Clinical evaluation of droplet digital pcr for suspected ascites infection in patients with liver cirrhosis.

BACKGROUND: Droplet digital PCR (ddPCR) is increasingly used in diagnosing clinical pathogens, but its effectiveness in cirrhosis patients with suspected ascites infection remains uncertain. METHODS: The diagnostic performance of ddPCR was assessed in 305 ascites samples, utilizing culture and clinical composite standards. The quantitative value and potential clinical impact of ddPCR were further analyzed in patients with spontaneous bacterial peritonitis. RESULTS: With culture standards, ddPCR demonstrated a sensitivity of 86.5% and specificity of 83.2% for bacterial or fungal detection. After adjustment of clinical composite criteria, specificity increased to 96.4%. Better diagnostic performance for all types of targeted pathogens, particularly fungi, was observed with ddPCR compared to culture, and more polymicrobial infections were detected (30.4% versus 5.7%, p < 0.001). Pathogen loads detected by ddPCR correlated with white blood cell count in ascites and blood, as well as polymorphonuclear cell (PMN) count in ascites, reflecting infection status rapidly. A positive clinical impact of 55.8% (43/77) was observed for ddPCR, which was more significant among patients with PMN count ≤ 250/mm3 in terms of medication adjustment and new diagnosis. ddPCR results for fungal detection were confirmed by clinical symptoms and other microbiological tests, which could guide antifungal therapy and reduce the risk of short-term mortality. CONCLUSIONS: ddPCR, with appropriate panel design, has advantages in pathogen detection and clinical management of ascites infection, especially for patients with fungal and polymicrobial infections. Patients with atypical spontaneous bacterial peritonitis benefited more from ddPCR.

Clinical Evaluation of Metagenomic Next-Generation Sequencing Method for the Diagnosis of Suspected Ascitic Infection in Patients with Liver Cirrhosis in a Clinical Laboratory.

Metagenomic next-generation sequencing (mNGS), mostly carried out in independent clinical laboratories, has been increasingly applied in clinical pathogen diagnosis. We aimed to explore the feasibility of mNGS in clinical laboratories and analyze its potential in the diagnosis of infectious ascites. Two reference panels composed of 12 strains commonly appearing in peritonitis were constructed to evaluate the performance metrics based on in-house mNGS protocols. The mNGS clinical detection value was analyzed in 211 ascitic samples and compared with culture and composite standards. Finally, eight patients with cirrhosis were prospectively enrolled to verify the clinical value of mNGS in peritoneal infection diagnosis. The mNGS analytical performance showed that the assay had great linearity, specificity, stability, interference, and limits of detection of 33 to 828 CFU/mL. The sensitivity and specificity of mNGS for bacterial or fungal detection using culture standards were 84.2% and 82.0%, respectively. After adjustment using digital PCR and clinical judgment, the sensitivity and specificity increased to 87.2% and 90.1%, respectively. Compared with culture, mNGS detected a broad range of pathogens and more polymicrobial infections (49% versus 9%, P < 0.05). The pathogen results were obtained within 24 h using mNGS in eight prospective cases, which effectively guided antibiotics therapy. mNGS testing in clinical laboratories affiliated with a hospital has certain advantages. It has unique superiority in pathogens detection, particularly in patients with polymicrobial infections. However, considering spectrum characteristics and test cost, pertinent pathogen panels should be developed in clinical practice. IMPORTANCE This study established and evaluated a complete metagenomics next-generation sequencing assay to improve the diagnosis of suspected ascitic infection in a clinical laboratory affiliated with a hospital. The assay is superior to traditional culture testing and will aid in the early and accurate identification of pathogens, particularly in patients with polymicrobial infections. This assay is also essential for precision therapy and can reduce the incidence of drug resistance stemming from irrational use of antibiotics.

Clinical evaluation of bacterial DNA using an improved droplet digital PCR for spontaneous bacterial peritonitis diagnosis.

