Q105407295
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BACKGROUND: Borrelial specific DNA was examined in a group of 62 patients with different forms of Lyme borreliosis (LB) (32 patients suffered from neuroborreliosis, 19 manifested erythema migrans, and 11 joint involvement). METHODS: Nested-PCR system with five newly derived primers was used in parallel. The study was organized prospectively, the presence of DNA was tested for plasma, CSF, joint fluid and urine before treatment, and plasma, joint fluid and urine were examined after treatment. RESULTS: Before therapy, 36 patients (58.1%) were DNA positive on the whole; 21 positive patients (65.6%) were found in the group of neuroborreliosis, 8 (42.1%) showed signs of skin involvement, and 7 (63.6%) were positive in arthritis. After treatment, 11 patients (36.7%) were positive in neuroborreliosis, 3 (17.6%) in skin form, and 6 (54.5%) in joint form of LB. Among 97 positive amplifications the most frequent target was found in primer corresponding with 16S rDNA (50 samples, 51.5%). Lower but very similar results were obtained with primers for OspA (18 positive amplifications; 18.6%), OspC (13 positive amplifications; 13.4%), and flagellin (13 positive amplifications; 13.4%). There were 11 patients in whom only DNA and no specific antibodies were found. CONCLUSIONS: Specific DNA was found in all clinical groups of LB with similar sensitivity. Examination of the borrelial DNA in urine displayed the same sensitivity as in CSF and had a two times higher sensitivity than in plasma.
- MeSH
- Borrelia burgdorferi komplex genetika MeSH
- DNA bakterií analýza genetika MeSH
- DNA primery MeSH
- infekční artritida mikrobiologie MeSH
- kůže mikrobiologie MeSH
- lidé MeSH
- lymeská nemoc genetika imunologie MeSH
- polymerázová řetězová reakce MeSH
- prospektivní studie MeSH
- protilátky bakteriální analýza MeSH
- senzitivita a specificita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Both the human leucocyte antigen (HLA) DRB1 and the HLA DQB1 gene loci play a role in the development and progression of autoimmune diabetes mellitus (T1DM). Similarly, the insulin promoter variable number tandem repeats (INS-VNTR) polymorphism is also involved in the pathogenesis of diabetes mellitus (DM). We studied the association between each of these polymorphisms and DM diagnosed in patients older than age 35 years. Furthermore, we analysed possible interactions between HLA DRB1/DQB1 and INS-VNTR polymorphisms. Based on C-peptide and GADA levels we were able to distinguish three types of diabetes: T1DM, latent autoimmune diabetes in adults (LADA) and T2DM. INS-VNTR was genotyped indirectly by typing INS-23HphI A/T polymorphism. The genotype and allele frequencies of INS-23HphI did not differ between each of the diabetic groups and group of healthy subjects. We did, however, observe an association between the INS-23HphI alleles, genotypes and C-peptide secretion in all diabetic patients: A allele frequency was 86.2% in the C-peptide-negative group vs. 65.4% in the C-peptide-positive group (P(corr.) < 0.005); AA genotype was found to be 72.4% in the C-peptide-negative group vs. 42.6% in the C-peptide-positive groups (P(corr.) < 0.01). The HLA genotyping revealed a significantly higher frequency of HLA DRB1*03 allele in both T1DM and LADA groups when compared to healthy subjects: T1DM (25.7%) vs. control group (10.15%), odds ratio (OR) = 3.06, P < 0.05; LADA (27.6%) vs. control (10.15%), OR = 3.37, P < 0.01. The simultaneous presence of both HLA DRB1*04 and INS-23HphI AA genotype was detected in 37.5% of the T1DM group compared to only 9.2% of the healthy individuals group (OR = 5.9, P(corr.) < 0.007). We summarize that in the Central Bohemian population of the Czech Republic, the INS-23HphI A allele appears to be associated with a decrease in pancreatic beta cell secretory activity. HLA genotyping points to at least a partial difference in mechanism, which leads to T1DM and LADA development as well as a more diverse genetic predisposition in juvenile- and adult-onset diabetes. The simultaneous effect of HLA and INS-VNTR alleles/genotypes predispose individuals to an increased risk of diabetes development.
