Samples Of Cystic Fibrosis Patients Research Articles

Evaluation of mixed biofilm production by Candida spp. and Staphylococcus aureus strains co-isolated from cystic fibrosis patients in northwest Algeria

Cystic fibrosis patients' lungs are chronically colonized by multiple microbial species capable of forming biofilms. This study aimed to characterize the polymicrobial biofilm formed by Candida spp. and S. aureus, co-isolated from sputum samples of cystic fibrosis patients regarding microbial density, metabolic activity, and structure. 67 samples from 28 patients were collected with a 96% alteration rate. 34% showed alterations by both Candida spp. and Gram-positive bacteria, predominantly Candida spp. and S. aureus in 77% of cases, accounting for 6 associations. Biofilm biomass was quantified using the crystal violet assay, and metabolic activity was assessed using the MTT reduction assay. Scanning electron microscopy analyzed the C. tropicalis/S. aureus24 biofilm architecture. Candida spp. isolates demonstrated the ability to form mixed biofilms with S. aureus. The C. tropicalis/S. aureus24 association exhibited the highest production of biofilm and metabolic activity, along with the C. albicans17/C. rugosa/S. aureus7 in both single and mixed biofilms.

P393 Physical activity participation and mental health status of patients with cystic fibrosis during the COVID-19 pandemic: a single centre experience

The COVID-19 pandemic has posed a significant mental health challenge to individuals worldwide. Little is known about the association between mental health and physical activity participation in patients with Cystic Fibrosis (CF) who are encouraged to exercise as part of their treatment regimen. This exploratory study aimed to determine physical activity levels, mental health status (anxiety and depression symptoms) and fear of illness (Fear of Illness and Viral Evaluation; FIVE) score among a sample of CF patients from our academic health center.

Study of human ribosomal gene complex in cystic fibrosis

Increasing the life expectancy of patients with CF is an urgent healthcare task all over the world. According to the Register of patients with cystic fibrosis in the Russian Federation (2020), the number of patients over 18 years of age is 26.5%. Assumably, cystic fibrosis can be used as a model of accelerated aging to study the aging process in general.Aim of the study was to analyze the number of rDNA copies in a sample of cystic fibrosis patients at different ages and with lethal outcome in relation to lung function, complications, and respiratory tract infections.Methods. We studied DNA samples isolated by the standard method from peripheral blood leukocytes of 277 patients diagnosed with cystic fibrosis. 998 DNA samples from healthy volunteers were used as a control group.Results. The study showed that the genomes of patients with CF contain more rDNA copies than those of control patients. The greatest number of copies of ribosomal genes was observed in DNA samples from deceased patients (p < 0.001) and was associated with more severe disease course. Among all CF patients, the largest number of rDNA copies in the genome was registered in patients with the lowest FEV1 values (less than 40%). It was found that patients with chronic Burkholderia cepacia complex infection had a significantly higher number of copies of ribosomal repeats than the total sample (p = 0.001) and the adults (p = 0.014). The number of ribosomal repeats did not differ between patients with other chronic respiratory tract infections.Conclusion. In the group of deceased patients, the patients with low respiratory function and Burkholderia cepacia complex infection had the highest number of rDNA copies in the genome, and the differences were significant. It can be assumed that the number of rDNA copies in the genome of CF patients is an additional prognostic marker that is associated with the patient’s life expectancy.

Cumulative Antimicrobial Susceptibility Data of Pseudomonas Aeruginosa Isolates from Cystic Fibrosis Patients: 4-Year Experience

