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

BackgroundOnce 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.MethodsSince 2001, 6109 respiratory samples from 299 CF patients were examined not only by conventional culture, but also by PCR, detecting Bcc directly in sputum.ResultsDiagnosis 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.ConclusionA 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.     

Successful Lung Re-transplant in a Patient with Cepacia Syndrome due to Burkholderia ambifaria

Chronic airway inflammation and infection drive morbidity and mortality among patients with cystic fibrosis (CF). While Haemophilus influenzae and Staphylococcus aureus predominate in children, the prevalence of Pseudomonas aeruginosa increases as patients age. Other bacteria, including species within the Burkholderia cepacia complex (Bcc), are also more prevalent among adults with CF. Species within the Bcc accelerate lung function decline and can trigger development of “cepacia syndrome,” both before and after lung transplantation. As a result, some centers advise against lung transplantation for Bcc-infected patients; however, little is known about the relative virulence of uncommon Bcc species. We describe a successful lung re-transplant in a patient with CF, chronic Burkholderia ambifaria airway infection, and cepacia syndrome.     

Inhaled amiloride and tobramycin solutions fail to eradicate Burkholderia dolosa in patients with cystic fibrosis

BackgroundBurkholderia dolosa can result in chronic airway infection and rapid decline in lung function in patients with cystic fibrosis (CF). Amiloride has antibacterial properties that may be synergistic with aminoglycosides against other species belonging to the Burkholderia cepacia complex (Bcc). We attempted to eradicate B. dolosa using a combination of nebulized tobramycin and nebulized amiloride in infected CF patients.MethodsA 6-month, open-label trial of continuous inhaled amiloride, delivered via nebulization four times daily, and continuous inhaled tobramycin (TIS or TOBI®) nebulized twice daily, was offered to all CF patients at our institution who are chronically infected with B. dolosa.ResultsTwenty two of 27 patients with B. dolosa were eligible and twelve elected to participate. Eradication of B. dolosa was not noted in any study subject. While patients tolerated treatment with no adverse effects, there was also no apparent impact on other secondary outcome measures.ConclusionsConcurrent, continuous inhalation of amiloride and tobramycin for 6 months was not effective for the eradication of chronic B. dolosa airway infection in CF patients.     

Recurrent Clostridium difficile colitis in cystic fibrosis: An emerging problem

ObjectiveTo examine the incidence of recurrent Clostridium difficile infection in patients with cystic fibrosis (CF), including patients who had undergone lung transplantation, and review clinical findings in hospitalized patients with C. difficile colitis.MethodsA retrospective chart review was performed to examine the clinical presentation and management of patients with cystic fibrosis (CF) who received care at the University of Wisconsin Hospital and Clinics (UWHC) from 1994 to 2011 and were prospectively identified with C. difficile colitis.ResultsTen cases of C. difficile associated disease (CDAD) occurred in patients with CF followed by our Adult CF Center over a period of 17 years, and 4 patients were bilateral lung transplant recipients. Two of the lung transplant recipients had recurrent CDAD that lead to fulminant pancolitis, surgical intervention, and shock. Two patients in the non-transplant group experienced recurrent C. difficile infection that led to fulminant pancolitis with associated systemic inflammatory response syndrome and required colectomy.ConclusionsC. difficile colitis can cause life threatening illness in patients with CF, and symptoms may be subtle and/or atypical and lead to significant delay in diagnosis. Patients with recurrent C. difficile colitis are at high risk of fatal outcome, and empiric therapy should be considered for patients with previous C. difficile colitis even in the absence of disease when broad-spectrum antibiotics are given to treat bacterial infection.     

Species distribution of Burkholderia cepacia complex isolates in cystic fibrosis and non-cystic fibrosis patients in New Zealand

Burkholderia cepacia complex (Bcc) isolates from 39 CF patients and 25 non-CF patients in New Zealand were speciated and characterised using the multilocus sequence typing (MLST) scheme for Bcc. B. multivorans predominated in CF patients (31/39, 79.5%) and in non-CF patients (7/25, 28%). Sequence types (ST) with an international distribution were identified (27/64, 42.2%) among the New Zealand Bcc isolates. MLST revealed a high level of diversity among Bcc isolates in CF patients indicating a lack of person-to-person transmission. Non-CF patients showed less diversity in MLST types, however, individuals with shared STs were geographically and chronologically separated. The use of MLST analysis allows continued surveillance of isolates with the potential to identify outbreaks. The identification of internationally distributed strains may provide an indicator of the relative transmissibility and infectivity of these strains and warrants further investigation.     

