Evaluation of TD test for analysis of persistence or tolerance in clinical isolates of Staphylococcus aureus.

Besides natural and acquired mechanisms of resistance, bacteria can cope with presence of antibiotics by using complex mechanisms such as persistence or tolerance. The main purpose of this study was to evaluate the suitability of newly developed Tolerance Disk Test (TDtest) (Gefen et al., 2017) to detect persistent or tolerant bacterial cells in clinical isolates of Staphylococcus aureus. The principle of the test is to resuscitate the subpopulation of persistent or tolerant bacterial cells following a disk diffusion test by glucose. Results of the TDtest were evaluated using time killing experiments for three pairs of consecutive S. aureus isolates from lower respiratory airway samples of three cystic fibrosis patients with chronic staphylococcal infections. TDtest enabled semi-quantitative detection of persistent or tolerant bacterial populations in all analyzed isolates for oxacillin, vancomycin, and ciprofloxacin to which isolates studied were susceptible. Therefore, TDtest is a promising method for rapidly determining persistence/tolerance in clinical isolates of S. aureus.

Distribution of CFTR mutations in the Czech population: positive impact of integrated clinical and laboratory expertise, detection of novel/de novo alleles and relevance for related/derived populations.

BACKGROUND: This two decade long study presents a comprehensive overview of the CFTR mutation distribution in a representative cohort of 600 Czech CF patients derived from all regions of the Czech Republic. METHODS: We examined the most common CF-causing mutations using the Elucigene CF-EU2v1™ assay, followed by MLPA, mutation scanning and/or sequencing of the entire CFTR coding region and splice site junctions. RESULTS: We identified 99.5% of all mutations (1194/1200 CFTR alleles) in the Czech CF population. Altogether 91 different CFTR mutations, of which 20 were novel, were detected. One case of de novo mutation and a novel polymorphism was revealed. CONCLUSION: The commercial assay achieved 90.7%, the MLPA added 1.0% and sequencing increased the detection rate by 7.8%. These comprehensive data provide a basis for the improvement of CF DNA diagnostics and/or newborn screening in our country. In addition, they are relevant to related Central European populations with lower mutation detection rates, as well as to the sizeable North American "Bohemian diaspora".

PCR detection of Burkholderia cepacia complex as one of key factors to handle a long-term outbreak.

BACKGROUND: Once the outbreak with Burkholderia cenocepacia ST32 was identified in the Prague cystic fibrosis (CF) centre, molecular tools were implemented into diagnostic routine in order to complement infection control measures with as accurate as possible microbiological service. In addition, genotyping techniques were applied as part of an infection surveillance program to assign species and strain status in samples positive for Burkholderia cepacia complex (Bcc). We sought to evaluate a usefulness of Bcc-specific PCR in infection control and to map evolution of the outbreak. METHODS: Since 2001, 6109 respiratory samples from 299 CF patients were examined not only by conventional culture, but also by PCR, detecting Bcc directly in sputum. RESULTS: Diagnosis of Bcc infection was established by culture in 54 patients already prior to 2001. As 39 more patients were diagnosed by culture and/or PCR during 2001-2010, this represented annual prevalence of 18.5%-28.9%. Twelve of 39 patients were culture negative at the time of their first PCR positivity. Although 2/3 of them became subsequently culture positive, the time delay in diagnostics of the infection by culture ranged from 1 to 22 months. New cases of Bcc infection were detected every year, however a dramatic drop was observed for the epidemic strain ST32. CONCLUSION: A likely factor contributing to the end of ST32 epidemic was segregation of Bcc infected patients that included also patients with no culture, but PCR positivity. The diagnostic PCR led to timely identification of cases with 'culture-invisible' infection.

Low-dose insulin therapy in patients with cystic fibrosis and early-stage insulinopenia prevents deterioration of lung function: a 3-year prospective study.

