RESUMEN
【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.
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The transfusion-transmission risk associated with the major transfusion-relevant pathogens, such as hepatitis B virus, hepatitis C virus and human immunodeficiency virus, has been gradually reduced and maintained at a low level. There has been a growing awareness of the threat to blood safety from emerging andre-emerging infectious diseases, a number of which are known to be, such as plasmodium, human parvovirusB19, hepatitis E virus, dengue virus, human T-lymphocytic leukemia virus and West Nile virus, or are potentially transfusion transmissible, such as novel coronavirus. Among them, dengue virus was regional epidemic in China, and may outbreak again unpredictably in the future. Its threat to blood safety cannot be underestimated. At present, there are only four pathogen screening items for routine blood screening in China. Duetoan increasing number of emergingand re-emerging pathogens and the absence of screening strategies for new pathogens in blood centers, it would be a great challenge to blood safety in China. In this review, we documented the prevalence of several pathogens emerged in China, and discussed the impact on blood safety and the viability of personalized screening strategies foremerginginfectiouspathogens in blood centers.
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The postmortem room is a source of potential hazards to the health of forensic experts, pathologists and mortuary technicians. Infectious pathogens in cadavers that present particular risk include mycobacterium tuberculosis, HBV, HCV, HIV and prions that cause transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease (CJD) and gerstmann-strausster-sheinker syndrome (GSS). This review focuses specifically on these hazards associated with the autopsy of infected patients.