Cystic Fibrosis Workup

The diagnosis of cystic fibrosis (CF) is based on typical pulmonary manifestations, GI tract manifestations, a family history, and positive sweat test results.

Requirements for a CF diagnosis include either positive genetic testing or positive sweat chloride test findings (>60 mEq/L) and 1 of the following:

• Typical chronic obstructive pulmonary disease

• Documented exocrine pancreatic insufficiency

• Positive family history (usually affected sibling)

Prenatal

Prenatal diagnosis allows the clinician to prepare for the medical and psychological needs of the parents, fetus, and newborn before, during, and after delivery.

Noninvasive CFTR analysis involves a technique for recovering DNA from cells obtained by buccal brushing. This technique can be used to determine the carrier status of the parents of a fetus with suspected CF based on sonographic findings of meconium ileus.

These tests are highly specific and are improving. One commercial test screens for 97 of the most common CF mutations. Although more than 1600 CF mutations exist, the 97 mutations covered by this test represent 98% of mutations responsible for the disease. In addition, results with this test are available in 5-8 days, versus 2-3 weeks with complete gene sequencing.

Screening tests do not screen for all possible mutations, and several types screen for just a few of the more common genetic mutations. Therefore, it is important to understand the implications of positive or negative results depending on the brand of screening test used.

Amniocentesis can provide subsequent fetal evaluation when both parents have identified CF mutations.

When only one or neither parent has an identified CF mutation but the couple has a previous child with CF, the status of the fetus can be predicted by restriction fragment length polymorphism (RFLP) analysis. Genetic material from both parents, the affected sibling, and the fetus must be available for RFLP testing. If the results predict CF in the fetus, referral to a tertiary care facility facilitates genetic counseling and consultation with specialists in maternal-fetal medicine.

If DNA analysis or amniocentesis tests are refused or if results are nondiagnostic, the authors recommend close sonographic follow-up at 6-week intervals.

Neonatal

Newborn screening for CF is universally required in the United States. All screening algorithms in current use in the United States rely on testing for immunoreactive trypsinogen (IRT) as the primary screen for cystic fibrosis. ^[31] The presence of high levels of IRT, a pancreatic protein typically elevated in infants with cystic fibrosis, warrants second level testing in the form of repeat IRT testing, DNA testing, or both.

A 2008 study from Massachusetts noted a decreasing incidence of cystic fibrosis identified by newborn screening, possibly resulting from more widespread preconception identification of cystic fibrosis carriers. ^[32] CFTR related metabolic syndrome (CRMS) is used to describe infants identified to have elevated levels of immunoreactive trypsinogen on newborn CF screening, have sweat chloride value of less than 60 mmol/L; have two CFTR mutations, at least one of which is not clearly categorized as CF causing and thus do not meet the Cystic Fibrosis Foundation guidelines for CF diagnosis. The Cystic Fibrosis Foundation published guidelines for the management of such infants. ^[33]

Postnatal

Suspect CF in patients with fetal or neonatal bowel obstruction and perform diagnostic tests as soon as possible.

From 75 to 80% of males with congenital bilateral absence of vas deferens (CBAVD) have been shown to possess a CFTR mutation for CF. With this condition, one may palpate the epididymis head; however, the structures derived from the Wolffian ducts under the control of the gonads, caudal epididymis, and vas deferens are absent. This anomaly may prove useful when looking for immediate support regarding a diagnosis of CF.

In adult males, obstructive azoospermia, in the absence of any other obvious cause (eg, vasectomy), provides additional corroborative evidence for the diagnosis of CF. Confirm results from semen analysis by obtaining a testicular biopsy.

A diagnosis of CF should be confirmed or refuted by a sweat test that meets all National Committee for Clinical Laboratory Standards (NCCLS) criteria. A sweat test may be performed any time after the first 48 hours of life if the neonate is not edematous.

Mutation analysis, performed on buccal or on blood cells using a Guthrie card, helps confirm the diagnosis if it yields at least one known CF mutation. Refer patients with confirmed CF to a regional or satellite CF center for counseling and education about this complex chronic disease. CF center physicians can also assist in postoperative management of nutritional or respiratory problems. To obtain a list of accredited centers, call 1-800-FIGHT CF or see the Cystic Fibrosis Foundation Web site.

