Detection of nitrate and nitrite in human saliva and other biofluids after nitrate ingestion, and optimisation of an electrochemical sensor for saliva

Detection of nitrate and nitrite in human saliva and other biofluids after nitrate ingestion, and optimisation of an electrochemical sensor for salivary nitrite

Introduction: Infective gastroenteritis patients display extreme increases in serum nitrate concentration due to high nitric oxide synthesis. We will test whether serum nitrate concentration reveals the cause of acute diarrhoea in patients admitted as emergency cases to hospital, and whether a novel electro-chemical sensor facilitates rapid bedside measurements of serum nitrate. Immune system activation by bacterial pathogens causes inducible nitric oxide synthase expression, thereby increasing nitric oxide (NO) synthesis. NO, nitrate (NO3-) and nitrite (NO2-) are inflammatory markers. Infective gastroenteritis patients display far greater increases in plasma nitrate concentration than in non-infective gastroenteritis (Dykhuizen et al, 1996). We hypothesise that serum nitrate is useful in determining the cause of acute diarrhoea in patients admitted as emergency cases to hospital.

Whilst diarrhoea can be caused by infective gastroenteritis (bacterial/viral pathogen), diarrhoea can also be caused by non-infective processes, e.g. inflammatory bowel disease. Distinguishing the causes is critical in the hospital setting, as treatments differ. Infectious diarrhoea patients must be isolated quickly, to prevent infections spreading. Currently, identifying infectious diarrhoea is difficult in the acute setting. Stool samples can be tested by microbiological culture, but this takes 2-3 days and has low sensitivity for the detection of infections. Polymerase chain reaction (PCR) can be used, for a faster and accurate diagnosis, but is expensive and not widely available in hospitals. A cheap/simple test is needed to distinguish rapidly between non-infective and infective gastroenteritis.

Hypothesis/Research question(s):
1. Does serum nitrate concentration reveals the cause of acute diarrhoea in patients admitted as emergency cases to hospital?
2. Can a novel electro-chemical sensor facilitate rapid bedside measurements of serum nitrate?

Experimental design: We will investigate serum nitrate concentration as a differential biomarker of pathogenic infection in patients with acute diarrhoea. Serum nitrate will be compared with a "gold standard" PCR-based assay, as an unequivocal identifier of bacterial infections including Campylobacter, C. difficile, salmonella, and viral infections e.g. norovirus. At Torbay Hospital, there are about 50 severe diarrhoea stools samples sent monthly to the microbiology department. We will test 300, of which about 100 will be positive, allowing a good estimate of sensitivity/specificity. Patients have stool samples and blood taken routinely and no blood will be taken surplus to requirements for normal clinical care.

To demonstrate the potential to transfer the diagnostic measurement of serum nitrate into the clinical environment, the student will compare our established spectrophotometric nitrate assay with a novel electrochemical sensor developed in Bath (see Gross et al, 2014, below). Further into the project (after obtaining ethics approval), we will compare plasma with salivary/urinary nitrate, alongside chemiluminescence determination of nitrite and electrochemiluminescence measurement of protein-bound 3-nitrotyrosine in isolated blood cells - all methods established in our laboratory. This will provide: (1) insight into metabolism of the large amounts of nitrate generated during infective gastroenteritis, and (2) contingencies for areas to explore in the event that serum nitrate has unexpectedly poor diagnostic potential.