Cystic fibrosis related diabetes (CFRD) is an insulinopenic condition. We aimed to detect insulinopenia early and to evaluate the impact of low dose insulin on nutritional status and forced expiratory volume in first second (FEV1). Out of 142 cystic fibrosis patients (CFpts) older than 10 years, 28 with abnormal oral glucose tolerance test in spite of normal fasting glycemia were found to have decreased first phase insulin release and started low dose insulin therapy (median age 15.4 years). Sex and age matched CFpts with normal glucose tolerance (NGT) were observed for comparison. Whereas nutritional status improved following 3 years of insulin administration, FEV1 stabilized in insulin-treated insulinopenic subjects (73.8 +/- 4.3% vs. 73.5 +/- 4.4%), but decreased in the parallel group with NGT who remained without insulin treatment (71.1 +/- 3.8% vs. 61.0 +/- 4.0%; p = 0.001). We conclude that low dose insulin improves nutritional status and stabilizes pulmonary functions. Regular estimation of stimulated insulin secretion in CFpts may allow optimizing treatment.

Polymorphisms of TGF-beta1 in cystic fibrosis patients.

There is a significant phenotypic variance among cystic fibrosis (CF) patients. Due to the role of TGF-beta1 in fibrotic processes we investigated its role in CF pathogenesis. TGF-beta 1 codons 10 and 25 were genotyped in 118 Czech CF patients and 268 controls by PCR-ARMS. Difference between CF and controls was found at codon 10, lower frequency of T/T homozygotes, and codon 25, higher frequency of G/C heterozygotes. We did not prove the association of TGF-beta1 polymorphisms and lung function in CF, however, the TT (codon 10)/GG (codon 25) genotype was preferentially associated with CF-related liver disease and diabetes. Independent of the TGF-beta1 genotype, production of cytokine was higher in patients than in controls with the notable exception of very low levels in Burkholderia cepacia complex colonized patients. In CF, both extremes, highest or lowest TGF-beta 1 production, were associated with impaired lung function.

Widespread clone of Burkholderia cenocepacia in cystic fibrosis patients in the Czech Republic.

The morbidity and mortality rates in patients with cystic fibrosis (CF) are significantly affected by infections with Burkholderia cepacia complex. In a Czech CF Centre, the prevalence of the infection reached up to 30 %, with the majority of patients found to be infected with Burkholderia cenocepacia (formerly genomovar III of the Burkholderia cepacia complex). Since B. cenocepacia is associated with patient-to-patient transmission and epidemic outbreaks among CF patients, this study sought to examine the epidemiological relatedness between the Czech isolates belonging to the genomovar-homogeneous group. Eighty-three clinical isolates recovered from 67 CF patients were analysed using a random amplified polymorphic DNA (RAPD) assay and macrorestriction typing (SpeI and XbaI) followed by PFGE. A single predominant banding pattern shared by multiple isolates was detected, although SpeI-generated PFGE results yielded a higher rate of inter-pattern variability in comparison to the more uniform RAPD and XbaI-generated PFGE results for this clone. Both typing systems also showed that only three out of 67 patients harboured strains distinct from the major strain type. The dominant clone was characterized by PCR positivity for the B. cepacia epidemic strain marker, PCR negativity for the cable pilin subunit gene and close genetic relatedness to the epidemic strain of RAPD 01 type previously identified in Canada.

Antineutrophil cytoplasmic antibodies directed against bactericidal/permeability-increasing protein detected in children with cystic fibrosis inhibit neutrophil-mediated killing of Pseudomonas aeruginosa.

Antineutrophil cytoplasmic antibodies (ANCA) directed against bactericidal/permeability-increasing protein (BPI) were repeatedly found in cystic fibrosis (CF) patients. We analyzed the effect of BPI-ANCA in inhibiting neutrophil-mediated killing of Pseudomonas aeruginosa. The bactericidal effect expressed as percentage of killed bacteria after 1 h incubation with neutrophils was 55% when the neutrophils were pretreated with normal human serum, ranged from 49 to 63% with the sera from control BPI-ANCA-negative groups and sharply decreased to the mean 30.5% (range 8-51%) in the presence of BPI-ANCA. Furthermore, the effect mediated by BPI-ANCA was dose dependent and reflected the titer of BPI-ANCA in tested sera.

Direct PCR detection of Burkholderia cepacia complex and identification of its genomovars by using sputum as source of DNA.

We developed a nested PCR assay that detects the recA gene of the Burkholderia cepacia complex in sputum. The product of the first PCR round is also used to identify the genomovar of the pathogen. The protocol achieves high sensitivity and specificity with simple interpretation of genomovar status.