Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is located on the long arm of human chromosome 7 [1]. All disease-causing mutations in the CFTR gene prevent the chloride ion channel from functioning properly, leading to a blockage of the movement of salt and water into and out of cells. As a result of this blockage, cells that line the passageways of the lungs, pancreas, and other organs produce abnormally thick, sticky mucus which obstructs the airways and glands, causing the characteristic signs and symptoms of CF. The most life-threatening complications, in CF patients, are pulmonary inflammation and infection, from bacteria and fungi becoming trapped in the thick, tenacious secretions in the airways, resulting in a vicious cycle of infection and inflammation. Chronic lung infection is responsible for more than 90% of deaths in adults with CF [2].

Although the predominant infections associated with the CF lung are associated with bacteria, yeasts and filamentous fungi are frequently recovered from respiratory specimens from these patients, especially with the increased use of B. cenocepacia selective agars, which support fungal detection. These bacterial selective media contain high concentrations of several antibiotics, enhancing growth of highly antibiotic-resistant bacterial species, including B. cenocepacia, which will inadvertently support the growth of fungi. However, it is important that laboratories do not rely solely on these for the isolation of fungi, but instead employ fungal selective agar to attempt the isolation of these eukaryotes.

There is a growing awareness amongst CF physicians of the importance of fungal infections in patients with CF, largely due to: (1) the increasing incidence of fungal disease in CF patients; (2) emerging novel mechanisms of fungal pathophysiological disease, for example Aspergillus bronchitis; (3) the limited number of effective therapies that are available or their association with dose-limiting toxicities; (4) the fact that fewer symptoms bring the infection to the attention of the patient and physician early on; and (5) the difficulties to make an early diagnosis because of the lack of sensitive tests for the detection of invasive fungal infections [3]. Culturing Aspergillus fumigatus, per se, is not an indication for treatment, but this fungus has a wide range of clinical presentations and, when combined with the presence of co-habiting bacterial pathogens, such as Pseudomonas aeruginosa, makes it more difficult to attribute clinical significance to the presence of the fungus. Reliable laboratory detection of fungi is thus the cornerstone of subsequent clinical considerations.

Currently, it is difficult to estimate the prevalence of fungal infections in patients with CF. This is due mainly to a lack of monitoring of the presence of fungi in microbiological cultures of sputum, in most CF registries/databases in the UK and Europe. The US Cystic Fibrosis Foundation (CFF) Registry data, however, indicate an approximate doubling of prevalence of fungi being detected in the sputum of patients with CF, from 1995 to 2005, rising from approximately 6% in 1995 to approximately 13% in 2005. Such a rise as this could be the cumulative effect of the ad hoc introduction of novel and improved laboratory methods, as well as greater awareness of fungi disease in CF. In the absence of such data, the clinical significance of fungi is indicated mainly through reports in the scientific/medical literature. To date, the majority of reports have included clinically significant fungi such as Aspergillus spp., Scedosporium species, and Exophilia dermatitidis [3].

Evolving laboratory technology and methodologies aids in the isolation, identification, and characterization of aetiological agents of fungal disease in CF patients. Coupled with this, various laboratory guidelines exist to guide Clinical Microbiology Laboratories, in the employment of suitable techniques to employ, to support laboratory workup of respiratory specimens, and to guide clinicians in patients’ management. It was therefore the aim of this study to examine how a cohort of NHS Clinical Microbiology Service Laboratories, supporting CF centres in the UK, were performing CF fungal diagnosis.

A 19-question anonymous electronic questionnaire was developed and posted on the SurveyGizmo platform for completion. This questionnaire is also available at the following link: www.surveygizmo.eu/s3/90008906/Fungal-Laboratory-Questionnaire-for-Cystic-Fibrosis.

The questionnaire was designed in four sections, exploring NHS service laboratory aspects of: (1) fungal isolation; (2) fungal identification; (3) fungal characterization; and (4) promoting best practice. The questionnaire was distributed amongst NHS Consultant Respiratory Physicians and NHS Consultant Paediatricians involved in the clinical care of patients with CF at recognized UK CF centres. A request was made to 94 paediatric and adult CF consultants in the UK to forward the questionnaire onto their Consultant Microbiologist, who supports the CF centre, in terms of NHS Microbiology Laboratory Service provision. Questionnaires were duly completed and returned via the SurveyGizmo platform for analyses.

Responses to the questionnaire were received from 11 publically funded NHS Clinical Microbiology laboratories in the UK, supporting either a CF Adult Service or a CF Paediatric Service. Collated responses to the specific questions asked are shown in Table 1.