Factors influencing the acquisition of Stenotrophomonas maltophilia infection in cystic fibrosis patients

BackgroundStenotrophomonas maltophilia is one of the most common multi-drug resistant organisms causing pulmonary infections in CF patients. It is unknown whether S. maltophilia infection follows the same pattern and shares similar risk factors for acquisition as described for Pseudomonas aeruginosa.MethodsWe examined all clinical events from 1997 to 2008 in the Toronto CF Database to identify risk factors for the acquisition of S. maltophilia and to define distinct patterns of infection.ResultsWe followed 601 patients over 12 years, during which time one quarter of subjects had at least one positive culture for S. maltophilia; the incidence rate was slightly higher in children (11.6/100 person years) compared with adults (10.6/100 person years). Using multi-variable Cox proportional hazards models, steeper rate of FEV₁ decline was a significant risk factor for S. maltophilia acquisition, whereas new infections were less likely to occur with greater oral antibiotic use and a history of Burkholderia cepacia complex infection.ConclusionsThis study illustrates the evolution of S. maltophilia infection over time in a large cohort of adults and children with CF. Younger CF patients, and those with greater lung function decline were at increased risk of S. maltophilia infection. The use of oral antibiotics to maintain lung function may be a way of decreasing the risk of infection. However, the optimal management of CF patients with persistent S. maltophilia infection is not yet known and requires further studies.     

The prevalence,clinical status and genotype of cystic fibrosis patients living in Cuba using national registry data

BackgroundWe aimed to establish a national cystic fibrosis (CF) registry for Cuba, a developing country.MethodsRegional centres that deliver care for all CF patients provided information for a national database.FindingsThe prevalence of CF in Cuba is 26.3 cases per 1,000,000 population. The median age at diagnosis is 2 years, and the median age of the total population was 15 years. Of those aged 16 years or older, the prevalence of Pseudomonas aeruginosa infection was 46%, the prevalence of Staphylococcus aureus infection was 36%, and 80% of individuals were receiving oral azithromycin. The commonest gene mutation was F508del which was observed in 50% of patients.InterpretationThese data demonstrate that it is possible to establish a national CF registry in a developing country such as Cuba. This provides baseline data to permit evaluation of health care delivery enable the spread of good clinical practice nationally.     

A review of pathophysiology and management of fetuses and neonates with meconium ileus for the pediatric surgeon

PurposeMeconium ileus (MI) is the earliest clinical manifestation of cystic fibrosis (CF), occurring in up to 20% of patients with CF. Our aim was to review and integrate current knowledge about the diagnosis and management of fetuses and neonates with MI that may aid the pediatric surgeon in caring for these patients.MethodsWe identified areas of interest including pathophysiology, prenatal diagnosis, nonoperative and operative management, postoperative management, and prognosis. We performed a Medline search using the search term meconium ileus for English language articles published in the last 20 years. We reviewed reference lists to identify other articles of historical significance.ResultsMeconium ileus is primarily associated with CF transmembrane (conductance) regulator mutations F508del, G542X, W1282X, R553X, and G551D, and modifier genes have been found to explain approximately 17% of the phenotypic variability. Mouse, pig, and ferret models for CF demonstrate neonatal bowel obstruction mimicking MI. Sonographic findings of hyperechoic masses and dilated bowel in a high-risk fetus are suggestive of MI. Less than 7% of low-risk fetuses with hyperechoic bowel will have MI. Contemporary series of noninvasive management with Gastrografin enema report success rates of 36% to 39%, significantly lower than historical values. The optimal surgical technique remains controversial, although primary anastomosis results in surgical complication rates between 21% and 31%, higher than those noted with delayed anastomosis. Pulmonary function for patients with CF and MI at 15 and 25 years old is similar to those without MI, although height and weight percentiles may be lower.ConclusionsThis review for pediatric surgeons presents an examination of the literature and synthesizes current information about the pathophysiology, prenatal diagnosis, nonoperative and operative management, postoperative management, and prognosis of the patient with CF and MI.     

Cystic fibrosis,gastroduodenal inflammation,duodenal ulcer,and H. pylori infection: The “cystic fibrosis paradox” revisited

BackgroundIn cystic fibrosis (CF) patients a duodenal impaired bicarbonate secretion and unbuffered gastric acid are always described and the development of duodenal ulceration is uncommon (CF paradox). Helicobacter pylori (HP) infection is the main cause for duodenal ulceration and its prevalence in CF patients is controversial.AimThe objective of this study is to evaluate HP prevalence, gastric histology, and duodenal ulceration in adult FC patients.Methods32 adult CF patients were submitted to ¹³C-urea breath test and serum immunoblotting test for HP diagnosis. Among them, 20 patients were submitted to endoscopy.Results19/32 (68%) patients showed positive serology. Endoscopy showed erosive duodenitis (15%), and duodenal ulcer scar in 10%. On duodenal histology, 94.5%, showed active inflammation and 66.7% gastric metaplasia.ConclusionHP infection prevalence in adult CF patients was similar to that of general Brazilian population. CF patients have all the duodenal spectrum of alterations, including duodenal ulcer. CF paradox may not exist.     

