ABSTRACT
【Objective】 To investigate the effectiveness of multilink real-time fluorescence quantitative PCR (qPCR) in the detection of common pathogens in transplantation. 【Methods】 The primers of the qPCR detection system were designed for 24 common infectious pathogens after clinical transplantation, and the standard plasmids of each pathogen were used to verify the qPCR reaction.After the primer probe effect and concentration of each pathogen reaction system in this experiment was optimized, the sensitivity, correlation coefficient (R2) and amplification efficiency (E) of qPCR method were analyzed and confirmed.Twenty-two samples from patients, who underwent liver and kidney transplantation in transplant ICU of Sichuan Provincial People′s Hospital, were used to verify the application of the detection system.The total nucleic acid of 100 μL was extracted from each individual and divided into two aliquots, which were detected by multi-link qPCR reaction system and analyzed by high-throughput sequencing method (NGS). At the same time, samples (2 mL each) were taken from the transplanted patients for microbial culture.The results of the three detection methods were compared, and the NGS method was taken as the gold standard to analyze the positive detection rate of the multi-link qPCR method and its difference with the culture method and NGS. 【Results】 The lower limit of qPCR detection for 24 pathogens in the established qPCR detection system was 101cp/μL(R2>0.99), with the positive rate of pathogens at 59.1% (13/22), showing significant difference versus microbial culture (18.2%, 4/22)(P<0.05), but not versus NGS (63.6%, 14/22)(P>0.05). Percentage of pathogens detected was as follows: human herpetic virus type 6 (HHV-6) 30.8% (4/13), cytomegalovirus (HCMV) 23.1% (3/13), Epstein-Barr virus (EBV) 23.1% (3/13), human parvovirus B19 15.4% (2/13), Haemophilus influenzae (Hin) 15.4% (2/13), Enterococcus faecium (EFM) 15.4% (2/13), Clostridium difficile 15.4% (2/13), Escherichia coli 7.7% (1/13), Stenotrophomonas maltophilia (Sma) 7.7% (1/13), Klebsiella pneumoniae (Kpn) 7.7% (1/13), Enterococcus faecalis (Efa) 7.7% (1/13) and Streptococcus pneumoniae (Spn) 7.7% (1/13). The consistency rate of pathogens detected by the three methods was 32% (7/22), among which the consistency rate of multi-link qPCR with NGS method was 59% (13/22), and multi-link qPCR with microbial culture was 41% (9/22). 【Conclusion】 Compared with the microbial culture, the multi-link qPCR method demonstrated high sensitivity, accurate quantification, short time and low cost for the detection of common pathogens in clinical transplantation.Multi-link qPCR combined with NGS and microbial culture is helpful to quickly predict the pathogen infection status of patients after transplantation.
ABSTRACT
Objective:To describe the characteristics of liver damage in severe coronavirus disease 2019 (COVID-19) patients in Sichuan area and the effect of antiviral drugs on liver function.Methods:The clinical data of severe COVID-19 patients admitted to Chengdu Public Health Clinical Medical Center from January 21 to February 24, 2020 were retrospectively collected, including demographic data, clinical manifestations and liver function changes within 1 week after admission to intensive care unit (ICU). The changes of liver function during the course of disease in severe COVID-19 patients were analyzed and summarized, and group analysis was performed.Results:A total of 30 COVID-19 patients with complete clinical data were enrolled. The incidence of severe COVID-19 in elderly men was higher (60.0%), with median age of 61 (47, 79) years old, and those aged 80 or above accounted for 23.3%. The severe COVID-19 patients mainly presented with respiratory symptoms such as fever (96.7%), cough (80.0%) and dyspnea (66.7%). The alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil) and prothrombin time (PT) of 30 patients were increased to various degrees within 1 week after ICU admission, and albumin (ALB) was decreased. ① The patients were divided into two groups according to whether to take lopinavir/ritonavir (kaletra). It was shown that the incidence of liver dysfunction in patients taking kaletra was significantly higher than those who did not take kaletra (7-day abnormal rate of ALT was 54% vs. 33%, the abnormal rate of AST was 38% vs. 33%, the abnormal rate of TBil was 8% vs. 0%), but there were no statistical differences (all P > 0.05). ② The patients were divided into normal dose group (500 mg, twice a day, n = 19) and reduced dose group (250 mg, twice a day, n = 5) according to the dosage of kaletra. It was shown that patients taking low-dose kaletra had a smaller effect on liver function within 1 week after ICU admission than those receiving normal dosage, and ALB, TBil in the reduced dose group were significantly lower than those in the normal dose group on the 2nd day after ICU admission [ALB (g/L): 33.3±2.0 vs. 37.5±4.0, TBil (μmol/L): 6.3±3.3 vs. 11.3±4.8, both P < 0.05]. Conclusions:Severe COVID-19 patients in Sichuan area suffered obvious liver damage in the early course of the disease and have a slower recovery. It is important to pay attention to avoid using drugs that can aggravate liver damage while treating the disease. If there is no alternative drug, liver protection treatment should be considered appropriately.