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Article describes new diagnostic fronts for cystic fibrosis

Researchers use silica plate that selects disease biomarkers

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Article by Unicamp researchers published in the Swiss magazine Frontiers in Pediatrics, of high impact in the field of Pediatrics, suggests a new form of diagnosis for the most serious mutation in cystic fibrosis. The technique uses a small silica plate that, when pressed lightly on the skin, selects specific biomarkers for the disease, based on patients' skin impressions, which are extracted with solvents and read in mass spectrometers. It only takes 30 seconds for the equipment to obtain the sample results.

The publication is entitled “Skin biomarkers for cystic fibrosis: a potential non-invasive approach for patient screening”, which translates into Portuguese as “A potential non-invasive approach for patient screening”. It is the result of Cibele Zanardi Esteves' doctoral thesis, supervised by Rodrigo Catharino and developed at the Faculty of Medical Sciences (FCM).

Cystic fibrosis, explained Rodrigo Catharino, is a hereditary genetic disease caused by changes in a defective gene, CFTR. This defect, or mutation, alters the production of sweat by the skin, mucus by the lungs and digestive secretions. “This is a serious condition that affects one in every eight thousand people in Brazil.”

According to the professor, spectrometers can detect metabolites of high sensitivity and specificity used as markers for this diagnosis. In the study, chemical markers of various molecular classes were chosen, represented by bile acids, a glutaric acid derivative, thyrotropin-releasing hormone, an inflammatory mediator, a phosphatidic acid and diacylglycerol isomers.

Photo: Scarpa
Cibele Zanardi Esteves, author of the doctoral thesis: “The procedures and analyzes require very strict protocols”

These markers signal metabolic disorders due to cystic fibrosis. The method is already patented by the National Institute of Industrial Property (Inpi) and its use proves to be advantageous, as it does not cause pain to the patient, in addition to being simple and quick. Therefore, early detection of the disease – starting with specific therapies – provides the patient with a better prognosis and a better quality of life. “And, through statistical analysis, it is verified whether or not the patient has the disease, in its most serious form”, commented Cibele.

Combined with this set of biomarkers, the sample collection method represents a useful tool for managing the disease and monitoring its evolution. This exploratory approach already points to new perspectives for the development of other diagnostic assays that cover other mutations that cause the disease.


Quadro

In general, children who have cystic fibrosis cough a lot, have excessive mucus retention and, sometimes, repeated lung infections. Many undergo lung transplantation and others are unable to fully digest food. Some still need to ingest digestive enzymes and may grow up malnourished. This condition increasingly worsens the child's condition and, depending on the degree of gene mutation, can lead to death. “The treatment of cystic fibrosis is only palliative”, said the professor.

Cibele commented that the first test that babies undergo, at birth, is the heel prick test. In it, some abnormalities indicative of this disease can now be screened. In most cases, the heel prick test is normal, but some symptoms (such as a persistent cough with secretions, difficulty gaining weight and dehydration without a determinable cause) make us suspect cystic fibrosis. The child then undergoes many examinations and tests until the disease is diagnosed.

The test traditionally indicated to confirm cystic fibrosis is the sweat test. However, the patient does not always achieve an accurate result, as there are different degrees of the disease. “The procedures and analyzes require very strict protocols, since methodological flaws are common, and the test does not have clear sensitivity and specificity data”, highlighted the author.

Added to this, the sweat test collection can cause discomfort to the patient, as it can take up to two hours to complete. To stimulate sweating on the patient's forearm (mostly children in early childhood), a low-voltage electrical stimulus is performed with the substance pilocarpine for 30 minutes.

Sweating is stimulated and, in about an hour, sweat is produced, which is collected and sent to determine chlorine levels, confirming the laboratory diagnosis. Typically, people with cystic fibrosis have chlorine levels in sweat higher than healthy people.

“Also, genetic tests may not be conclusive, as more than 1.500 disease-causing mutations have already been detected. Only the 300 most frequent mutations are searched. Furthermore, these tests are restricted to some diagnostic centers and research centers, and are not accessible to the public”, revealed the researcher.


Innovation

Rodrigo Catharino reported that, at Unicamp, a great partnership was formed between Cibele and a Pediatrics group that studies cystic fibrosis at the Center for Research in Pediatrics (Ciped) and that has produced innovative work, led by professors Fernando Ribeiro and Dirceu Ribeiro. “We took a leap in innovation, as there is practically no solo career today. Scientifically important research involves the efforts of many actors.”

Photograph; Scarpa
Professor Rodrigo Catharino, research advisor: spectrometers can detect metabolites with high sensitivity and specificity

Now we need to disseminate this information so that other diagnostic centers can recognize this great potential discovered here. “We studied the mutation more severe of the CFTR gene in 40 children. But more mutations occur and, if we disseminate this finding to other partners, we will be able to work with different types of mutations that also cause the disease, often in milder degrees, given that the technique is highly sensitive and can detect small changes related to the disease.” , the teacher clarified.

The markers found in the research, he emphasized, are not specific to this mutation, but rather to the condition that the mutation induces, that is, the characteristics observed in the patient. 


Other patents

In the thesis, two patents were obtained in total for the approach to cystic fibrosis and one for neonatal cholestasis. The two patents related to cystic fibrosis are related to two types of biological material: skin and saliva.

The same group of patients (aged between five and 19 years) evaluated for the skin test, with the same mutation, underwent saliva collection. “Saliva is also an easy material to collect and, in the case of children, approaching it is not as scary. A cotton ball is placed in your mouth and molecules are extracted from the saliva for analysis”, described Cibele.

Rodrigo Catharino stated that the intention was to develop a less invasive, simpler and cheaper technique because the chosen materials are easy to collect, can be transported to the analysis center and, as a result, can more quickly assist the patient who arrives at the outpatient clinic. .

Another patent obtained in this investigation was related to neonatal cholestasis, a symptom that can be associated with several diseases with multifactorial causes (genetic or viral) and that draws attention for causing jaundice that begins within two months of the baby's life. In addition to the yellowish physiognomy, he may present dark urine and light stools.

When neonatal cholestasis appears, the case is generally surgical, requiring immediate intervention for the child to survive; or, as the cause is unknown, the symptoms must be individually treated.

Instead of working with saliva and skin, in this case the heel prick test filter is used. Thus, it is possible to diagnose the disease during the initial screening. From the heel prick test, it is possible to find out whether the child will have the disease. She does not need to be symptomatic at that moment for the anomaly to be discovered.

“The most interesting thing about this work is to predict in neonatal screening what the child may show in a few months, which allows them to be treated and improve their chances of life”, stated Rodrigo Catharino. “This study helps save lives and is ensuring that the ICMS incentive is directly applied to activities relevant to society.”

 

 

JU-online cover image
Audio description: In the internal area, close-up and frontal image, fingers of the right hand, using a blue glove, hold, with the thumb and index finger, a small silver rectangular silica plate, measuring approximately one centimeter by one centimeter. In the background, on the left, the shadow of that same image appears. Image 1 of 1.

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