Table 1 Responses to CF laboratory fungal questionnaire from 11 UK NHS microbiology service laboratories
Full size table

The goal of any clinical microbiology laboratory supporting the routine processing of sputum and other respiratory specimens from CF patients is to provide a robust and effective service, in a timely and cost-effective manner. Any assays that are performed need to add clinical value and aid the physician in the clinical decision-making process. Driving forces, namely the development of novel techniques of isolation and characterization of fungi and new insights into the pathophysiology of fungal disease in CF patients, make the methodological techniques to be in a constant state of evolution, thus requiring periodic rationalization to ensure NHS routine service fungal assays are keeping pace with methodological innovation, as well as emerging knowledge on disease driving what assays to optimally employ.

Currently, there are at least two laboratory standards in the UK and USA, respectively, namely the UK CF Trust Laboratory Standards for Processing Microbiological Samples from People with Cystic Fibrosis [4] and the US Cumulative Techniques and Procedures in Clinical Microbiology (Cumitech) Guidelines 43 Cystic Fibrosis Microbiology [5]. Many countries may have their own national standards in place. A comparison of these standards is shown in Table 2. When we compare the findings of this questionnaire, against these current guidelines for the laboratory processing of sputum for fungi from CF patients, responses to this questionnaire were generally within compliance of these guidelines. This study indicated that many laboratories are currently performing several more assays that are presently listed in the laboratory guidelines, with a high degree of non-standardization in assays not defined in the guidelines. Our questionnaire showed that most laboratories are employing basic fungal detection media, mainly SDA with or without antibiotics. Most laboratories are cautious about employing enhanced specific fungal culture media, such as DRBC-benomyl, Sce-Sel+ or Scedo-Select III, to aid with the isolation of fungi from CF sputum; none were using these recently described media [6].

Table 2 Comparison of UK CF Trust Laboratory Standards for Processing Microbiological Samples from People with Cystic Fibrosis and the US Cumulative Techniques and Procedures in Clinical Microbiology (Cumitech) Guidelines 43 Cystic Fibrosis Microbiology, for the examination of fungi from patients with CF
Full size table

Whilst this study received responses from 11 NHS Clinical Microbiology laboratories, this should not be interpreted as being fully reflective of practice in all NHS laboratories. However, we believe that the responses received from these 11 laboratories are a microcosm of UK laboratory practice and a reasonable reflection of what UK laboratories are currently doing.

The biggest challenge resulting from analysis of this questionnaire is the lack of standardization of methods across these laboratories. Previously, Borman et al. [7] investigated the consequences of the lack of standardization of fungal methodologies across eight laboratories and concluded that without more sophisticated molecular methods, the aetiological role of ‘rarer’ filamentous fungi in pulmonary exacerbations will remain hidden.

Most recently, the February 2018 issue of Mycopathologia (Volume 183; Issue 1) contains 25 articles which are highly relevant to this area. In particular, the paper by Chen et al. [8] discusses the challenges in laboratory detection of fungal pathogens in the airways of CF patients. In this article, the authors highlight and discuss in detail the repertoire of available mycological laboratory techniques (cultural and molecular methods) to support accurate isolation, identification, and characterization of fungal organisms from CF respiratory specimens and conclude that guidelines for standardized processing of respiratory specimens are urgently needed. Following on from this call for urgent standardization of methods, the paper by Coron et al. [9] takes on this challenge to standardize cultural/isolation methods with the ‘MucoFong’ programme, whereby sputa from 243 CF patients from seven CF centres in France were studied over a 15-month period. Six fungal culture media were compared, and the study concluded that four of these media, namely CHROMAgar Candida medium incubated at 37 °C, Sabouraud dextrose agar with chloramphenicol and gentamicin at 25 °C, Sabouraud dextrose agar with chloramphenicol and cycloheximide at 37 °C and erythritol agar at 27 °C, should be employed to optimally recover fungal pathogens from CF respiratory specimens. Initiatives such as the MucoFong programme are extremely valuable as they present an evidence base for laboratories to move forward confidently in the knowledge that they are providing an optimal service for the patients.

The current study highlights the following:

  1. (1)

    that current UK laboratory practice, in general, follows the current guidelines, but that the scope and diversity of what is currently being delivered by laboratories far exceeds what is detailed in the guidelines;

  2. (2)

    there is a lack of standardization of fungal tests amongst laboratories, outside of the current guidelines;

  3. (3)

    as a result, both the UK CF Trust Laboratory Standards for Processing Microbiological Samples from People with Cystic Fibrosis [4] and the US Cumulative Techniques and Procedures in Clinical Microbiology (Cumitech) Guidelines 43 Cystic Fibrosis Microbiology [5] need to be updated to reflect both new methodological innovations, as well as better knowledge of fungal disease pathophysiology in CF;

  4. (4)

    there is a need for clinical medicine to decide upon a stratification strategy for the provision of new fungal assays with added value to guide physicians for an optimal management of CF patients;

  5. (5)

    there is a need to rationalize what assays may be performed at local laboratory level and those which are best served at National Mycology Reference Laboratory level and;

  6. (6)

    further research is required in developing laboratory assays, which will help ascertain the clinical importance of ‘old’ fungal pathogens, as well as ‘emerging’ fungal pathogens.