, EBV, HHV-6, HHV-7, and CMV) and bacterial (Chlamydia pneumoniae, Helicobacter pylori, Borrelia burgdorferi, Porphyromonas gingivalis, and Treponema spp.) pathogens in serum, cerebrospinal fluid (CSF) and brain tissues of AD patients. We have used an in-house multiplex PCR kit for simultaneous detection of five bacterial and five viral pathogens in serum and CSF samples from 50 AD patients and 53 healthy controls (CTRL). We observed a significantly higher frequency rate of AD patients who tested positive for Treponema spp. compared to controls (AD: 62.2 %; CTRL: 30.3 %; p-value = 0.007). Furthermore, we confirmed a significantly higher occurrence of cases with two or more simultaneous infections in AD patients compared to controls (AD: 24 %; CTRL 7.5 %; p-value = 0.029). The studied pathogens were detected with comparable frequency in serum and CSF. In contrast, Borrelia burgdorferi, human herpesvirus 7, and human cytomegalovirus were not detected in any of the studied samples. This study provides further evidence of the association between microbial infections and AD and shows that paralleled analysis of multiple sample specimens provides complementary information and is advisable for future studies.

Natural compound cudraflavone B shows promising anti-inflammatory properties in vitro.

Cudraflavone B (1) is a prenylated flavonoid found in large amounts in the roots of Morus alba, a plant used as a herbal remedy for its reputed anti-inflammatory properties. The present study shows that this compound causes a significant inhibition of inflammatory mediators in selected in vitro models. Thus, 1 was identified as a potent inhibitor of tumor necrosis factor α (TNFα) gene expression and secretion by blocking the translocation of nuclear factor κB (NF-κB) from the cytoplasm to the nucleus in macrophages derived from a THP-1 human monocyte cell line. The NF-κB activity reduction resulted in the inhibition of cyclooxygenase 2 (COX-2) gene expression. Compound 1 acts as a COX-2 and COX-1 inhibitor with higher selectivity toward COX-2 than indomethacin. Pretreatment of cells by 1 shifted the peak in an regulatory gene zinc-finger protein 36 (ZFP36) expression assay. This natural product has noticeable anti-inflammatory properties, suggesting that 1 potentially could be used for development as a nonsteroidal anti-inflammatory drug lead.

Importance of thiopurine S-Methyltransferase gene polymorphisms for prediction of azathioprine toxicity.

OBJECTIVES: Our study aims to find the relationship between metabolic enzyme thiopurine S-methyltransferase (TPMT) gene polymorphisms and clinical output of the therapy with azathioprine. We focused on patients who experienced leucopenia caused by high blood levels of active azathioprine metabolites. DESIGN: Our group consists of 87 patients who have been treated by azathioprine. 21 individuals experienced leucopenia during treatment with standard dose of azathioprine. We have used PCR-REA and "real-time" PCR methods for genotype detection G238C, G460G and A719G substitutions in TPMT gene. RESULTS: We have found statistical association between the presence of non-standard TPMT alleles and adverse event associated with azathioprine treatment - leucopenia (p=0.0033). CONCLUSION: Our results confirm that TPMT genotyping prior to the treatment with azathioprine could predict patients with genetic predisposition for serious leucopenia and seems to be a useful genetic marker for individualisation of the therapy.

Comprehensive proteomic analysis of exoproteins expressed by ERIC I, II, III and IV Paenibacillus larvae genotypes reveals a wide range of virulence factors.