Abstract Objective Pseudomonas aeruginosa is the most important cause of lung infection among cystic fibrosis (CF) patients, and to reduce the severity of the infection, facility-specific cumulative antibiograms could help clinicians in empirical treatment. Methods Respiratory samples of CF patients between January 2015 and December 2018 were scanned through Laboratory Operating System retrospectively. Demographical data of patients, culture results, and antibiotic susceptibilities are recorded using Microsoft Excel 2010. Cumulative antibiogram data were obtained according to the CLSI M39A4 document. Results The number of registered patients increased in 4 years from 154 to 253. The mean age of patients varied from 9 to 11.7 (range, 2–42). The ratio of patients with a positive culture for P. aeruginosa increased from 32 to 40%, and the mean patients' age decreased from 16.6 to 11.1 (p <0.05). A total number of 4,146 respiratory samples were analyzed. Sputum samples consisted of 42.5% (n: 1,767) of the samples with a 58.4% isolation rate of P. aeruginosa (n: 1,034). A notable increase of resistance was seen almost all antimicrobials tested by years. The ratio of multidrug-resistant (MDR) P. aeruginosa was 4.1, 10.2, 4.5, and 8.6% in 2015, 2016, 2017, and 2018. Conclusion Antimicrobial resistance is a challenging problem in CF patients, and surveillance should be done regularly.

Citrullination as a novel posttranslational modification of matrix metalloproteinases

Matrix metalloproteinases (MMPs) are enzymes with critical roles in biology and pathology. Glycosylation, nitrosylation and proteolysis are known posttranslational modifications (PTMs) regulating intrinsically the activities of MMPs. We discovered MMP citrullination by peptidyl arginine deiminases (PADs) as a new PTM. Upon hypercitrullination, MMP-9 acquired a higher affinity for gelatin than control MMP-9. Furthermore, hypercitrullinated proMMP-9 was more efficiently activated by MMP-3 compared to control MMP-9. JNJ0966, a specific therapeutic inhibitor of MMP-9 activation, inhibited the activation of hypercitrullinated proMMP-9 by MMP-3 significantly less in comparison with control proMMP-9. The presence of citrullinated/homocitrullinated MMP-9 was detected in vivo in neutrophil-rich sputum samples of cystic fibrosis patients. In addition to citrullination of MMP-9, we report efficient citrullination of MMP-1 and lower citrullination levels of MMP-3 and MMP-13 by PAD2 in vitro. In conclusion, citrullination of MMPs is a new PTM worthy of additional biochemical and biological studies.

Extracellular polymeric substance‐mediated tolerance of Pseudomonas aeruginosa biofilms to atmospheric pressure nonthermal plasma treatment

AbstractPseudomonas aeruginosa is an opportunistic disease‐causing bacterium, with a number of strains exhibiting a mucus‐forming (mucoid) phenotype during infection, producing biofilms with a surrounding matrix containing alginate. Atmospheric pressure nonthermal plasmas (APNTP) are an emerging, potential approach to control biofilms across a range of medical and industrial applications. In this study, we examine the effect of plasma treatment on P. aeruginosa biofilms from clinical samples of cystic fibrosis patients, exhibiting both mucoid and non‐mucoid types. Biofilms of mucoid strains exhibit significantly elevated APNTP tolerance (p < .05). Endogenous alginate overproduction, as well as supplementation of P. aeruginosa cultures with exogenous alginate, results in significantly increased APNTP tolerance. Overall, this study shows how extracellular polymeric substance components mediate tolerance to APNTP, with significantly greater effects observed in mucoid strains.

Evaluation of a New Culture Protocol for Enhancing Fungal Detection Rates in Respiratory Samples of Cystic Fibrosis Patients.

Cystic fibrosis (CF) can be complicated by fungal infection of the respiratory tract. Fungal detection rates in CF sputa are highly dependent on the culture protocol and incubation conditions and thus may lead to an underestimation of the true prevalence of fungal colonization. We conducted a prospective study to evaluate the additional value of mucolytic pre-treatment, increased inoculum (100 μL), additional fungal culture media (Sabouraud agar; SAB, Medium B+, Scedosporium selective agar; SceSel+ and Dichloran-Glycerol agar; DG18) and longer incubation time (3 weeks) compared with our current protocol. Using the new protocol, we prospectively analyzed 216 expectorated sputum samples from adult and pediatric CF patients (n = 77) and compared the culture yield to a three year retrospective cohort that used direct 10 μL loop inoculation on SAB with 5 days incubation (867 sputum samples/103 patients). Detection rates for molds increased from 42% to 76% (p < 0.0001). Twenty-six percent of cultures were polymicrobial in the prospective cohort as opposed to 4.7% in the retrospective cohort (p < 0.0001). Colonization rate with A. fumigatus increased from 36% to 57%. SAB and DG18 showed the highest detection rates for all molds (SAB 58.6%; DG18 56.9%) and DG18 had the best performance for molds other than A. fumigatus. The larger sample volume and longer incubation also contributed to the increased recovery of molds. The introduction of a modified fungal culture protocol leads to a major increase in detection rate and the diversity of molds, which influences fungal epidemiology and may have implications for treatment decisions.

