With Technology Advances, Saliva Tests Hold Greater Promise

Advancements in proteomics and gene expression analysis are providing researchers sensitive enough tools to consider saliva as an ideal, noninvasive sample for a host of new diagnostic tests. Two recent studies highlight the potential of salivary marker analysis to potentially improve the diagnosis of autism spectrum disorder (ASD) and assess feeding readiness in premature babies. Saliva is functionally equivalent to blood in terms of reflecting the physiological state of the body, according to a review of the emerging use of mass spectrometry for evaluation of salivary biomarkers, published in the January issue of Clinica Chimica Acta. It is estimated, the authors say, that saliva contains approximately 2,000 proteins of which 597 are also identifiable in the blood. “With the fast development of mass spectrometry and proteomic technologies, saliva is a growing area for basic and clinical research with substantial potential for disease diagnosis,” write the authors led by Qihui Wang from Sichuan University (China).
Proteomic Assessment for Autism-Related Markers Protein markers found in saliva can differentiate children diagnosed with autism spectrum disorders (ASDs) from a group of controls, according to results of a pilot study published online ahead of print Jan. 24 in Autism Research. ASD diagnosis is currently based on behavioral assessments. But, experts say that the lack of definitive biomarkers hampers accurate diagnosis, treatment monitoring, and reliable prognosis. The emergence of more than 1,000 ASD-associated genes indicates, likely, multiple causes and subtypes, which may be further defined with the addition of protein-level analysis. “ASD biological testing is already here in a sense, since gene arrays are being used increasingly, requested by parents and physicians,” lead study author Alisa Woods, Ph.D., tells DTET. “The challenge is that often the tests do not come up with ASD-associated markers, and even when they do, it is not clear that this in- formation alters treatment.... I think proteomic work needs to be more seriously considered from a basic science perspective, to supplement genomic data.” Woods, along with proteomics expert Costel Darie, Ph.D., analyzed the salivary proteome in six males (aged 5 years to 18 years) diagnosed with ASD and in six neurotypical control subjects without any major medical condition. Samples were processed both individually and in pooled analysis (to amplify the possibility of finding markers for quick initial screening). Using nanoliquid chromatography-tandem mass spectrometry, statistically significant differences were seen in several salivary proteins, including elevated levels of eight salivary proteins and lower levels of two proteins in the saliva of ASD patients, compared with the controls. Most of the proteins found dysregulated in the pooled samples were also found in the individual samples. Furthermore, there was not dramatic individual variability for most of the statistically significant dysregulated proteins. The authors say the findings support the possibility that immunological responses are present in some forms of ASD and could provide a first step toward a diagnostic test for ASD. Woods tells DTET larger validation studies are being planned. The published study was partially funded by Shire pharmaceutical company.
Gene Expression Assay for Preemie Feeding Readiness A separate study, published in the February issue of the Journal of Pediatrics, has identified salivary biomarkers related to oral feeding success in premature babies. The markers noninvasively assess the mechanisms underlying the development of oral feeding readiness (a newborn’s brain, sensory, and facial development) and may improve clinical decision-making, and possibly cutting length of stays. Prior to discharge, infants must demonstrate mature oral feeding skills, according to American Academy of Pediatrics guidelines. However, the determination of oral feeding readiness is a significant clinical challenge, as it is based on subjective assessments. Oral feeding delays and complications increase the risk for poor growth and developmental disabilities and are additionally tied to increased length of hospital stay and millions of dollars in health care expenditure the authors say. In the present study, the Tufts University researchers utilized whole-transcriptome microarrays to compare saliva pre- and post-oral feeding success in 12 preterm newborns (postconceptional age [PCA] range at pre-success measurement, 33 5/7 to 36 2/7 weeks; post-success range 34 2/7 to 37 3/7 weeks). Gene expression biomarkers were selected from computational modeling (n=15); evidence-based (n=6); and reference markers (n=3). Then, 400 salivary samples were evaluated by reverse-transcription quantitative polymerase chain reaction amplification. Models assessed genes, alone and in combination, controlling for sex and PCA. The researchers found that advancing PCA and female sex both significantly, positively predicted an infant’s ability to feed orally. The five genes most predictive of feeding success represent sensory integration systems (NPHP4, PLXNA1), hunger signaling (NPY2R), facial development (WNT3), and energy expenditure (AMPK). A mature oral feeding pattern was predicted by negative expression of three genes (NPHP4, NPY2R, and WNT3), and positive expression of two genes (AMPK, PLXNA1). The combination of the five genes in addition to PCA and sex, demonstrated “good” accuracy for determining feeding success. “Ultimately, these models have the potential to allow caregivers to develop individualized treatment modalities based upon each infant’s gene expression profile to improve quality of care,” write the authors led by Jill Maron, M.D., from Tufts University Medical Center (Boston). “Compared with subjective cue-based feeding algorithms, this approach represents a significant advance to supplement clinical decision making and establishes the foundation for the development of a point-of-care assay.” Maron tells DTET by incorporating the results of this low-cost test (approximately $50 to run) into feeding decisions, it would be “realistic” to expect a reduction in one day to three days in the NICU (average daily NICU stay $3,500) per child. First, further validation in a multicenter trial is necessary. In general, though, Maron says, salivary testing is particularly appealing in pediatric populations, where repetitive blood sampling is untenable in very low birth weight infants due to their low blood volume and is undesirable in young children. Takeaway: While these tests are likely years away from clinical use, saliva represents an important specimen that is increasingly recognized for containing diagnostically significant markers of disease and human development.