American foulbrood is a quarantine disease of the honeybee Apis mellifera L. in many countries and contributes greatly to colony losses. We performed a label-free proteomics study of exoprotein fractions produced in vitro by Paenibacillus larvae reference strains of the ERIC I-IV genotypes. A quantitative comparison was performed of previous studied protein-based virulence factors and many newly identified putative virulence factors. Among the multiple proteases identified, key virulence factors included the microbial collagenase ColA and immune inhibitor A (InhA, an analog of the Bacillus thuringiensis protein InhA). Both of these virulence factors were detected in ERICs II-IV but were absent from ERIC I. Furthermore, the different S-layer proteins and polysaccharide deacetylases prevailed in ERICs II-IV. Thus, the expression patterns of these virulence factors corresponded with the different speeds at which honeybee larvae are known to be killed by ERICs II-IV compared to ERIC I. In addition, putative novel toxin-like proteins were identified, including vegetative insecticidal protein Vip1, a mosquitocidal toxin, and epsilon-toxin type B, which exhibit similarity to homologs present in Bacillus thuringiensis or Lysinibacillus sphaericus. Furthermore, a putative bacteriocin similar to Lactococcin 972 was identified in all assayed genotypes. It appears that P. larvae shares virulence factors similar to those of the Bacillus cereus group. Overall, the results provide novel information regarding P. larvae virulence potential, and a comprehensive exoprotein comparison of all four ERICs was performed for the first time. The identification of novel virulence factors can explain differences in the virulence of isolates.

Evaluation of miRNA detection methods for the analytical characteristic necessary for clinical utilization.

miRNAs are promising biomarkers but methods for their measurement are not clear. We therefore examined three miRNA detection technologies and considered the analytical characteristics essential for clinical utilization. TaqMan assays, SplintR-qPCR and miREIA were compared for their absolute quantification bias, conformity and robustness. Absolute concentrations of miR-142-5p, miR-23a-3p and miR-93-5p were measured with all three methods using 30 samples. Robustness was evaluated by measurement of miR-21-5p in five uniform experiments. Correlations were miRNA-specific, but we observed a different absolute concentration range in RT-qPCR (fmol/µl) and methods evading the RT process (amol/µl). Consistently, RT-less methods reported better robustness (CV 8-19%) than RT-qPCR (CV 39-50%). The calibration curve in TaqMan Advanced assay was influenced by dilution media. Methods avoiding RT seem to be a promising future alternative for miRNA measurement.

A New Combination of Substrates: Biogas Production and Diversity of the Methanogenic Microorganisms.

Agriculture, food industry, and manufacturing are just some of the areas where anaerobic technology can be used. Currently, anaerobic technologies are mainly used for wastewater treatment, solid waste treatment, or for the production of electrical and thermal energy from energy crops processing. However, a clear trend is towards more intensive use of this technology in biomass and biodegradable waste processing and hydrogen or biomethane production. An enormous number of anaerobic digesters are operating worldwide but there is very little information about the effect of different substrate combinations on the methanogens community. This is due to the fact that each of the anaerobic digesters has its own unique microbial community. For the most effective management of anaerobic processes it would be important to know the composition of a consortium of anaerobic microorganisms present in anaerobic digesters processing different input combinations of raw material. This paper characterizes the effect of the input raw materials on the diversity of the methanogen community. Two predominant microorganisms in anaerobic digesters were found to be 99% identity by the sequences of the 16S rRNA gene to the Methanoculleus and Thermogymnomonas genera deposited in GenBank.

Nontuberculous mycobacteria in the environment of Hranice Abyss, the world's deepest flooded cave (Hranice karst, Czech Republic).

Nontuberculous mycobacteria (NTM) are widely distributed in the environment. On one hand, they are opportunistic pathogens for humans and animals, and on the other hand, they are effective in biodegradation of some persistent pollutants. Following the recently recorded large abundance of NTM in extreme geothermal environments, the aim of the study was to ascertain the occurrence of NTM in the extreme environment of the water zone of the Hranice Abyss (HA). The HA mineral water is acidic, with large concentrations of free CO2, and bacterial slimes creating characteristic mucilaginous formations. Both culture and molecular methods were used to compare the mycobacterial diversity across the linked but distinct ecosystems of HA and the adjacent Zbrasov Aragonite Caves (ZAC) with consideration of their pathogenic relevance. Six slowly growing NTM species (M. arupense, M. avium, M. florentinum, M. gordonae, M. intracellulare) and two rapidly growing NTM species (M. mucogenicum, M. sediminis) were identified in the water and in the dry zones at both sites. Proteobacteria were dominant in all the samples from both the HA and the ZAC. The bacterial microbiomes of the HA mineral water and HA slime were similar, but both differed from the microbiome in the ZAC mineral water. Actinobacteria, a phylum containing mycobacteria, was identified in all the samples at low proportional abundance. The majority of the detected NTM species belong among environmental opportunistic pathogens.