The Phagocytosis of Blood Leukocytes from Cystic Fibrosis Patients is not Impaired in General.

Impaired phagocytosis of Pseudomonas aeruginosa was found in isolated monocytes of peripheral blood of cystic fibrosis patients, but not in their neutrophils, as reported some years ago. In the present study, we analysed the phagocytic capacity of peripheral blood neutrophils and monocytes of cystic fibrosis patients and of healthy controls. Phagocytosis was determined using a commercial phagocytosis "in whole blood" assay on the basis of fluorescence-labelled opsonized Escherichia coli bacteria and flow cytometry. Venous blood of cystic fibrosis patients and of healthy controls was collected and the phagocytosis assay was performed. No differences in the percentage of phagocytic cells or in the overall phagocytic capacity were found between samples of cystic fibrosis patients and healthy controls either in monocytes or in neutrophils. Thus, our results did not support the hypothesis of a generally reduced phagocytic ability in the peripheral blood immune cells of cystic fibrosis patients.

Isolation and identification of Pandoraea\xa0spp. From bronchoalveolar lavage of cystic fibrosis patients in Iran

BackgroundPandoraea species are gram negative, motile, non-spore forming, rod shaped and oxidase positive, obligate aerobes bacteria, and have one polar flagellum. Most of Pandoraea species are associated with lung infections in cystic fibrosis patients. Cystic fibrosis is the most prevalent autosomal recessive hereditary disease in the world that affects various organs of the body. The main important cause of death in these patients is lung involvement. This study was conducted to isolate and identify Pandoraea bacterium from bronchoalveolar lavage and sputum samples of cystic fibrosis patients in Shiraz, Iran.MethodsIn this research 31 samples of bronchoalveolar lavage and sputum were examined by culture and PCR method. Then confirmed isolates were evaluated for susceptibility to different antibiotics and ability to produce biofilm.ResultsThe results of this study after cultivation, purification and DNA extraction led to the isolation of 4 Pandoraea bacterium by PCR using specific primers. Antibiotic susceptibility test were indicated all isolates were resistant to gentamicin, amikacin and imipenem and susceptible to ciprofloxacin, trimethoprim-sulfumethoxazole, piperacillin and tetracycline. Ability to create biofilm was indicated by some of Pandoraea isolates. According to findings of this study, ability to synthesis biofilm by Pandoraea isolates and resistance to some antibiotics are very important.ConclusionsOur study notes the role of P. pnomenusa as an emerging pathogen that can cause chronic lung colonization in CF patients. Identification tools need to be accurate and must be based on molecular techniques. Also our findings should raise awareness about antibiotic resistance in cystic fibrosis patients in Iran and ability of including bacterial agents to produce biofilm is an alarm for public health. Thus clinicians should exercise caution about finding of clinical relevance of this pathogen to the infection and prescribing antibiotics, especially in cases of children infections.

Molecular Identification of Gram-Negative Bacteria in Respiratory Samples of Cystic Fibrosis Patients from a Children Referral Hospital in Tehran

Background: Gram-negative bacteria are a major cause of pulmonary infection in patients with cystic fibrosis (CF). Objectives: The study aimed to conduct the molecular identification of Gram-negative bacterial flora causing pulmonary infection in children with CF. Methods: In this study, sputum samples were taken from 64 CF children undergoing treatment as outpatients or inpatients at a referral children’s hospital in Tehran. The PCR technique was used to detect the presence of Gram-negative bacteria, namely Pseudomonas spp., Acinetobacter baumannii, Stenotrophomonas maltophilia, and Burkholderia cepacia. Results: All samples were positive for 16srRNA. Pseudomonas spp. and A. baumannii were detected in 47% and 14% of the studied samples, respectively. Co-colonization by Pseudomonas spp. and A. baumannii was observed in three (5%) samples. Conclusions: According to this survey, Pseudomonas spp. were the most prevalent Gram-negative bacteria isolated from CF patients with pulmonary infection by molecular assays.