Prevalence of Helicobacter pylori infection in patients with cystic fibrosis

IntroductionHelicobacter pylori (H. pylori) is one of the most common bacterial infections worldwide. The prevalence of Hp infection in cystic fibrosis (CF) is unclear. Thus, the aim of our study was to determine the prevalence of H. pylori infection in CF patients and to correlate H. pylori presence with CF expression.Material and methodsThe presence of H. pylori infection was assessed using a breath test with isotope-labeled urea in CF 79 patients compared to 302 healthy control subjects (HS).ResultsFifteen (19.0%) CF patients were H. pylori positive. No statistical differences in the basic clinical parameters or in their distribution were documented. No clinical factor was an independent risk factor of H. pylori infection. The corrected prevalence of H. pylori infection in pediatric CF patients and HS was 14.4% and 9.8%, respectively.ConclusionThe prevalence of H. pylori infection in CF patients is not different from that in healthy subjects.     

Exopolysaccharides produced by clinical strains belonging to the Burkholderia cepacia complex

BackgroundIn the frame of a research line dedicated to better clarify the role of exopolysaccharides (EPS) in bacterial virulence, EPS produced by species of the Burkholderia cepacia complex (Bcc), namely Burkholderia multivorans, Burkholderia cenocepacia, and a Bcc member of undetermined genomovar, all isolated at the Cystic Fibrosis Regional Centre of Florence (Italy), were investigated for they structural properties.MethodsThree strains of B. multivorans, three of B. cenocepacia and one of a Bcc member of undetermined genomovar were isolated from CF patients. The reference strains C1576 and J2315, for genomovar II and III, respectively, were included in the study. The bacteria were grown on solid media, the exopolysaccharides produced were purified, and their structures were determined. In addition, sugar analysis of sputum samples was accomplished to search for EPS produced in vivo.ResultsSix strains out of seven produced the exopolysaccharide cepacian, while one strain of B. multivorans produced a completely different polymer, previously known in the literature as PS1. Two strains synthesised very small amounts of EPS. No definitive evidence for the presence of cepacian in sputum samples was found.ConclusionsMost strains examined produced abundant amounts of polysaccharides. Cepacian was the most common EPS isolated and its production was not associated to a particular genomovar.     

Defective immunometabolism pathways in cystic fibrosis macrophages

BackgroundMitochondria play a key role in immune defense pathways, particularly for macrophages. We and others have previously demonstrated that cystic fibrosis (CF) macrophages exhibit weak autophagy activity and exacerbated inflammatory responses. Previous studies have revealed that mitochondria are defective in CF epithelial cells, but to date, the connection between defective mitochondrial function and CF macrophage immune dysregulation has not been fully elucidated. Here, we present a characterization of mitochondrial dysfunction in CF macrophages.MethodsMitochondrial function in wild-type (WT) and CF F508del/F508del murine macrophages was measured using the Seahorse Extracellular Flux analyzer. Mitochondrial morphology was investigated using transmission electron and confocal microscopy. Mitochondrial membrane potential (MMP) as well as mitochondrial reactive oxygen species (mROS) were measured using TMRM and MitoSOX Red fluorescent dyes, respectively. All assays were performed at baseline and following infection by Burkholderia cenocepacia, a multi-drug resistant bacterium that causes detrimental infections in CF patients.ResultsWe have identified impaired oxygen consumption in CF macrophages without and with B. cenocepacia infection. We also observed increased mitochondrial fragmentation in CF macrophages following infection. Lastly, we observed increased MMP and impaired mROS production in CF macrophages following infection with B. cenocepacia.ConclusionsThe mitochondrial defects identified are key components of the macrophage response to infection. Their presence suggests that mitochondrial dysfunction contributes to impaired bacterial killing in CF macrophages. Our current study will enhance our understanding of the pathobiology of CF and lead to the identification of novel mitochondrial therapeutic targets for CF.     