Comparative evaluation of positive tests to Mycobacterium avium subsp. paratuberculosis in clinically healthy sheep and goats in south-west Greece using molecular techniques, serology, and culture.

Mycobacterium avium subsp. paratuberculosis (MAP) is the cause of paratuberculosis, which affects mainly ruminants although there is a growing concern about its possible implication in Crohn's disease in humans especially in connection with environmental spread and risks to the food chain. Retail cheese may represent a significant source of human exposure to MAP and the aim of this study was to assess MAP status in clinically healthy sheep and goats in Greece, comparing techniques routinely used in the positive diagnosis of the disease. From a total of 30 flocks, 632 sheep and goats had faecal, serum, and whole-blood samples examined by culture, complement fixation test (CFT), and polymerase chain reaction (PCR) targeted at IS900, IS1245, and IS6110. PCR produced positive results in 21% of the animals tested, with 5.6%, 3.9%, and 11.5% being identified as MAP, Mycobacterium avium subsp. avium, and Mycobacterium tuberculosis complex, respectively. CFT produced positive and suspicious results in 4.4% and 14.4% of the cases. Faecal cultures were negative in all but a single case that was identified as restriction fragment length polymorphism (RFLP)-type BC1. Agreement between results obtained by PCR and CFT was poor with isolated cases although an assessment of the MAP positive tests produced similar results for both methods. The findings indicate the need for additional measures of control, although the costs may be substantial if public health protection justifies elimination of MAP from livestock.

Identification of members of Mycobacterium avium species by Accu-Probes, serotyping, and single IS900, IS901, IS1245 and IS901-flanking region PCR with internal standards.

From Mycobacterium avium species Mycobacterium avium subsp. paratuberculosis (n=961), Mycobacterium a. avium (n=677), Mycobacterium a. silvaticum (n=5), and Mycobacterium a. hominissuis (n=1566) were examined, and from Mycobacterium tuberculosis complex M. tuberculosis (n=2), Mycobacterium bovis (n=13), M. bovis BCG (n=4), and Mycobacterium caprae (n=10) were examined. From other mycobacterial species Mycobacterium intracellulare (n=60) and atypical mycobacteria (n=256) including Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium scrofulaceum, Mycobacterium gastri and other species of conditionally pathogenic mycobacteria were analysed. The internal standard molecules corresponding to insertion sequences IS900, IS901, IS1245, and flanking region (FR300) of IS901 were produced by PCR of alfalfa genome segment and inserted into plasmid vector. The resulting recombinant plasmid molecules were used as internal standards in coamplification with a total of 4729 mycobacterial collection strains and field isolates between 1996 and 2003. The size differences between amplicons obtained from IS900 (258 bp), IS901 (1108 bp), IS1245 (427 bp), and FR300 (300 bp) and from corresponding internal standard molecules ISIS900 (591 bp), ISIS901 (1 336 bp), ISIS1245 (583 bp), and IS901 flanking region of 300 bp ISFR300 (488 bp), respectively, allowed easy discrimination. The internal amplicons were visible by naked aye on agarose gel when 10(1), 10(3), 10(2), and 10(2) molecules for ISIS900, ISIS901, ISIS1245, and ISFR300 were used in the PCR, respectively, when no bacterial DNA was added to the reaction. The system was tested to define the amount of internal standards that could be used in the PCR without affecting the amplification of the specific segment. Non-specific amplifications were observed in M. fortuitum with IS1245 PCR and mixed infections with M. a. avium and M. a. hominissuis from pigs and cattle were found. PCR results of typing were compared with serotyping and Accu-Probes analyses in selected field isolates.

Detection of Mycobacterium avium subsp. paratuberculosis in two brown bears in the central European Carpathians.