Objective: Bacterial DNA (bactDNA) detection can be used to quickly identify pathogenic bacteria and has been studied on ascitic fluid. We aimed to retrospectively analyze the diagnostic value and applicational prospect of the bactDNA load in spontaneous bacterial peritonitis (SBP). Method: We extracted viable bactDNA from ascitic samples of 250 patients with decompensated cirrhosis collected from October 2019 to April 2021 and detected the bactDNA by droplet digital polymerase chain reaction (ddPCR). We used ascitic samples of a baseline cohort of 191 patients to establish diagnostic thresholds for SBP and analyze the patients' diagnostic performance based on ascites polymorphonuclear (PMN) and clinical manifestation. We performed bactDNA quantification analysis on 13 patients with a PMN less than 250 cells/mm3 but with clinical symptoms. The dynamic changes of the bactDNA load from eight patients (before, during, and after SBP) were analyzed. Results: After the removal of free DNA, the bactDNA detected by ddPCR was generally decreased (1.75 vs. 1.5 log copies/µl, P < 0.001). Compared with the traditional culture and PMN count in the SBP diagnosis, the bactDNA showed that the ddPCR sensitivity was 80.5%, specificity was 95.3%, positive predictive value was 82.5%, and negative predictive value was 94.7%, based on clinical composite criteria. In patients with a PMN less than 250 cells/mm3, the bactDNA load of 13 patients with symptoms was significantly higher than those without symptoms (2.7 vs. 1.7 log copies/µl, P < 0.001). The bactDNA in eight patients had SBP that decreased by 1.6 log copies/µl after 48 h of antibiotic treatment and by 1.0 log copies/µl after 3 days of continued treatment. Conclusion: BactDNA detection can be used to further enhance the diagnostic efficiency of SBP. Therefore, the application of ddPCR assay not only can be used to discriminate and quantify bacteria but also can be used in the clinical assessment for antibiotics treatment.

Pinching spine: A potential treatment for depression.

OBJECTIVE: To investigate whether pinching spine (PS, i.e. , a traditional Chinese manipulative therapy) is beneficial to ameliorating the depressive state (including behavioral deficit, retardative weight gain and decreased sucrose consumption) in a rat model of depression induced by chronic unpredictable stress (CUS) and to explore the candidate mechanism of action. METHODS: PS was performed on rats' spine once daily for 1 week after exposure to CUS. The open-field test, body weight measuring, and sucrose intake test were applied on different dates: before stress (d0), at the end of stress (d21) and after PS treatment (d28), respectively. Then the rats' hippocampuses were performed genome-wide microarray analysis, and the expression levels of several genes were evaluated by real-time polymerase chain reaction (PCR). RESULTS: Exposure to CUS resulted in decreases of behavioral activity and sucrose consumption, which were reversed significantly after PS treatment. The expression of several genes relevant to energy metabolism, anti-oxidation, and olfactory receptor, etc., were down-regulated, while the expression of those relevant to hemostasis, immunity-inflammation, and restriction of activities and ingestion, etc., were up-regulated in hippocampuses of rats exposed to CUS. PS treatment significantly inverted these changes. Furthermore, increase or decrease in gene expression evaluated by realtime PCR was concordant with up-regulated or down-regulated expression evaluated by microarray analysis. CONCLUSION: PS showed a potential antidepressant-like effect, of which the action mechanism might be due to gene expression regulation in hippocampus.

[Experimental study on Buyang Huanwu decoction ([Chinese characters: see text]) for promoting functional recovery of crushed common peroneal nerve in rats].

OBJECTIVE: To study the effects of Buyang Huanwu Decoction ([Chinese characters: see text]) on promoting functional recovery of crushed common peroneal nerve in rats. METHODS: Thirty Sprague-Dawley rats were subjected to produce common peroneal nerve injuries model,and the length of injury was 5 mm. All the rats were divided into 3 groups: BYHWD group, mecobalamin group and model group. The drugs were given by gavage daily for 18 days. Footprint test was performed at the 18th day after surgery to evaluate toe spread function (TSF). Electrophysiology was performed at the 18th day after operation to determine the nerve conduct velocity (NCV). The wet weight ratio and section area of tibial muscle were also measured. RESULTS: (TSF:At the 18th day after operation, the TSF in BYHWD group (-0.15 +/- 0.07) increased significantly compared with that of model group (-0.25 +/- 0.07) (P < 0.01); the TSF in mecobalamin group (-0.17 +/- 0.08) also increased notably compared with that of model group (P < 0.01).(2) NCV: the NCV in BYHWD group [(18.36 +/- 2.74) m/s] (P < 0.01l) and in mecobalamin group [(16.32 +/- 3.54) m/s] (P < 0.05) also increased significantly compared with that of model group [(9.08 +/- 2.56) m/s]; there was striking variation between model group and mecobalamin group (P < 0.05). (3) Wet weight ratio: the wet weight ratio in BYHWD group [(64.21 +/- 2.92)%] (P < 0.01)and in mecobalamin group [(62.43 +/- 3.21)%] (P < 0.01) all increased significantly compared with that of model group [(54.27 +/- 2.05)%]. (4) The section area of tibial muscle: the section area of tibial muscle in BYHWD group [(654.21 +/- 42.92) cm2] (P < 0.01) and in mecobalamin group [(638.43 +/- 93.21) cm2] (P < 0.01) all increased significantly compared with that of model group [(574.27 +/- 52.05) cm2]; there was also striking variation between model group and mecobalamin group (P < 0.05). CONCLUSION: BYHWD can promotes functional recovery of crushed nerve as a result of accelerating recovery of TSF, raising NCV and delaying the decrease of tibial muscle section area and wet weight ratio.