Achromobacter species in cystic fibrosis: Cross-infection caused by indirect patient-to-patient contact

Background and methodsAchromobacter species leads to chronic infection in an increasing number of CF patients. We report 2 cases of Achromobacter ruhlandii cross-infection between patients after well-described indirect contact.ResultsBoth cases were young, stable, CF patients without chronic infections and with normal FEV₁, but experienced clinical deterioration after visits to the home of a CF patient with A. ruhlandii infection and after sharing facilities with an A. ruhlandii infected CF patient on a skiing vacation, respectively. Both cases became positive for A. ruhlandii in airway secretions and were colonized with A. ruhlandii in their sinuses.Aggressive, long-term antibiotic treatment led to clinical stability. One of the cases developed chronic A. ruhlandii infection.ConclusionA. species can cause cross-infection even after a short period of indirect contact between infected and non-infected CF patients. Patients should be followed closely for several months before the possibility of cross-infection is ruled out.     

Depletion of BAFF cytokine exacerbates infection in Pseudomonas aeruginosa infected mice

BackgroundCystic fibrosis (CF) is a genetic disease characterized by chronic inflammation of the lungs that is ineffective at clearing pathogens. B-cell activating factor (BAFF), a cytokine involved in the development of B-cells, is known to be elevated in CF patients with subclinical infections. We postulate that the elevated BAFF levels in CF patients might be triggered by Pseudomonas aeruginosa infection and it might play a protective role in the regulation of lung responses to infection.MethodsTo address this hypothesis, we used a well characterized model of CFTR.KO mice infected with a clinical strain of P. aeruginosa (PA508). We quantified cell types with flow cytometry, concentration of cytokines by ELISA tests, bacterial load by colony counting and lung physiology by metacholine-induced lung resistance.ResultsOur data demonstrates that BAFF is not elevated in uninfected CF mice, and infection with Pseudomonas leads to significant induction of this regulatory cytokine. We also demonstrate that the maintenance of BAFF levels and its induction during the infection is important for clearance of Pseudomonas infection as its depletion during the course of infection leads to decrease in the resolution of infection both in WT and CFTR-KO mice. Interestingly, the depletion of BAFF not only results in a depletion of B cells numbers but also to a significant decrease in the number of regulatory T cells in the non-infected lungs.ConclusionsOverall, our data demonstrate for the first time that BAFF is an important regulatory molecule helping to maintain the immunological response to infection and clearance of lung infection.     

Reclassifying inconclusive diagnosis after newborn screening for cystic fibrosis. Moving forward

BackgroundNewborn screening for Cystic Fibrosis (CF) is associated with situations where the diagnosis of CF or CFTR related disorders (CFTR-RD) cannot be clearly ruled out.Materials/patients and methodsWe report a case series of 23 children with unconclusive diagnosis after newborn screening for CF and a mean follow-up of 7.7 years (4–13). Comprehensive investigations including whole CFTR gene sequencing, in vivo intestinal current measurement (ICM), nasal potential difference (NPD), and in vitro functional studies of variants of unknown significance, helped to reclassify the patients.ResultsExtensive genetic testing identified, in trans with a CF causing mutation, variants with varying clinical consequences and 3 variants of unknown significance (VUS). Eighteen deep intronic variants were identified by deep resequencing of the whole CFTR gene in 13 patients and were finally considered as non-pathogenic. All patients had normal CFTR dependent chloride transport in ICM. NPD differentiated 3 different profiles: CF-like tracings qualifying the patients as CF, such as F508del/D1152H patients; normal responses, suggesting an extremely low likelihood of developing a CFTR-RD such as F508del/TG11T5 patients; partial CFTR dysfunction above 20% of the normal, highlighting a remaining risk of developing CFTR-RD such as F508del/F1052V patients. The 3 VUS were reclassified as variant with defective maturation (D537N), defective expression (T582I) or with no clinical consequence (M952T).ConclusionThis study demonstrates the usefulness of combining genetic and functional investigations to assess the possibility of evolving to CF or CFTR-RD in babies with inconclusive diagnosis at neonatal screening.     

A survey of the prevalence,management and outcome of infants with an inconclusive diagnosis following newborn bloodspot screening for cystic fibrosis (CRMS/CFSPID) in six Italian centres