Several methods are used to conduct a sweat test. Performed properly, the quantitative pilocarpine iontophoresis test (QPIT) to collect sweat and perform a chemical analysis of its chloride content is currently considered to be the only adequately sensitive and specific type of sweat test. ^[1]

For reliable results, collect at least 50 mg or, preferably, 100 mg of sweat, a quantity that may be difficult to obtain from young infants. This test can be inaccurate in very young infants or if an inadequate volume is collected. ^[34] Never pool sweat from multiple sites to obtain the required quantity because the rate of sweating determines electrolyte content.

Current macroduct collection methods allow adequate analysis with smaller volumes of sweat. If a macroduct coil is used for collection, then sweat must be stimulated with a disposable Pilogel electrode using the Webster Sweat Inducer for 5 minutes. After 30 minutes, the minimum acceptable sample is 15 µL.

The sweat chloride reference value is less than 30 mmol/L. A value of more than 60 mmol/L of chloride in the sweat is consistent with a diagnosis of cystic fibrosis. ^[35]  The values of 30-60 mEq/L may represent heterozygous carriers, these carriers cannot be accurately identified with a sweat chloride test. ^[34]

The sweat test must be performed at least twice in each patient, preferably several weeks apart. Values of 40-60 mmol/L are considered borderline, and the test must be repeated because these values have been found to be consistent with the diagnosis in some patients with typical features.

Repeat a sweat test to confirm positive results. Repeat a sweat test with negative results if clinical features suggestive of cystic fibrosis are present. Some patients with genetically documented cystic fibrosis and typical symptoms have consistently negative results on sweat tests.

Other causes of elevated levels of sweat chloride include the following:

• Untreated adrenal insufficiency

• Glycogen storage disease

• Type I fucosidosis

• Hypothyroidism

• Vasopressin-resistant diabetes insipidus

• Ectodermal dysplasia

• Malnutrition

• Mucopolysaccharidosis

• Panhypopituitarism

• Familial cholestasis

• Familial hypoparathyroidism

• Atopic dermatitis

• Iatrogenic causes (ie, infusion of prostaglandin E₁, improper technique)

Radiography

On chest radiography, initial changes are hyperinflation and peribronchial thickening. Progressive air trapping with bronchiectasis may be apparent in the upper lobes. With advancing pulmonary disease, the following findings may be noted:

• Pulmonary nodules resulting from abscesses

• Infiltrates with or without lobar atelectasis

• Marked hyperinflation with flattened domes of the diaphragm

• Thoracic kyphosis

• Bowing of the sternum

Pulmonary artery dilatation and right ventricular hypertrophy associated with cor pulmonale is usually masked by marked hyperinflation. Several radiologic scoring systems are recognized. An example of chest radiography in cystic fibrosis is shown in the image below.

On sinus radiography, panopacification of the sinuses is present in almost all patients with cystic fibrosis, and its presence is strongly suggestive of the diagnosis. Conversely, absence of panopacification strongly suggests that cystic fibrosis is not present.

Abdominal radiography

In about 71% of uncomplicated meconium ileus cases, abdominal radiography reveals a characteristic pattern of unevenly dilated loops of bowel with variable air-fluid levels. Air-fluid levels may be absent because of the viscid nonliquid nature of the inspissated meconium.

Bubbles of gas may become evident as air mixes with the tenacious meconium. While this soap bubble appearance (or Neuhauser sign) depends on the viscosity of the meconium and is not a constant feature, this radiographic feature is highly suggestive of meconium ileus. Although none of these features alone is diagnostic for meconium ileus, they strongly suggest the diagnosis when combined with a family history of CF.

Radiologic findings in complicated meconium ileus vary based on the associated complication. Speckled calcification visible on abdominal plain radiography strongly suggests intrauterine intestinal perforation and meconium peritonitis. Visible obstruction and a large dense mass with a rim of calcification suggest a pseudocyst. In 1970, however, Leonidas et al reported no radiologic findings that suggest a complication in a third of patients with complicated meconium ileus. ^[36]

In utero perforation can lead to meconium peritonitis or meconium pseudocyst formation; only postoperative evaluation may differentiate between CF-related and non–CF-related meconium peritonitis or meconium pseudocyst formation.

