Establishment of PCR assays and genetic polymorphism analysis of genes encoding Clostridium perfringens β2 toxin from different sources / 中华流行病学杂志

Objective: To establish and optimize PCR methods for the gene encoding of Clostridium perfringens β2 toxin (cpb2) and atypical-cpb2 (aty-cpb2), analyze the epidemiological characteristics and genetic polymorphism of the cpb2 of Clostridium perfringens in 9 Chinese areas from 2016 to 2021. Methods: The cpb2 of 188 Clostridium perfringens strains were examined by PCR; the cpb2 sequences were acquired by whole-genome sequencing to analyze the genetic polymorphism. Using Mega 11 and the Makeblastdb tool, a phylogenetic tree, and cpb2-library based on 110 strains carrying the cpb2 were produced. Using the Blastn technique, a comparison was made to discover sequence similarity between consensus-cpb2 (con-cpb2) and aty-cpb2. Results: The specificity of PCR assay for the cpb2 and aty-cpb2 was verified. The PCR results for cpb2 amplification were highly consistent with the whole-genome sequencing approach (Kappa=0.946, P<0.001). A total of 107 strains from nine regions in China carried cpb2, 94 types A strains carried aty-cpb2, 6 types A strains carried con-cpb2, and 7 types F strains carried aty-cpb2. The nucleotide sequence similarity between the two coding genes was 68.97%-70.97%, and the similarity between the same coding genes was 98.00%-100.00%. Conclusions: In this study, a specific PCR method for cpb2 toxin was developed, and the previous PCR method for detecting aty-cpb2 was improved. aty-cpb2 is the primary gene encoding of β2 toxin. There is a significant nucleotide sequence variance between the various cpb2 genotypes.

Study on the features of Clostridioides difficile infection among diarrhea patients in Kunming from 2018 to 2020 / 中华流行病学杂志

Objective: We analyze the characteristics of Clostridioides difficile (C. difficile) infection among diarrhea patients in Kunming from 2018 to 2020 and provide evidence for follow-up surveillance and prevention. Methods: A total of 388 fecal samples of diarrhea patients from four sentinel hospitals in Yunnan Province from 2018 to 2020 were collected. Real-time quantitative PCR was used to detect the fecal toxin genes of C. difficile. The positive fecal samples isolated the bacteria, and isolates were identified by mass spectrometry. The genomic DNA of the strains was extracted for multi-locus sequence typing (MLST). The fecal toxin, strain isolation, and clinical patient characteristics, including co-infection with other pathogens, were analyzed. Results: Among the 388 fecal samples, 47 samples with positive reference genes of C. difficile were positive, with a total positive rate of 12.11%. There were 4 (8.51%) non-toxigenic and 43 (91.49%) toxigenic ones. A total of 18 strains C. difficile were isolated from 47 positive specimens, and the isolation rate of positive specimens was 38.30%. Among them, 14 strains were positive for tcdA, tcdB, tcdC, tcdR, and tcdE. All 18 strains of C. difficile were negative for binary toxins. The MLST results showed 10 sequence types (ST), including 5 strains of ST37, accounting for 27.78%; 2 strains of ST129, ST3, ST54, and ST2, respectively; and 1 strain of ST35, ST532, ST48, ST27, and ST39, respectively. Fecal toxin gene positive (tcdB+) results were statistically associated with the patient's age group and with or without fever before the visit; positive isolates were only statistically associated with the patient's age group. In addition, some C. difficile patients have co-infection with other diarrhea-related viruses. Conclusions: The infection of C. difficile in diarrhea patients in Kunming is mostly toxigenic strains, and the high diversity of strains was identified using the MLST method. Therefore, the surveillance and prevention of C. difficile should be strengthened.

Polymorphism analysis of virulence-related genes among Candida tropicalis isolates / 中华医学杂志(英文版)