The incidence of mycobacterial infections was monitored in brown bears (Ursus arctos) in the National Park Low Tatras in the central European Carpathians in Slovakia. Tissue samples of 20 brown bears were examined microscopically and by culture for the presence of mycobacteria. Acid-fast rods were detected by Ziehl-Neelsen staining in a smear from the kidney of one brown bear, although the culture was negative for mycobacteria. Mycobacterium avium subsp. paratuberculosis, the causative agent of paratuberculosis in ruminants, was isolated from the intestinal mucosa of another two brown bears. The isolates were identified by polymerase chain reaction for the specific insertion sequence IS900. Using standardized IS900 restriction fragment length polymorphism (RFLP) analysis, the M. a. paratuberculosis isolates were classified as RFLP type B-C1, which also were detected in the infected cattle in surrounding area. This study describes the first isolation of M. a. paratuberculosis from a brown bear. Our results confirm that animal species other than ruminants can become infected with M. a. paratuberculosis and can act as potential vectors and/or reservoirs of the infection.

Model-based Characterization of the Parameters of Dissimilatory Sulfate Reduction Under the Effect of Different Initial Density of Desulfovibrio piger Vib-7 Bacterial Cells.

The objective of this study was to design a model of dissimilatory sulfate reduction process using the Verhulst function, with a particular focus on the kinetics of bacterial growth, sulfate and lactate consumption, and accumulation of hydrogen sulfide and acetate. The effect of the initial density (0.12±0.011, 0.25±0.024, 0.5±0.048 and 1.0±0.096 mg cells/ml of medium) of the sulfate-reducing bacteria Desulfovibrio piger Vib-7 on the growth and dissimilatory sulfate reduction was studied. The exponential growth phase of the D. piger Vib-7 was observed for 72 hours of cultivation at the (0.12 and 0.25 mg/ml) initial concentration of bacterial cells. Sulfate and lactate were consumed incompletely during this time. The increase in the initial concentration of cells to 0.5 and 1 mg/ml led to a shortening of the exponential bacterial growth phase and a shift to the stationary phase of the growth. In the case of 0.5 mg/ml seeding, the stationary growth phase was observed in the 36(th) hour of cultivation. The increase in the initial concentration of cells to 1 mg/ml led to the beginning of the stationary growth phase in 24th hours of cultivation. Under these conditions, sulfate and lactate were consumed completely in the 48th hour of cultivation. The kinetic analysis of the curves of bacterial growth and the process of dissimilatory sulfate reduction by D. piger Vib-7 was carried out.

Expression of Actinobacillus pleuropneumonia gene coding for Apx I protein in Escherichia coli.

This study presents cloning and expression of Actinobacillus pleuropneumoniae Apx I toxin in Escherichia coli expression system to produce fusion protein for the subsequent immunological studies. The gene coding Apx I toxin was amplified from the A. pleuropneumoniae serotype 10 DNA using polymerase chain reaction and cloned to vector under the control of strong, inducible T7 promoter. The presence of insert was confirmed by PCR screening and sequencing after the propagation of recombinant DNA in E. coli cells. The gene coding A. pleuropneumoniae Apx I toxin was extended with a segment to encode a polyhistidine tag linked to its C-terminal sequence allowing a one-step affinity purification of the complex with Ni-NTA resin. Expression of the Apx I coding sequence in E. coli resulted in the formation of insoluble inclusion bodies purified according to a standard purification protocol. The ease of this expression system, the powerful single-step purification and low costs make it possible to produce Apx I in large amounts to further study the role of Apx I in physiological processes.

Comparative evaluation of PCR assays for the robust molecular detection of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) can cause a very serious, often-fatal disease, namely paratuberculosis, in several animal species, especially ruminants. Recently, it has also been implicated in the pathogenesis of Infectious Bowel Disease of man. The aim of this study was to develop a molecular method for the routine detection and identification of MAP, from tissue samples of animal origin. The proposed assay would have to combine optimum performance and cost, with high reproducibility. To this goal, three laboratories in Greece and the Czech Republic undertook different parts of a study that involved evaluation of DNA extraction procedures, and PCR assays, for MAP detection. For DNA extraction we used one in-house, and one commercial method, and for the PCR we assessed a number of different assays, starting with the evaluation of primer specificity with an extended GenBank database search. Based on these results, we chose to assess a one-tube nested, 2 two-tube nested, and a single PCR assay, targeted to different genomic regions of the IS900 element. These four methods were applied on positive and negative control samples, consisted of pure bacterial cultures and formalin-fixed paraffin-embedded (FFPE) tissue samples collected from cattle with paratuberculosis and chickens with M. avium subsp. avium infection. Based on the criteria of reliability and cost, the procedure that performed better was the one-tube nested PCR assay combined with the in-house DNA extraction method. The agreement of the results obtained by the three collaborating laboratories indicates the reliability of the proposed assay even under different laboratory conditions.