Chest CT scanning

Though chest CT scanning is not yet advised as a routine modality in patients with CF and there are concerns about exposure to radiation and the high cost of the procedure, chest CT scanning has been used to diagnose lung involvement, such as an onset of bronchiectasis. High-resolution chest CT scans are reported to be more sensitive than traditional spirometry in detecting changes in lung disease severity. In a recently published prospective study of 81 children with CF, Brody chest scan scores and Wisconsin and Brasfield chest radiograph scores were significantly associated with future lung disease severity. ^[37] Such quantitative chest imaging has a potential to help clinicians to identify patients at risk of future lung disease progression. CT mosaic attenuation is associated with lung disease. ^[38]

Ultrasonography

Prenatal sonographic characteristics associated with meconium ileus include hyperechoic masses (ie, inspissated meconium in the terminal ileum), dilated bowel, and inability to visualize the gallbladder. Normal fetal meconium, when visualized in the second and third trimesters, is usually hypoechoic or isoechoic to adjacent abdominal structures. (Hyperechoic mass is defined as a mass with greater sonographic density than liver or bone.)

The sensitivity of intra-abdominal echogenic masses for CF detection reportedly ranges from 30%-70%. This finding as a sonographic marker of meconium ileus is plagued by difficulties, including subjective assessment of echogenicity and extensive differential diagnoses.

In addition to meconium ileus, hyperechoic bowel may occur with Down syndrome, intrauterine growth retardation, prematurity, in utero cytomegalovirus (CMV) infection, intestinal atresias, abruptio placenta, and fetal demise. The importance of hyperechoic fetal bowel relates to gestational age at detection, ascites, calcification, volume of amniotic fluid, and presence of other fetal anomalies.

Furthermore, a prenatal diagnosis of meconium ileus using the sonographic feature of hyperechoic bowel must consider the parents' a priori risk. The positive predictive value of hyperechoic masses in a high-risk fetus is estimated at 52%, while the predictive value for a low-risk fetus is just 6.4%.

While reviews of pregnancies with 1-in-4 risk of CF show a 25%-60% association between hyperechoic bowel and CF, this association is less prevalent in the general population. In 1992, Dicke and Crane reviewed 12,776 fetal sonograms performed after 14 weeks' gestation and noted hyperechoic bowel in 30 (0.2%) of these patients. Of these, 13.3% had CF. This team also reported a 16.7% associated risk of perinatal death, a 23.3% risk of growth retardation, and a 3.3% risk of genetic abnormality. ^[39]

Note that hyperechoic bowel is a normal variant in both the second and third trimesters. Hyperechoic bowel, when it occurs as an isolated event early during the second trimester, may represent a normal variant and indicates the need for follow-up prenatal examinations. Although an increased risk of meconium ileus and CF is associated with hyperechoic bowel, the prevalence, degree of risk, and decisions involving prenatal management remain uncertain.

Prenatal ultrasonographic findings of dilated bowel in association with CF have been reported less frequently than findings of hyperechoic bowel.

In meconium ileus, bowel dilatation is caused by a meconium obstruction but mimics similar findings in the following conditions:

• Midgut volvulus

• Congenital bands

• Bowel atresia

• Intestinal duplication

• Internal hernia

• Meconium plug syndrome

• Hirschsprung disease

However, studies that show correlation between dilated fetal bowel and meconium ileus suggest that dilated fetal bowel warrants parental testing for CF and continued sonographic surveillance of the fetus.

In addition to the findings of increased abdominal echogenicity and bowel dilation, the inability to visualize the gallbladder on fetal ultrasonography is associated with CF. Combined with other sonographic features, nonvisualization of the gallbladder can help detect the disease prenatally. However, exercise caution in interpreting an absent gallbladder because the differential diagnosis includes biliary atresia, omphalocele, and diaphragmatic hernia.

Sonographic characteristics of fetal bowel obstruction are neither sensitive nor specific for meconium ileus. In general, a rate of sonographic detection for meconium ileus or meconium peritonitis can be up to 19%. Interpretation of these sonographic findings must consider the fetus' risk of CF. While ultrasonographic findings that suggest meconium ileus in a high-risk fetus indicate a high probability of CF, similar suspicious findings in a low-risk fetus warrant consideration of DNA testing or, at the very least, serial follow-up examinations.