Updating Diagnostic Spectrum of Recurring CFTR Mutations

Cystic fibrosis (CF) is a frequent hereditary disease widely observed in Europeans. A correlation of the mutation profile in the CFTR gene with the geographical location and ethnic origin represents an important peculiarity of this disease. In order to identify the most frequent mutations typical for Russian CF patients and create a highly informative diagnostic panel, an analysis of 40 CFTR mutations in a sample of CF patients from different regions of the Russian Federation was performed. The selection of mutations was based on the published data together with the data of the CF Registry of Russian Patients in 2014. The frequencies of analyzed mutations were distributed as follows: F508del (53.6%), E92K (6.6%), CFTRdele2,3(21kb) (5.3%), 1677delTA (3%), 3849+10kbC>T (2.8%), W1282X (2.3%), 2143delT (2.2%), N1303K (2.1%), 2184insA (1.8%), L138ins (1.8%), 3272-16T>A (1.3%), 394delTT (1.0%), G542X (1.0%), W1282R (0.9%), S466X (0.6%), R1066C (0.6%), 2789+5G>A (0.6%), R334W (0.5%), W1310X (0.5%), 1898+1G>A (0.4%), 4015delA (0.4%), S1196X (0.3%), S945L (0.3%), R347P (0.3%), and 1367del5 (0.3%); an additional nine mutations had a frequency less than 0.1%, and the remaining six were unobserved. The total frequency of 40 examined mutations was 91.8%.

Comparison of the RGM medium and the mycobacterial growth indicator tube automated system for isolation of non-tuberculous mycobacteria from sputum samples of cystic fibrosis patients in Belgium

PurposePulmonary infections due to non-tuberculous mycobacteria (NTM) are an emerging issue in the cystic fibrosis (CF) population. Due to bacterial and fungal overgrowth, isolation of mycobacteria from the sputum samples of these patients remains challenging. RGM medium, a novel agar-based culture medium was evaluated for the isolation of NTM from sputum samples of CF patients. MethodologySputum samples were inoculated onto RGM medium and conventional Mycobacterial Growth Indicator Tube (MGIT™, Becton Dickinson, USA). Agar plates were incubated at 35 °C and growth was recorded once a week during 42 days. We compared the yield of the two media. Results217 samples were obtained from 124 CF patients. 20 samples (13 patients) had a positive culture for NTM. 79/217 (36.4%) MGIT had to be discontinued due to contamination compared to 18/217 (8.3%) for RGM. We reported equivalent NTM detection performances for RGM and MGIT (P = 0.579): these media enabled the isolation of 15 and 12 NTM strains respectively. ConclusionRGM medium increases the proportion of interpretable results and the number of NTM cultured. Taking into account the non-inferiority compared to conventional methods and ease of use of RGM medium, we estimate that this test can replace current approaches for the screening of NTM among people with CF. Additionally, RGM provides semi-quantitative results (number of colonies) and information on the morphology of colonies, which may be clinically relevant information.

