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At present, many infectious pathogens, especially emerging/re-emerging pathogens, exist in the blood of voluntary blood donors and may be transmitted through blood transfusions. However, most of Chinese blood centers only routinely screen for HBV, HCV, HIV, and syphilis. We employed metagenomic next-generation sequencing (mNGS) to investigate the microbiome in healthy voluntary blood donors to help assess blood safety in China by identifying infectious pathogens presented in donations that could lead to transfusion-acquired infections. We collected 10,720 plasma samples from voluntary blood donors from seven blood centers in different cities during 2012-2018 in China. A total of 562 GB of clean data was obtained. By analyzing the sequencing data, it was found that the most commonly identified bacteria found in the healthy blood were Serratia spp. (5.0176%), Pseudomonas spp. (0.6637%), and Burkholderia spp. (0.5544%). The principal eukaryote were Leishmania spp (1.3723%), Toxoplasma gondii (0.6352%), and Candida dubliniensis (0.1848%). Among viruses, Human Parvovirus B19 (B19V) accounts for the highest proportion (0.1490%), followed by Torque teno midi virus (0.0032%) and Torque teno virus (0.0015%). Since that B19V is a non-negligible threat to blood safety, we evaluated the positive samples for B19V tested by mNGS using quantitative polymerase chain reaction, Sanger sequencing, and phylogenetic analysis to achieve a better understanding of B19V in Chinese blood donors. Subsequently, 9 (0.07%) donations were positive for B19V DNA. The quantitative DNA levels ranged from 5.58 × 102 to 7.24 × 104 IU/ml. The phylogenic analyses showed that prevalent genotypes belonged to the B19-1A subtype, which disclosed previously unknown regional variability in the B19V positivity rate. The investigation revealed that many microbes dwell in the blood of healthy donors, including some pathogens that may be dormant in the blood and only cause disease under specific conditions. Thus, investigating the range and nature of potential pathogens in the qualified donations provided a framework for targeted interventions to help prevent emerging and re-emerging infectious diseases.
RESUMO
【Objective】 To explore the influence of common methods of reducing non-viral nucleic acid on the abundance of plasma virus group. 【Methods】 Three kinds of library construction, five kinds of centrifugation conditions, two kinds of filters, four kinds of enzymes and four concentrations of chloroform were used to treat plasma samples added quantitatively 2.16 mL of pseudorabies virus(PRV) and 2.16 mL of porcine parvovirus(PPV). A total of 21.6 mL of plasma samples were processed, including 54 samples. Subsequently, nucleic acid was extracted, mitochondrial DNA(mtDNA) and two viruses were quantitated, the library of the next generation sequencing was constructed, Illumina NovaSeq 6000 was used for the next generation sequencing. The sequencing data were compared with Kraken Py 2.0 software, and the species annotation analysis was conducted. The corresponding species classification information of each segment was obtained to analyze the impact of different reducing non-viral nucleic acid methods on the relative abundance of microorganisms and two indicator viruses. 【Results】 After sequencing by Illumina NovaSeq 6000, 306.27 GB raw data and 193.17 GB clean data were obtained, with Q20>90%, Q30>85%, Error Rate of 0.03%, and average GC Content of 45.02%. The DNA library construction process significantly increased the proportion of microbial sequences and the PRV abundance [(91.8±0.5)%](P<0.05); RNA library construction and combined library construction can increase the abundance of Pestivirus, an RNA virus, and the PRV abundance was(17.7±3.3)% and(8.1±1.5)% respectively. The Ct value of mtDNA was increased and the proportion of human sequence decreased to less than(89.5±1)%, while the proportion of microbial sequence increased to (2.4±0.03)% after treatment of five centrifugation conditions(P<0.05); After centrifugation at 4℃, 100 g, 30 min, the PRV abundance was increased to (40.6±6)%, and centrifugation at 4℃, 4 000 g, 45 min reduced the PRV abundance to (4.1±0.01)%(P<0.05). Both of 0.22-μm filter and 0.45-μm filter increased the Ct value of mtDNA to above 25.56±0.13, decreased the proportion of human sequence to less than (86.1±0.6)%, increased the proportion of microbial sequence to (3.1±0.1)% and (3.4±0.2)%, and decreased the PRV abundance to (1.6±0.3)% and (4.1±0.7)%(P<0.05), while there was no statistical difference in the effect on PPV concentration and abundance. DNase Ⅰ and Benzonase increased the Ct value of PPV to 25.65±0.06 and 25.36±0.45, decreased the proportion of human sequence to (81.7±5.6)% and (72.8±6.7)%, and increased the proportion of microbial sequence and PRV abundance to (11.0±4.1)% and (16.1±4.7)%, (55.8±2.3)% and (39.0±8.9)%, respectively(P<0 05); After treatment with RNase A, the Ct value of PRV increased to 25.20±0.11, and the human sequence proportion decreased to (85.4±5.6)%(P<0 05); Lysozyme had no effect on removing non-viral nucleic acid. The chloroform of 1%, 5%, 10% and 20% increased Ct value of PRV and mtDNA to no less than 27.17±0.21 and 25.68±0.04; Only 10% chloroform increased the proportion of microbial sequences to (3.1±1.2)%(P<0.05); The abundance of PRV with 1% and 5% chloroform treatment was increased to (48.7±13.3)% and (42.1±5.5)%(P<0.05), while 10% and 20% chloroform reduced PRV abundance to (1.0±0.5)% and (3.4±2.8)%(P<0.05). There was no statistical difference in the effect of chloroform with four contents on PPV abundance. 【Conclusion】 Centrifugation at 4℃, 5 000 g, 10 min is suitable for increasing the overall abundance of virus, and centrifugation at 4℃, 100 g, 30 min is suitable for increasing the content of virus similar to PRV. 0.45-μm filter, DNase Ⅰ, Benzonase and low concentration chloroform can effectively reduce the proportion of non-viral nucleic acid sequence in plasma to increase the abundance of the indicated virus group. Thus, the enrichment effect of plasma meta-virome is closely related to the nature of the virus, and the appropriate virus enrichment method should be selected according to the research purpose to establish the corresponding enrichment strategy.