ObjectiveWe evaluated the prevalence, Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene profile, clinical data, management and outcome for infants with a CFTR-related metabolic syndrome/CF Screen Positive, Inconclusive Diagnosis (CRMS/CFSPID) designation from six Italian centres.MethodsAll newborn bloodspot screening (NBS) positive infants born from January 2011 to August 2018 with a CF diagnosis or a CRMS/CFSPID designation were enrolled. Data on sweat testing, genetics, clinical course and management were collected.ResultsWe enrolled 257 CF patientsand 336 infants with a CRMS/CFSPID designation (CF: CRMS/CFSPID ratio of 1:1.30).Blood immuno-reactive trypsinogen (IRT) was significantly lower in CRMS/CFSPID infants and the F508del variant accounted for only 20% of alleles. Children with CRMS/CFSPID showed a milder clinical course, pancreatic sufficiency compared to CF infants. Varied practice across centres was identified regarding sweat testing, chest radiograph (8-100%) and salt supplementation (11-90%). Eighteen (5.3%) CRMS/CFSPID infants converted or were reclassified to diagnosis of CF. Four infants (1.3%) developed a clinical feature consistent with a CFTR-related disorder (1.2%). Twenty-seven were re-classified as healthy carriers (8.0%) and 16 as healthy infants (4.8%).ConclusionsWe have identified considerable variability in the evaluation and management of infants with an inconclusive diagnosis following NBS across six Italian centres. CRMS/CFSPID is more regularly seen in this population compared to countries with higher prevalence of F508del.Conversion to a CF diagnosis was recorded in 18 (5.3%) of CRMS/CFSPID infants and in 16 was as a result of increasing sweat chloride concentration.     

Evaluating the alginate oligosaccharide (OligoG) as a therapy for Burkholderia cepacia complex cystic fibrosis lung infection

OligoG has previously shown potentiation of aztreonam against Burkholderia cepacia complex (Bcc) through biofilm disruption. A randomized, double-blind, placebo-controlled cross-over design was used to evaluate safety and efficacy of inhaled OligoG as a therapy for Bcc-infected CF patients taking aztreonam. Subjects received OligoG (1050 mg daily) or matching placebo for 28-days. Of 14 subjects completing the study, 8 showed a mean decrease in total bacterial CFU's (0.82 log10) after OligoG treatment. There was a reduction in mean Bcc CFU's (2.19 log10) after OligoG treatment but this was not statistically significant. Rheology analysis showed improvements in phase-angle after OligoG, but there was no statistically significant improvement in lung function parameters. Six out of 12 QoL summary scores showed relative improvement after OligoG treatment compared to placebo. There was a favourable safety profile for OligoG. Potential for reducing Bcc warrants further investigation of OligoG for the treatment of infection in CF.     

Distribution and outcomes of infection of Mycobacterium avium complex species in cystic fibrosis

BackgroundThe majority of nontuberculous mycobacterial (NTM) pulmonary infections in people with cystic fibrosis (CF) are caused by Mycobacterium avium complex (MAC) species. Data on MAC species distribution and outcomes of infection in CF are lacking.MethodsThis was a single center, retrospective study. MAC isolates had species identification with MLSA of rpoB and the 16S23S ITS region. Clinical data were compared between species.ResultsTwenty-three people with CF and 57 MAC isolates were included. Infection with M. avium was the most common (65.2%). M. intracellulare was associated with higher rates of NTM disease, younger age, and steeper decline in lung function prior to infection.ConclusionsWe observed worse clinical outcomes in people with M. intracellulare infection relative to other MAC species. Further investigation of clinical outcomes of MAC infection among CF patients is warranted to better define the utility of species-level identification of MAC isolates in CF.     

Clinician variability in the diagnosis and treatment of aspergillus fumigatus-related conditions in cystic fibrosis: An international survey

BackgroundThe diagnosis and treatment of Aspergillus fumigatus (Af)-related conditions remain a challenge in cystic fibrosis (CF) due to overlapping features of disease and absence of clinical guidelines for Af-related conditions outside of ABPA.ObjectiveTo investigate the differences of clinical practice in the diagnosis and management of Af-related conditions in CF.MethodsWe conducted an international survey to CF clinicians to ascertain the screening, diagnostic, and treatment practices for Af-related conditions in CF. Respondents were grouped into geographical regions and regional comparisons using chi-square tests of independence or Fisher's tests were performed.ResultsA total of 319 survey responses from 35 countries were analyzed. We observed differences in use and frequency of fungus culture, Aspergillus-specific IgE and IgG, skin prick testing, and pulmonary function testing as screening for Af-related conditions between the geographical regions. ABPA and Aspergillus bronchitis diagnostic criteria selection differed by region; significantly greater proportion of United States (US) and Canadian clinicians were unable to define Aspergillus bronchitis compared to Europe and other regions. Decision to treat ABPA was uniform across regions, but the consideration of Aspergillus bronchitis as a clinical disease warranting therapy differed between regions. The use of glucocorticoid and itraconazole was the first-line treatment of ABPA among clinicians; however, prednisone monotherapy was more common in US and Canada.ConclusionsSignificant variability in the diagnosis and management of Aspergillus-related conditions in CF was observed. Future studies are necessary to better harmonize the approach to Af-related disease in CF.