Cystic fibrosis is usually diagnosed when at least one typical cystic fibrosis clinical symptom is present with biochemical or genetic markers of CFTR dysfunction [De Boeck et al., 2006; Farrell et al., 2008]. In countries where newborn screening is performed, risk of cystic fibrosis will be identified in the absence of clinical symptoms and the screen-positive infant will be referred to a cystic fibrosis centre for clinical evaluation and follow-up.
The gold standard biochemical marker is the sweat test, which measures the chloride concentration in a sweat sample. Sweat is produced within specialised glands, consisting of a secretory coil and a reabsorptive duct. Fluid that is isotonic with interstitial fluid is expressed into the secretory coil, and it then passes through the reabsorptive duct, where NaCl is actively reabsorbed. This process means that the sweat that emerges from the duct is hypotonic relative to plasma. CFTR is the protein responsible for the reabsorption of NaCl, so in cystic fibrosis, sweat has a significantly higher concentration of NaCl [Quinton, 2007].
Raised sweat chloride concentrations in cystic fibrosis were first described in 1953 [Di Sant’Agnese et al., 1953]. The test has since been standardised and should be performed according to the guidelines [LeGrys et al., 2007; LeGrys et al., 2010; Royal College of Paediatrics and Child Health, 2014]. It is the cheapest and most readily available way of making a diagnosis of cystic fibrosis, and consists of three steps:
The sweat test is a highly sensitive and specific test for cystic fibrosis diagnosis. Sweat chloride concentration ≥60 mmol/L indicates cystic fibrosis. The disease is considered unlikely if sweat chloride concentration is below 30 mmol/L in the first six months of life (or below 40 mmol/L after 6 months of life). Between 30 (40) and 59 mmol/L, the sweat test is considered borderline and further investigations are required [Smyth et al., 2014].
The diagnosis of cystic fibrosis by CFTR genotyping requires the identification of two disease-causing mutations. CFTR genetic analysis begins with the analysis of a limited CFTR mutation panel that recognises at least one abnormal allele in 90% of the individuals with cystic fibrosis in a local population. When only one mutation is identified with this panel, an extended exon DNA analysis (gene sequencing) should be undertaken to identify a second mutation. While many CFTR mutations have been characterised and are well known to be associated with the disease, the majority of mutations have not been functionally characterised and for most of them, the pathogenic potential is unclear. Thus, mutation analysis is not the answer to every diagnostic dilemma: there are several limitations and difficulties in interpretation of the results that require cautious evaluation in a specialist CF centre [Castellani et al., 2008; Smyth et al., 2014].
Newborn screening for cystic fibrosis is supported by evidence showing benefits in terms of lowered disease severity, decreased burden of care, and reduced costs. Risks are mainly associated with disclosure of carrier status and diagnostic uncertainty [Castellani et al., 2016]. In Europe more than 25 screening programmes have been developed. All current programmes rely on the measurement of immunoreactive trypsinogen (IRT) in a blood spot performed at birth, and on a sweat test if the IRT is elevated. An intermediate test is required to achieve an acceptable combination of sensitivity and specificity. This test may consist of CFTR mutation analysis on the first blood spot and a second IRT test on another blood spot collected later, or various combinations of these two. In infants, the sweat test plays a pivotal role in the diagnosis. Programmes include arrangements for counselling and management of infants when the diagnosis is not clear. All children identified by newborn screening are then managed according to internationally accepted guidelines [Castellani et al., 2009].
When the sweat test or CFTR genotyping show some abnormalities but do not allow a clear result, unrelated CFTR disease or CFTR-related disorder may be diagnosed. Further follow-up is necessary and exploration of CFTR function in other tissues such as the nasal epithelium by nasal transepithelial potential difference, and/or the rectal tissue with intestinal current measurement, may be of use. Algorithms have been developed which apply these functional diagnostic tests along with genetic tests to provide differential diagnosis [De Boeck et al., 2006; Goubau et al., 2009; Bombieri et al., 2011].