Test promises to diagnose cystic fibrosis more accurately

Brazilian researchers develop a novel noninvasive method to diagnose a genetic disease that creates a chloride and sodium imbalance in glands producing bodily secretions.

By Elton Alisson  |  Agência FAPESP – Pediatricians will soon be able to use a new technique to diagnose newborns with suspected cystic fibrosis, a genetic disease that creates an imbalance in the chloride and sodium levels in the exocrine glands, which produce secretions such as mucus and sweat. 

A group of researchers at the State University of Campinas (UNICAMP) in São Paulo State, Brazil, have developed a novel noninvasive diagnostic technique that promises to be simpler and more accurate than the methods used today.

The new test resulted from research supported by FAPESP and is described in a paper published in the journal Frontiers in Pediatrics.

“It’s a new screening methodology capable of diagnosing cystic fibrosis by skin markers, with no need to cause suffering for the baby or to use other procedures for collecting biological samples,” said Rodrigo Catharino, a professor in UNICAMP’s School of Pharmaceutical Sciences, head of the Innovare Biomarker Laboratory, and principal investigator for the project.

According to the Cystic Fibrosis Foundation, approximately 70,000 people have cystic fibrosis worldwide. The disease causes the production of thick, sticky mucus, which is not properly eliminated by the organism and builds up in several organs, such as the lungs, pancreas, liver and intestine. Although it is found in all ethnic groups, white people are the most affected. In Europe, its prevalence is in the range of 1 per 2,000-3,000 live births.

“The most widely used lab tests today for the diagnosis of cystic fibrosis are DNA analysis of the blood and the sweat test. Both have limitations,” said Cibele Zanardi Esteves, lead author of the paper.

The DNA analysis test focuses on recognizing known mutations that cause the disease. The mutations affect a gene called CFTR (short for cystic fibrosis transmembrane conductance regulator). The test is expensive, takes time, and has to be performed by labs that specialize in molecular genetics, all of which limit patient access.

The sweat test, widely seen as the gold standard in cystic fibrosis diagnostics, detects the level of chloride in the patient’s perspiration. More than 60 millimoles per liter (mmol/l) is considered positive for the disease. This test can also take time, and above all, it is distressing for newborns and children because it involves inducing perspiration on the skin of the forearm by the local administration of pilocarpine nitrate and then applying an electric current for approximately 30 minutes to boost sudoresis.

Another drawback of the sweat test is its relative lack of sensitivity, in that patients with chloride levels between 30 and 60 mmol/L are classified as indeterminate, requiring further evaluation. Its design is based on the diagnosis of patients with symptoms in multiple organs, and the researchers note that patients with single-organ symptoms may have inconclusive sweat tests.

“The sweat test’s relatively narrow accuracy range makes diagnosing cystic fibrosis imprecise in some borderline cases, which require complementary tests,” Esteves said.

Because the mutations affecting CFTR and known to cause cystic fibrosis produce many metabolic alterations in patients, the researchers decided to prospect for biochemical markers of these alterations using skin and other biological samples to develop a simpler, cheaper and more precise diagnostic method.

Skin biomarkers

The researchers selected 16 patients diagnosed with cystic fibrosis who were treated at UNICAMP’s general and teaching hospital. All had the F508del mutation, the most common of the more than 2,000 CFTR mutations associated with the disease. The control group comprised 16 healthy individuals of the same age and gender who were recruited from schools and universities.

Skin metabolites were collected from both groups using plates of silica gel placed on the patient’s back for 1 minute. All collected samples were analyzed in a mass spectrometer, a device that acts as a type of molecular weighing scale, sorting molecules according to their mass. 

Statistical and metabolic analyses were performed to compare the skin molecules from cystic fibrosis patients with those taken from the control group of healthy patients. The results pointed to seven different molecules associated with metabolic dysfunctions observed in cystic fibrosis and hence considered potential skin biomarkers.

“The biomarkers we identified aren’t linked only to sweat glands but also to the ion imbalance in the organism affected by the disease due to the genetic mutation. This makes the new method more specific than the sweat test,” Esteves said.

The researchers now plan to use the method, which they have patented, to evaluate other mutations associated with cystic fibrosis to validate and commercialize the test.

They hope the new test can be used to diagnose suspected cases of the disease in newborns, for example, requiring only the placing of a silica gel plate on the baby’s skin for a minute and dispensing with the need to stimulate sweating.

The plate would then be run through a mass spectrometer, which would analyze the molecules in 30 seconds and compare them with the skin biomarkers identified by the researchers. The disease could thereby be diagnosed faster and more accurately.

“Some hospitals have a mass spectrometer, which they use to measure vitamin D and perform the heel prick test, among others, but the type of device is different from the one we used to develop the technique. We intend to adapt the technique so that hospitals can use it with the equipment they already have,” Esteves explained.

The Frontiers in Pediatrics article “Skin biomarkers for cystic fibrosis: a potential non-invasive approach for patient screening” (doi: 10.3389/fped.2017.00290) by Cibele Esteves et al. can be read at frontiersin.org/articles/10.3389/fped.2017.00290/full.