Lung Clearance Index

Demonstration of ventilation heterogeneity by lung clearance index (LCI) is a sensitive marker for CF pulmonary disease even in young children and LCI findings have been correlated with CT lung findings, ^[40] however, LCI is mostly used in clinical trials and studies.

Hyperpolarized Gas Lung MRI

Ventilation MRI using hyperpolarized gas has been shown to detect ventilation heterogeneity. This technique could be used to detect and monitor early lung disease. ^{[41, 42]}

 

Genotype testing is recommended for individuals with a positive family history and for couples planning a pregnancy. It is not necessarily indicated for the general population. ^[18]

More than 1893 CF mutations have been identified. ^[12] In the commercially available CF gene sequencing method, the entire coding region, splice junction sites, and promoter region of the CFTR gene are amplified from genomic DNA by polymerase chain reaction (PCR) and then subjected to nucleotide sequence analysis on an automated capillary DNA sequencer.

A finding of 2 CFTR mutations in association with clinical symptoms is diagnostic. This test can detect more than 98% of disease-causing mutations in whites; the detection rate is lower in black, Hispanic, and Asian populations. Therefore, failure to find 2 abnormal genes does not exclude the disease.

In November 2013, the FDA approved 4 next-generation gene sequencing devices for clinical use in CF. Two of the devices are used to screen and diagnose CF by detecting DNA changes in the CF transmembrane conductance regulator (CFTR) gene ^{[43, 44]} : the Illumina MiSeqDx Cystic Fibrosis 139-Variant Assay, which checks specific points in the patient's CFTR gene sequence to detect known variants in the gene, and the Illumina MiSeqDx Cystic Fibrosis Clinical Sequencing Assay, which sequences a large portion of the CFTR gene to detect any difference in the CFTR gene compared with a reference CFTR gene.

The other 2 FDA-approved devices are the Illumina MiSeqDx instrument platform, which analyzes the genes, and the Illumina Universal Kit reagents, which isolate and create copies of the genes of interest from patient blood samples. ^{[43, 44]} These 2 devices comprise the first FDA-regulated test system that allows laboratories to develop and validate sequencing of any part of a patient’s genome. ^{[43, 44]}

Potential difference (PD) in voltage measured from nasal mucosa and the reading obtained by a reference electrode inserted into the forearm correlates with the movement of sodium across cell membranes, which is a physiologic function rendered abnormal by a CFTR mutation. The nasal PD (NPD) is a sensitive test of electrolyte transport that can be used to support or refute a diagnosis of CF.

A normal mean value standard error (SE) is 0.9-24.7 mV; an abnormal value is 1.8-53 mV. When measurements are repeated after mucosal perfusion with amiloride to block an epithelial sodium channel, the drop in PD is greater in patients with cystic fibrosis (73%) than in control subjects (53%). Subsequent perfusion with chloride-free solution and isoproterenol produces a sharp increase in the PD in normal subjects but has little effect when CFTR function is abnormal.

As a result of the lack of commercially available equipment and the practical difficulties with NPD measurement, this test is performed in only a few research centers to diagnose CF in patients in whom making a diagnosis is difficult or a sweat test is not technically possible because of skin problems.

Infant lung function testing using raised volume rapid thoracic compression (RVRTC) whole body plethysmography is gaining wider acceptance; however, its use is mostly confined to specialized and research centers. This testing has been successfully used to demonstrate airway obstruction in young infants with CF. ^{[45, 46]}

Standard spirometry may not be reliable until patients are aged 5-6 years; however, some younger patients can be taught to do reproducible maneuvers. Partial flow-volume curves may show abnormalities in addition to an elevated airway resistance and hyperinflation.

The forced oscillation technique (FOT), which uses the impulse oscillometry system (IOS), can be used successfully in younger children. Airway resistance measured by IOS has been found to be similar to the airway resistance measured by body plethysmography, and this technique has been successfully used to measure lung function in young patients with CF who are unable to perform spirometry. ^[47]

Typically, peripheral airway involvement resulting from CF manifests as an obstructive defect with airtrapping and hyperinflation; oxyhemoglobin desaturation may occur because of a ventilation-perfusion mismatch. In the early stages, forced expiratory volume in 1 second (FEV₁) may be normal, and forced expiratory flow (FEF) after 25-75% of vital capacity has been expelled (FEF 25-75) is reduced, suggesting small airway involvement.

