Clinical Evaluation of Bacterial DNA Using an Improved Droplet Digital PCR for Spontaneous Bacterial Peritonitis Diagnosis

Objective Bacterial DNA (bactDNA) detection has been studied on ascitic fluid. However, there is insufficient data to support its use in clinical practice. We improved a novel droplet digital PCR (ddPCR) method and enhanced its diagnostic efficiency for spontaneous bacterial peritonitis (SBP). Method A total of 250 patients were included in this retrospective study. Extra cell-free DNA was depleted using Benzonase before pathogen DNA extraction to obtain viable bacterial DNA. The threshold value of bactDNA quantitation and its diagnostic performance were established based on ascites-polymorphonuclear (PMN) and clinical manifestation. The bactDNA quantification analysis were detailedly performed on patients who were symptomatic and had a PMN < 250 cells/mm3. Results This study enrolled 191 patients with liver cirrhosis and ascites. After the removal of free DNA, bactDNA detected by ddPCR were generally decreased(1.75 vs 1.5 copies/µl, P<0.001), while the area under the curve for diagnosing SBP was increased, which 0.98 for total, 0.91 for gram-positive, 0.95 for gram-negative bactDNA. Compared with traditional culture and PMN count, results based on composite diagnostic standard showed that the sensitivity of ddPCR testing was 80.5% for total,72% for Gram-positive, and 93.9% for Gram-negative bactDNA while the specificity was 95.3%, 93.9%, and 89.3%, respectively. In patients with PMN <250 cells/mm3, the bactDNA quantitation of 13 patients who were symptomatic was significantly higher than those asymptomatic(2.7 vs 1.7 copies/µl, P<0.001). Conclusion BactDNA quantitation in ascites by ddPCR is a promising approach to improve the diagnostic accuracy of SBP, especially for symptomatic patients with PMN < 250 cells/mm3.