Publications (3)0 Total impact
Article: [First experience with detecting bacterial DNA in the cerebrospinal fluid of patients with purulent meningitis using broad spectrum multiplex nested PCR].[show abstract] [hide abstract]
ABSTRACT: To propose and verify a PCR assay for detecting Escherichia coli, 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. DNA from the cerebrospinal fluid was isolated using the QIAamp DNA Mini Kit. PCR was performed as two-step amplification (nested PCR). For E. coli, H. influenzae, L. monocytogenes, S. species and S. pneumoniae, universal and species-specific 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. 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. 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.Klinicka mikrobiologie a infekcni lekarstvi 09/2007; 13(4):160-4.
Article: [The demonstration of tuberculosis using molecular genetic methods polymerase chain reaction (PCR) and Amplified Mycobacterium tuberculosis direct (AMTD) test].[show abstract] [hide abstract]
ABSTRACT: Tuberculosis is a communicable disease, in most instances with a chronic course. The aetiological agent is Mycobacterium tuberculosis. Its demonstration is based on microscopic investigations and cultures. Microscopy is not sufficiently sensitive, while cultures are lengthy. One of the possibilities of speeding up diagnosis are molecular genetic methods. To compare the demonstration of mycobacteria using molecular genetic methods with the results of cultures. We used two methods to demonstrate the nucleic acid complex of Mycobacterium tuberculosis-the polymerase chain reaction (PCR) and the Amplified Mycobacterium tuberculosis direct test (AMTD). We investigated 647 samples. Out of these, 275 samples were tested with PCR and 372 were investigated with a AMTD set. At the same time we started for each sample a parallel culture. In 275 samples, out of a total of 647, which were analysed with PCR, mycobacterial DNA was demonstrated in 18 (6.5 %). Out of the 372 samples investigated with AMTD, mycobacterial RNA was demonstrated in 27 (7 %). Out of the 18 PRC positive samples, 6 (13 %) did not yield a positive mycobacterial culture. Out of the 27 positive results RNA with the AMTD method 17 did not yield positive cultures. On the other hand, a diagnosis of tuberculosis verified by cultures without a positive PCR was found in 2 patients (0.7 %). Disagreement between the results of AMTD and cultures was also found in 2 samples (0.5 %). Molecular genetic methods substantially speed up the diagnosis of tuberculosis. These methods are particularly important in cases of paucibacillary material and of unique and unrepeatable samples (tissues biopsies, nodes, cerebrospinal fluid). Given the possibility of false positive results, parallel verification by microscopy and cultures is essential.Klinicka mikrobiologie a infekcni lekarstvi 11/2006; 12(5):195-9.
Article: [Detection of anti-Ehrlichia antibodies and direct demonstration of Ehrlichia nucleic acid using the polymerase chain reaction (PCR) in patients in the Czech Republic].[show abstract] [hide abstract]
ABSTRACT: In patients presenting symptoms with a suspicion of ehrlichiosis we determined antiehrlichia antibodies and investigated the presence of Ehrlichia nucleic acid in the plasma. In our group were 46 patients with tick sucks in their case history, who presented symptoms compatible with ehrlichiosis. Anti-Ehrlichia antibodies were determined by an indirect immunofluorescent test with a commercial kit from MRL Diagnostics. Ehrlichia DNA was detected using a nested PCR - the target sequence was a part of the antigen Anaplasma phagocytophilum. Antibodies against HGE agents were demonstrated in 28 % of the patients; 10.5 % of the patients had in their serum antibodies reacting to the Ehrlichia chaffeensis antigen. The nucleic acid of A. phagocytophilum was detected in 11 % of the patients. The Czech population is relatively often exposed to Ehrlichia infections. Although most cases are asymptomatic, we should bear in mind this diagnosis, especially in immunodeficient patients, where early treatment may prevent a complicated course of the disease.Klinicka mikrobiologie a infekcni lekarstvi 03/2004; 10(1):25-9.