A simple, fast and inexpensive method for mutation scanning of CFTR gene

BackgroundMutation scanning methods in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene may not distinguish between a Cystic Fibrosis (CF) causing mutation and a benign variant. We have developed a simple and fast method for scanning 14 selected CF-causing mutations which have high frequency in Latin America.MethodsIn a group of 35 samples coming from CF patients previously characterized and using two allele-specific real-time multiplex PCRs targeting wild-type and mutant alleles respectively, we detect the presence of mutations by analyzing the Ct variation. Twenty-five samples without mutations considered non-carrier samples, were also included in this study. High Resolution Melting Analysis (HRMA) was performed to confirm the result of the scanning method and in most cases allowed the genotype determination.ResultsThe results validate this method for CF diagnosis. A least one CFTR gene mutation was detected in the samples of CF patients, as predicted by their ΔCt values. The ΔCt value also indicated the zygosity of the sample according to the distribution of CFTR gene mutations. In most cases, HRMA allowed the identification of the mutation(s), thereby confirming the efficiency of this scanning strategy.ConclusionsThis strategy simplifies the detection of CF, reducing the analysis of 14 CF-causing mutations to two parallel reactions and making the procedure compatible with the analysis of a large number of samples. As the method is fast, inexpensive and highly reliable, it is advisable for scanning CFTR gene mutations in newborns, patients with a clinical suspicion of CF as well as in the preconception carrier screening.

The Effect of Curcumin to the Several Virulance Factors of Pseudomonas aeruginosa Isolates Which Obtained From Respiratory Tract Samples of Cystic Fibrosis Patients

The Effect of Curcumin to the Several Virulance Factors of Pseudomonas aeruginosa Isolates Which Obtained From Respiratory Tract Samples of Cystic Fibrosis Patients

Accurate identification of members of the Burkholderia cepacia complex in cystic fibrosis sputum.

The Burkholderia cepacia complex (BCC) is a group of closely related species which includes opportunistic pathogens causing chronic respiratory infections in immunocompromised patients, or individuals affected by cystic fibrosis (CF). Other Burkholderia species causing infection in the CF population are Burkholderia gladioli and Burkholderia pseudomallei. Traditional phenotypic analyses have been demonstrated to be inadequate for reliable identifications of isolates of BCC and B.gladioli. A pan-genomic analysis approach was used to design species-specific probes for Burkholderia cenocepacia, B.cepacia, Burkholderia multivorans, Burkholderia vietnamiensis, Burkholderia ambifaria, Burkholderia dolosa, Burkholderia pyrrocinia and B.gladioli. Multiplex real-time PCR assay was developed and tested using sputum specimens collected from CF patients spiked with Burkholderia species. The assay exhibited 100% sensitivity for all eight target species and detected 10(2) to 10(3) CFUml(-1) when applied to spiked sputum. Our PCR assay resulted highly specific for each of the Burkholderia species tested, allowing discrimination among Burkholderia and non-Burkholderia pathogens. Analysis carried out on 200 sputa positive for the presence of Burkholderia revealed that PCR assay and recA sequencing were fully comparable for identification of Burkholderia at the level of species. Burkholderia cepacia complex (BCC) has a complex taxonomic organization and its identification is a challenge for microbiology laboratories. Nonidentification or misidentification of BCC isolates represent a problem in epidemiology and treatment of cystic fibrosis patients. The high specificity and sensitivity of the multiplex Real-time PCR assay developed in this study indicates its potential to be a rapid and reliable method for the detection of Burkholderia at the level of species from sputum samples of cystic fibrosis patients.

The role of 2,4-dihydroxyquinoline (DHQ) in Pseudomonas aeruginosa pathogenicity.

Bacteria synchronize group behaviors using quorum sensing, which is advantageous during an infection to thwart immune cell attack and resist deleterious changes in the environment. In Pseudomonas aeruginosa, the Pseudomonas quinolone signal (Pqs) quorum-sensing system is an important component of an interconnected intercellular communication network. Two alkylquinolones, 2-heptyl-4-quinolone (HHQ) and 2-heptyl-3-hydroxy-4-quinolone (PQS), activate transcriptional regulator PqsR to promote the production of quinolone signals and virulence factors. Our work focused on the most abundant quinolone produced from the Pqs system, 2,4-dihydroxyquinoline (DHQ), which was shown previously to sustain pyocyanin production and antifungal activity of P. aeruginosa. However, little is known about how DHQ affects P. aeruginosa pathogenicity. Using C. elegans as a model for P. aeruginosa infection, we found pqs mutants only able to produce DHQ maintained virulence towards the nematodes similar to wild-type. In addition, DHQ-only producing mutants displayed increased colonization of C. elegans and virulence factor production compared to a quinolone-null strain. DHQ also bound to PqsR and activated the transcription of pqs operon. More importantly, high extracellular concentration of DHQ was maintained in both aerobic and anaerobic growth. High levels of DHQ were also detected in the sputum samples of cystic fibrosis patients. Taken together, our findings suggest DHQ may play an important role in sustaining P. aeruginosa pathogenicity under oxygen-limiting conditions.