A standardised restriction fragment length polymorphism (RFLP) method for typing Mycobacterium avium isolates links IS901 with virulence for birds.

A standardised method for PvuII-PstI-IS901 restriction fragment length polymorphism (RFLP) typing was developed and evaluated against 173 isolates of Mycobacterium avium subsp. avium and M. avium subsp. silvaticum originating from birds (N=46) and their aviaries (N=5), pigs (N=85), cattle (N=18), reference serotype strains (N=9), humans (N=7), a horse (N=1), a nutria (N=1), and strain M. avium subsp. avium ST 18 (formerly M. avium subsp. paratuberculosis ST 18). PvuII-IS1245 RFLP typing was also performed on all isolates. DNA was digested in parallel by restriction endonucleases PvuII or PstI and hybridised to standard probes prepared by PCR. DNA fingerprints were scanned by CCD camera and analysed by the Gel Compar (Applied Maths, Version 4.1, Kortrijk, Belgium) software using a standard isolate control profile. A total of 52 PvuII-PstI RFLP profiles was described including 25 PvuII RFLP profiles designated A to Y and 25 PstI RFLP profiles designated A1-L3. Profiles were found to be stable in vivo and in vitro after multiple subcultures. High IS901 copy number was associated with a "bird" PvuII-IS1245 RFLP profile and low IS901 copy number with M. avium subsp. avium isolates from humans and the nutria. A virulence assay of 100 IS901-positive isolates using intramuscular infections of pullets showed 83 isolates differentiated into 32 RFLP types to be virulent and 17 isolates differentiated into 12 RFLP types as nonvirulent. Attenuation of virulence for pullets could be attributed to either multiple in vitro subculture, polyclonal infection or human passage and was not related to IS901 or IS1245 profiles.

Differentiation of Actinobacillus pleuropneumoniae by PCR-REA based on sequence variability of the apxIVA gene and by ribotyping.

During the period of 2001-2003, a total of 591 Actinobacillus pleuropneumoniae field isolates from the Czech Republic were serotyped with a high occurrence of cross-reactions. The cross-reactions were observed in 416 isolates. Most frequently, in 401 isolates (67.9%), cross-reactions with antisera specific for serotypes 9, 11, and/or 1 were observed. Two additional molecular methods, ribotyping and restriction analysis of PCR amplified apxIVA gene (PCR-REA), were therefore used for detailed characterisation of A. pleuropneumoniae. In this subsequent analysis, reference strains representing serotypes 1-12 and 25 field isolates showing the most frequent serotype cross-reactions were examined. PCR-REA enabled all reference strains to be distinguished except for the strains of serotypes 9 and 11. Ribotyping distinguished all reference strains except two pairs of serotypes: 3 versus 6, and 9 versus 11, respectively. Field isolates with serotype cross-reactivity 9, 11, and/or 1 could not be differentiated by either of these methods.

Frequencies of HLA-DQ2 and HLA-DQ8 haplotypes in Czech and Slovak coeliac patients and the healthy population.

Coeliac disease is an autoimmune disorder with genetic predisposition. The aim was to determine the frequency of HLA-DQ2 and HLA-DQ8 in Czech and Slovak patients and the healthy population. The study included 127 patients and 66 healthy volunteers. HLA-DQ2 was identified in 85.03% patients, and 24.24% healthy individuals (P=0.0001; OR17.7632; CI=8.4347-37.4088). HLA-DQ8 was identified in 11.81% patients and 15.5% healthy individuals. HLA-DQ8 occurred more often in HLA-DQ2-negative patients compared to HLA-DQ2-positive patients (P=0.0494; OR3.5; CI 1.0428-11.7468). At least one of the studied HLA-variants was found more often in patients than in healthy individuals (P=0.0001; OR58.8; CI 7.6856-449.8602).