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【Objective】 To explore the composition of culturable bacteria in platelets through bacterial culturomics and verify the results of culturomics and metagenomics to improve the detection rate of bacteria in platelets. 【Methods】 Platelet samples from 6 healthy people were collected. Eight kinds of culture media were placed in aerobic conditions and 12 kinds of culture media were placed in anaerobic conditions for large-scale culture and isolation of bacteria in platelets. The isolated single colony was identified by 16S rRNA gene sequencing. The bacterial abundance of healthy human platelet microbiome was analyzed by metagenomic sequencing, and the cultivable bacterial species in platelets was confirmed based on metagenomic and culturomics results. 【Results】 A total of 90 strains of bacteria belonging to 3 phylums, 5 classes, 5 orders, 7 families, 9 genus and 23 species were isolated from 6 platelet samples by culturomics. Among them, the strains with more monoclonal clones at the species level were Brevundimonas aurantiaca (16.7%), Bacillus sp. Y1 (15.6%), Cutibacterium acnes (14.4%) and Brevibacillus brevis (13.3%). The platelet samples sequenced by mNGS showed that the abundance values of Proteobacteria, Firmicutes and Actinobacteria were high. The bacteria detected by both culturomics and metagenomic sequencing methods were as follows: Firmicutes: Bacillus sp. Y1, B. thuringiensis, B. cereus, B. mobilis, B. velezensis, Staphylococcus epidermidis, and Brevibacillus brevis; Actinobacteria: Cutibacterium acnes; Proteobacteria: Escherichia coli and Delftia tsuruhatensis. 【Conclusion】 The mutual validation of culturomics and metagenomics has identified some bacteria, proving that bacteria exist in platelets.
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【Objective】 To investigate the distribution of Hepatitis B virus(HBV)genotypes and the genetic characteristics of genotype B HBV populations among voluntary blood donors in five regions of China. 【Methods】 A total number of 630 plasma samples from blood donors with positive HBV HBsAg neutralization test from 2014 to 2016 in Guangxi Blood Center, Chongqing Blood Center, Urumqi Blood Center, Mianyang Central Blood Station and Luoyang Central Blood Station were collected. The S-region sequence of the HBV genome was amplified by semi-nested PCR and followed with Sanger sequencing in order to investigate the HBV genotype distribution and population genetics. 【Results】 Among the voluntary blood donors in five regions, 55 cases of HBV S gene fragments were successfully amplified. Three genotypes were found in HBV typing, including 46 cases of type B(83.64%), 7 cases of type C(12.73%) and 2 cases of type D(3.63%). There were 15 cases of type B and 3 cases of type C in Guangxi; 10 cases of type B and 1 case of type C in Chongqing; 3 cases of type B, 1 case of type C and 1 case of type D in Luoyang; 15 cases of type B in Mianyang; 3 cases of type B, 2 cases of type C and 1 case of type D in Urumqi. The mismatch distribution of the HBVB type population with the largest population number showed a unimodal distribution, and the results of Tajima′s D test and Fu′s Fs test were both negative, indicating that the HBV population in these five regions was expanding. 【Conclusion】 The prevalent genotype of HBV in voluntary blood donors is type B, and the type B HBV population is experiencing a slow expansion, which should attract our attention. In the future, a thorough molecular epidemiological investigation of HBV should be carried out to ensure blood safety.
