NEW YORK – A team of researchers has demonstrated in a study that elevated amounts of the protein intestinal alkaline phosphatase (iAP) and low iAP enzyme activity may be useful biomarkers for detecting necrotizing enterocolitis (NEC), a condition of the gastrointestinal tract that is sometimes fatal in preterm infants.
Integrated into a laboratory-based commercial in vitro diagnostic platform, the biomarkers could help clinicians quickly differentiate NEC from other confounding conditions, and lower the rate of mortality, said Sunyoung Kim, a professor of biochemistry and molecular biology at LSU Health New Orleans School of Medicine and senior author of the study.
Kim, along with LSU colleagues and researchers at Washington University School of Medicine in St. Louis, Missouri, published the results of the study in JAMA Network Open, in which they describe measuring and analyzing the activity of iAP protein obtained from stool samples from 136 preterm babies at Children's Hospital of New Orleans, Touro Infirmary, and St. Louis Children's Hospital in St. Louis, Missouri.
"We've known about this disease for about 200 years, but we still need to develop an understanding of who is going to get it, why they're going to get it, and if they will survive it," Kim said.
She and her colleagues have developed a prototype of the test though a commercial version may be more than a year away because further validation of the biomarkers is needed in a larger multicenter trial, Kim said in an interview. Nonetheless, she has founded a company, Chosen Diagnostics, which has licensed the technology from LSU Health and obtained the right to license it to other companies.
The firm has been in discussion with laboratory and hospital clinicians about how to move forward with developing the test and is currently combining the biomarkers into a clinical chemistry platform and planning a later, more robust version that combines clinical chemistry with immunoassay technology, Kim said.
After future clinical trials have been completed in about one year, Chosen Diagnostics anticipates seeking US Food and Drug Administration clearance for the test, and it is currently planning a presubmission to the agency for marketing clearance, Kim said.
The New Orleans-based company anticipates presubmitting to the FDA in about six months. The outcomes from that meeting will shape parameters of future clinical trials, including the number of patients required, Kim said.
Diagnostic sensitivity
During NEC, inflammation of the intestine leads to bacterial invasion causing cellular damage and cell death, which causes necrosis of the colon and intestine. As NEC progresses, it can lead to intestinal perforation causing peritonitis, sepsis, and death. The likelihood of contracting NEC increases the earlier the preterm baby is born.
NEC almost exclusively affects neonates, and it presents in about 10 percent of preterm birth infants, Kim said, adding that mortality rates as a result of the condition vary between 30 percent and 70 percent per healthcare institution.
Clinicians use X-rays to diagnose the disease, but the level of diagnostic sensitivity can be as low as 44 percent, according to Kim.
Angelle Klar, a practicing neonatologist at the University of Mississippi Medical Center, said that the absence of overt findings associated with NEC in X-rays, which neonatologists are trained to recognize, does not rule out the potential for a baby having NEC. Babies can present with subtle symptoms of NEC and appear to be relatively healthy for weeks but then succumb to sudden and severe infection, said Klar, who is not involved in the work being conducted by Kim and her colleagues. Such characteristics make finding a diagnostic test that enables early detection and intervention more urgent.
In current practice, neonatologists are well aware of risk factors associated with NEC, with prematurity being the greatest, said Klar in an interview. Neonatologists implement "universal precautions in the management of all premature infants based on gestational age and birth weight," she said. "A biomarker test such as the intestinal alkaline phosphatase [assay under development] has the potential for allowing more individualized care for those premature infants at greatest risk."
Further, the test may have potential for differentiating sepsis cases associated with intestinal causes from those sepsis cases that are associated with non-intestinal causes, she said, adding that there may be potential to integrate the biomarker into screening programs that inform feeding protocols for preterm infants.
Kim noted that use of stool samples for testing is particularly suitable for preterm babies because it is non invasive. Providing blood samples can be problematic for premature babies whose red blood cell levels are low.
To diagnose NEC, investigators have been searching for proteins associated with inflammation, but those markers provide "late-stage signals triggered at a point of no return for the infant," Kim said. "We decided that if the disease was so gut-specific, then the protein biomarker causing the condition had to be something that a human infant was producing that was interacting with the gut microbiota."
For their study, the investigators collected clinical data on gestational age; birth weight; Apgar scores, a test of a baby's heart rate, muscle tone, and other signs; delivery type; race; gender; feeding; intake of antibiotics; and laboratory and radiology results, among other parameters. They classified 18 percent of the babies as having severe NEC; 14 percent as having suspected NEC; and 68 percent as NEC controls.
Because iAP activity precedes the chemical process triggering inflammation, they studied the abundance and enzyme activity of iAP shed in stool to assess the correlation of the two iAP biochemical measures with disease severity.
During research for the JAMA study, the investigators found that measuring amounts of iAP protein — the higher performing of the two biomarkers they tested during the study — led to detection of NEC four days earlier than X-rays, at 95 percent sensitivity and specificity, Kim said. The specific accuracy rates using iAP levels and iAP activity as markers for severe NEC were 97 percent and 76 percent, respectively. The accuracy values for suspected NEC were also 97 percent and 62 percent, respectively.
The study found no association of iAP measurements with non-gastrointestinal tract infections.
Kim said that a number of investors have said they are interested in backing the development of the test, but she anticipates that federal funding from the National Institutes of Health and National Science Foundation will adequately support the next phase of development and clinical studies. A number of neonatal care units in the US have agreed to participate in the next study, though she declined to identify them.