T2R38 genotype is correlated with sinonasal quality of life in homozygous ΔF508 cystic fibrosis patients.

Chronic rhinosinusitis (CRS) is very prevalent in the cystic fibrosis (CF) patient population, and leads to high morbidity and markedly decreased quality of life (QOL). Identification of genetic markers that contribute to CRS symptoms in these patients can allow for risk stratification and tailoring of medical and surgical treatments. T2R38 is a bitter taste receptor expressed in the sinonasal tract, and nonfunctional alleles of this receptor have been implicated in treatment-refractory CRS in non-CF patients. The purpose of this study is to investigate the significance of T2R38 genotype in the variability of sinonasal QOL and CRS disease severity in a sample of CF patients. ΔF508 homozygous CF patients were recruited from the University of Pennsylvania Cystic Fibrosis Center and were genotyped for the TAS2R38 locus. To assess sinonasal symptom severity, a 22-item Sino-Nasal Outcome Test (SNOT-22) was collected from each patient. Additional demographic and medical history data was obtained at the time of patient enrollment. A total of 49 ΔF508 homozygous CF patients aged 18 to 32 years were included in the final SNOT-22 score analysis. Individuals with 2 functional T2R38 alleles (PAV/PAV) had significantly lower SNOT-22 scores (n = 49, p < 0.05). On further breakdown of SNOT-22 subcategories, rhinologic symptoms specifically were less severe in PAV/PAV patients than patients with other genotypes (n = 47, p < 0.05). Our investigation indicates that T2R38 genotype correlates both with SNOT-22 scores and rhinologic-specific QOL in ΔF508 homozygous CF patients.

The development of the approaches for fungal pathogens identification directly in respiratory samples of cystic fibrosis patients

The development of the approaches for fungal pathogens identification directly in respiratory samples of cystic fibrosis patients

Effect that an educational program for cystic fibrosis patients and caregivers has on the contamination of home nebulizers.

OBJECTIVE:To describe the pathogens found in home nebulizers and in respiratory samples of cystic fibrosis (CF) patients, and to evaluate the effect that a standardized instruction regarding cleaning and disinfection of nebulizers has on the frequency of nebulizer contamination. METHODS: We included 40 CF patients (22 males), all of whom used the same model of nebulizer. The median patient age was 11.2 ± 3.74 years. We collected samples from the nebulizer mouthpiece and cup, using a sterile swab moistened with sterile saline. Respiratory samples were collected by asking patients to expectorate into a sterile container or with oropharyngeal swabs after cough stimulation. Cultures were performed on selective media, and bacteria were identified by classical biochemical tests. Patients received oral and written instructions regarding the cleaning and disinfection of nebulizers. All determinations were repeated an average of two months later. RESULTS: Contamination of the nebulizer (any part) was detected in 23 cases (57.5%). The nebulizer mouthpiece and cup were found to be contaminated in 16 (40.0%) and 19 (47.5%), respectively. After the standardized instruction had been given, there was a significant decrease in the proportion of contaminated nebulizers (43.5%). CONCLUSIONS: In our sample of CF patients, nebulizer contamination was common, indicating the need for improvement in patient practices regarding the cleaning and disinfection of their nebulizers. A one-time educational intervention could have a significant positive impact.

Sampling and Decontamination Method for Culture of Nontuberculous Mycobacteria in Respiratory Samples of Cystic Fibrosis Patients

We confirmed that chlorhexidine decontamination yielded more nontuberculous mycobacteria than did the N-acetyl-l-cysteine-NaOH-oxalic acid procedure from respiratory samples of cystic fibrosis patients on solid cultures. However, this improved recovery is mostly balanced if the latter is combined with liquid culture. Furthermore, none of the 145 cough swabs, used to sample young children, cultured positive, suggesting that swabs are low-quality samples.