- MeSH
- alely MeSH
- diabetes mellitus 1. typu genetika imunologie MeSH
- dospělí MeSH
- frekvence genu genetika imunologie MeSH
- genetická predispozice k nemoci MeSH
- HLA-DQ antigeny genetika imunologie MeSH
- HLA-DR antigeny genetika imunologie MeSH
- inzulin genetika imunologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- minisatelitní repetice genetika MeSH
- věk při počátku nemoci MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- multicentrická studie MeSH
- Geografické názvy
- Česká republika MeSH
Cíl práce: Sestavení a ověření postupu PCR na průkaz Escherichia coli, Haemophilus influenzae, Listeria monocytogenes, Staphylococcus species, Streptococcus pneumoniae a Neisseria menigitidis séroskupiny B a C z jednoho vzorku mozkomíšního moku u pacientů s hnisavou meningitidou. Materiál a metody: Na izolaci DNA z mozkomíšního moku byl použit QIAamp DNA Mini Kit. PCR probíhala jako dvoustupňová amplifikace (nested PCR). Pro E. coli, H. influenzae, L. monocytogenes, S. species a S. pneumoniae byly v první reakci použity univerzální a ve druhé druhově specifické primery kódující bakteriální 16S rDNA. Pro N. menigitidis séroskupiny B a C byl použit amplifikačni systém s primery pro gen SiaD. Výsledky. Z 25 vyšetřených pacientů na začátku léčby byla DNA bakterií prokázána v mozkomíšním moku u 17 (68 %). Šestkrát se jednalo o N. meningitidis séroskupiny B, 4x N. meningitidis séroskupiny C, 5x S. pneumoniae ,lx H, influenzae a Ix L. monocytogenes. Ze 7 pacientů, u kterých byla antibiotická terapie zahájena před diagnostickou lumbální punkcí, byla PCR pozitivní ve čtyřech případech. Závěr. Navržený postup nested PCR je rychlejší než klasická kultivace a hodí se pro včasnou laboratorní diagnostiku infekčního agens. v porovnání s kultivací poskytuje technika mírně vyšší pozitivitu (o 16 %), podobně je tomu i u vzorků vyšetřených po nasazení antibiotické terapie. Metodou PCR nebyla nikdy prokázána jiná bakterie než ta, která byla vykultivována.
Objectives: To propose and verify a PCR assay for detecting Escherichia coii, Haemophilus influenzae, Listeria monocytogenes, Staphylococcus species, Streptococcus pneumoniae and Neisseria meningitidis serogroups B and C in a single sample of the cerebrospinal fluid of patients with purulent meningitis. Material and methods: DNA from the cerebrospinal fluid was isolated using the QIAamp DNA Mini Kit. PCR was performed as twostep amplification (nested PCR). For E. coli, H. influenzae, L. monocytogenes, S. species and S. pneumoniae, universal and speciesspecific primers encoding bacterial 16S rDNA were used in the first and second reaction, respectively. For N. meningitidis serogroups B and C, an amplification system with primers for the SiaD gene was utilized. Resiilts: Of 25 patients examined at the beginning of their treatment, bacterial DNA was detected in the cerebrospinal fluid of 17 (68 %) of them. Those were six cases of N. meningitidis serogroup B, four of N. meningitidis serogroup C, five of S. pneumoniae, one of H. influenzae and one of L. monocytogenes. Of 7 patients in whom antibiotic therapy was initiated prior to diagnostic lumbar puncture, PCR was positive in four cases. Conclusions: The proposed nested PCR approach is faster than traditional culture methods and suitable for early laboratory diagnosis of infectious agents. When compared to culture methods, the technique offers slightly higher positivity (by 16 %). This is similar in samples analyzed after the initiation of antibiotic therapy. The PCR method never detected other bacteria than the cultured ones.
OBJECTIVES: DNA proof is the only widely available direct diagnostic tool in Lyme borreliosis. Sensitive PCR detecting of spirochetal DNA was prepared and a prospective study in neuroborreliosis was performed. MATERIALS AND METHODS: 57 hospitalised patients with active neuroborreliosis and proved CSF antibodies synthesis were examined. Nested-PCR (utilizing three targets) was used for the detection of specific DNA in plasma, CSF and urine. RESULTS: Before treatment 36 positive patients (63.1%) were found in all tested specimens in parallel, 28 patients (49.1%) were positive in urine, 20 in CSF (35.0%) and 16 in plasma 28.0%). Later only urine was tested and the following results were obtained: 17 positive patients (30.0%) immediately after treatment, 8 (14.0%) after 3 months and one patient persisted positivity after 6 months. CONCLUSIONS: The highest sensitivity of PCR was achieved in the acute period of neuroborreliosis - 63.1% in three body fluids comparing with CSF antibody synthesis.
- MeSH
- Borrelia burgdorferi genetika imunologie MeSH
- dítě MeSH
- DNA bakterií genetika MeSH
- dospělí MeSH
- financování organizované MeSH
- lidé středního věku MeSH
- lidé MeSH
- lymská neuroborelióza diagnóza mikrobiologie MeSH
- mladiství MeSH
- následné studie MeSH
- polymerázová řetězová reakce MeSH
- prospektivní studie MeSH
- protilátky bakteriální MeSH
- senioři MeSH
- senzitivita a specificita MeSH
- specificita protilátek MeSH
- tělesné tekutiny mikrobiologie MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