BACKGROUND@#Adhesion, biofilm formation, yeast-hyphal transition, secretion of enzymes, and hemolytic activity are all considered important factors in Candida tropicalis infection. However, DNA sequence data for this pathogen are limited. In this study, the polymorphism and heterogeneity of genes agglutinin-like sequences (ALS)2, Lipase (LIP)1, LIP4, and secretory aspartyl proteinase tropicalis (SAPT)1-4 as well as the relationship between phenotype and genotype were analyzed.@*METHODS@#This study started in August 2013, and ended in July 2017. The complete length of ALS2, LIP1, LIP4, and SAPT1-4 of 68 clinical C. tropicalis isolates was sequenced. Single nucleotide polymorphisms (SNPs) as well as insertions and deletions (indels) were identified within these genes. In addition, phenotypic characteristics of the virulent factors, including adhesion and the secretion of aspartyl proteinases and phospholipases, were determined.@*RESULTS@#There were 73, 24, 17, 16, 13, and 180 SNPs in the genes LIP1, LIP4, SAPT1, SAPT2, SAPT3, and SAPT4, respectively. Furthermore, 209 SNPs were identified in total for the gene ALS2. Interestingly, large fragment deletions and insertions were also found in ALS2. Isolate FXCT 01 obtained from blood had deletions on all 4 sites and showed the lowest adhesion ability on the polymethylpentene surface. In addition, isolates with deletions in the regions 1697 to 1925 and 2073 to 2272 bp displayed relatively low abilities for adhesion and biofilm formation, and this phenotype correlated with the deletions found in ALS2. LIP1, SAPT4, and ALS2 displayed great heterogeneity among the isolates. Large deletions found in gene ALS2 appeared to be associated with the low ability of adhesion and biofilm formation of C. tropicalis.@*CONCLUSION@#This study might be useful for deeper explorations of gene function and studying the virulent mechanisms of C. tropicalis.

A New High-throughput Real-time PCR Assay for the Screening of Multiple Antimicrobial Resistance Genes in Broiler Fecal Samples from China / 生物医学与环境科学（英文）

OBJECTIVE@#Antimicrobial resistance (AMR) has become a global concern and is especially severe in China. To effectively and reliably provide AMR data, we developed a new high-throughput real-time PCR assay based on microfluidic dynamic technology, and screened multiple AMR genes in broiler fecal samples.@*METHODS@#A high-throughput real-time PCR system with an new designed integrated fluidic circuit assay were performed AMR gene detection. A total of 273 broiler fecal samples collected from two geographically separated farms were screened AMR genes.@*RESULTS@#The new assay with limits of detection ranging from 40.9 to 8,000 copies/reaction. The sensitivity rate, specificity rate, positive predictive value, negative predictive value and correct indices were 99.30%, 98.08%, 95.31%, 99.79%, and 0.9755, respectively. Utilizing this assay, we demonstrate that AMR genes are widely spread, with positive detection rates ranging from 0 to 97.07% in 273 broiler fecal samples. blaCTX-M, blaTEM, mcr-1, fexA, cfr, optrA, and intI1 showed over 80% prevalence. The dissemination of AMR genes was distinct between the two farms.@*CONCLUSION@#We successfully established a new high-throughput real-time PCR assay applicable to AMR gene surveillance from fecal samples. The widespread existence of AMR genes detected in broiler farms highlights the current and severe problem of AMR.

Progress in Application of Measuring Skeleton by CT in Forensic Anthropology Research / 法医学杂志

Individual identification by measuring the human skeleton is an important research in the field of forensic anthropology. Computed tomography （CT） technology can provide high-resolution image of skeleton. Skeleton image can be reformed by software in the post-processing workstation. Different skeleton measurement indexes of anthropology, such as diameter, angle, area and volume, can be measured on section and reformative images. Measurement process is barely affected by human factors. This paper reviews the literatures at home and abroad about the application of measuring skeleton by CT in forensic anthropology research for individual identification in four aspects, including sex determination, height infer, facial soft tissue thickness measurement and age estimation. The major technology and the application of CT in forensic anthropology research are compared and discussed, respectively.

Correlation of bilateral testicular volume discrepancy with semen parameters in men with varicocele / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the influence of varicocele on the volume discrepancy of bilateral testes, and the relationship between testicular volume discrepancy and semen parameters.</p><p><b>METHODS</b>This study included 181 varicocele patients and 102 normal fertile men without varicocele. We retrospectively analyzed their clinical data, including the grades and locations of varicocele, testis volume and semen parameters.</p><p><b>RESULTS</b>Bilateral testicular volume discrepancy was found in 132 (72.9%) of the varicocele patients (including 117 cases of left testicular hypotrophy [88.6%]), and 35 (34.3%) of the non-varicocele fertile men. The rates of bilateral testicular volume discrepancy were 61.3%, 3.5%, 20.9% and 14.3% in the grade-III, grade-II, grade-I and non-varicocele groups, respectively (P < 0.05), with statistically significant differences among different age groups (P < 0.05). The percentage of morphologically normal sperm and sperm motility were reduced differently with different degrees of testicular volume discrepancy (P <0.05).</p><p><b>CONCLUSION</b>Testicular volume discrepancy is more common in men with left varicocele, and its prevalence and degree are correlated with the grade of varicocele. Semen quality decreases with the increase of testicular volume discrepancy.</p>