Progression of disease has been correlated with a change in FEV1. A 2012 Danish study, using a longitudinal modeling technique specifically aimed at analyzing long sequences of repeated measurements of FEV1 measurements in CF patients reported that on average a change in FEV1 of greater than 13% (ie, twice the error SD to give a 95% confidence range) is likely to represent a true within-patient variation over time, whereas a lesser change may be due to transient (recoverable) fluctuation. ^[48]

The associated air trapping results in an elevated ratio of residual volume to total lung capacity (RV/TLC). With hyperinflation, TLC is also increased. In patients with advanced disease, extensive lung changes with fibrosis are reflected as restrictive changes characterized by declining TLC and vital capacity.

Lung clearance index (LCI) calculated from multiple breath inert gas (sulfur hexafluoride-SF6/helium gas mixture) washout has been used to demonstrate ventilation inhomogeneity, an early marker of lung disease in young children with CF. ^{[49, 50, 51]} LCI is a sensitive early marker of CF in young children, comparable with high-resolution CT scanning (HRCT), and is gaining wider acceptance by clinicians and researchers. ^[52]

Airway inflammation is the hallmark of lung disease in patients with CF. Studies suggest that airway inflammation is present even in the absence of infection.

Bronchoalveolar lavage fluid usually shows a high percentage of neutrophils, and recovery of Pseudomonas aeruginosa from bronchoalveolar lavage fluid supports the diagnosis of CF in a clinically atypical case.

Sputum microbiology

The most common bacterial pathogens in the sputum of patients with cystic fibrosis are as follows:

• Haemophilus influenzae

• Staphylococcus aureus

• Pseudomonas aeruginosa

• Burkholderia cepacia

• Escherichia coli

• Klebsiella pneumoniae

Findings of P aeruginosa, especially the mucoid form, support the diagnosis of cystic fibrosis in children.

Immunoreactive trypsinogen (IRT) is a pancreatic enzyme that can help with diagnosing CF in neonates with meconium ileus when IRT relative ratios are elevated greater than the 99th percentile. IRT plus sweat test was shown to increase sensitivity and specificity in screening.

In a study by Steven et al, only 2 of 29 patients with intestinal obstruction from meconium ileus had a normal IRT relative ratio, supporting a false-negative rate of 7%; however, when checked at days 9 and 12, the IRT relative ratio was elevated above the 99th percentile. IRT levels cannot be used to differentiate between simple and complicated meconium ileus. ^[53]

Monitoring the detectability of IRT over the first 5 years of life has also shown that the eventual absence of this enzyme correlates with severe CF. This finding is also indicative of a negative correlation between the start of pancreatic enzyme replacement and the end of IRT detectability. ^[54]

When meconium ileus is suspected on the basis of clinical and radiographic evidence, a contrast barium enema may be performed for diagnosis. One study showed a barium enema to be diagnostic in 45 (52%) patients. If meconium ileus is likely, follow the contrast enema with a therapeutic water-soluble contrast (Gastrografin) enema.

Some physicians advocate water-soluble contrast initially for both diagnosis and treatment. Controlled dilutions of Gastrografin remain the agent of choice for diagnosis and evacuation of inspissated meconium.

Fluoroscopically monitor contrast instillation in patients with meconium ileus to visualize a small-caliber colon (described as the microcolon of disuse), which often contains small "rabbit pellets" (ie, scybala) of meconium.

Progression of the contrast proximally may also outline pellets of inspissated meconium. Contrast that is successfully refluxed proximal to the obstruction allows observation of the dilated loops of small bowel.

In addition, evidence supports performing contrast enema using fluoroscopy in very low birth weight infants (average gestation age and weight, 27 wk and 788 g) in order to ensure that contrast reaches the distal ileum. Lack of contrast reflux to the distal ileum was found to be associated with unsuccessful relief of the obstruction. ^[55]