Development and application of a multiplex PCR system for forensic salivary identification.

In forensics, accurate identification of the origin of body fluids is essential for reconstructing a crime scene or presenting strong evidence in court. Microorganisms have demonstrated great potential in body fluid identification. We developed a multiplex PCR system for forensic salivary identification, which contains five types of bacteria:Streptococcus salivarius, Neisseria subflava, Streptococcus. mutans, Bacteroides thetaiotaomicron, and Bacteroides. uniformis. And the validated studies were carried out following the validation guidelines for DNA analysis methods developed by the Scientific Working Group on DNA Analysis Methods (SWGDAM), which included tests for sensitivity, species specificity, repeatability, stability, and mixed samples, trace samples, case samples, and a population study. Our result depicted that the lowest detection limit of the system was 0.01 ng template DNA. Moreover, the corresponding bacteria can still be detected when the amount of saliva input is low to 0.1 µL for DNA extraction. In addition, the target bacteria were not detected in the DNA of human, seven common animals, and seven bacteria DNA and in nine other body fluid samples (skin, semen, blood, menstrual blood, nasal mucus, sweat, tears, urine, and vaginal secretions). Six common inhibitors such as indigo, EDTA, hemoglobin, calcium ions, alcohol and humic acid were well tolerated by the system. What is more, the salivary identification system recognized the saliva component in all mixed samples and simulated case samples. Among 400 unrelated individuals from the Chinese Han population analyzed by this novel system, the detection rates of N. subflava, S. salivarius, and S. mutans were 97.75%, 70.75%, and 19.75%, respectively, with 100% identification of saliva. In conclusion, the salivary identification system has good sensitivity, specificity, stability, and accuracy, which can be a new effective tool for saliva identification.

Integrating the salivary microbiome in the forensic toolkit by 16S rRNA gene: potential application in body fluid identification and biogeographic inference.

Saliva is a common body fluid with significant forensic value used to investigate criminal cases such as murder and assault. In the past, saliva identification often relied on the α-amylase test; however, this method has low specificity and is prone to false positives. Accordingly, forensic researchers have been working to find new specific molecular markers to refine the current saliva identification approach. At present, research on immunological methods, mRNA, microRNA, circRNA, and DNA methylation is still in the exploratory stage, and the application of these markers still has various limitations. It has been established that salivary microorganisms exhibit good specificity and stability. In this study, 16S rDNA sequencing technology was used to sequence the V3-V4 hypervariable regions in saliva samples from five regions to reveal the role of regional location on the heterogeneity in microbial profile information in saliva. Although the relative abundance of salivary flora was affected to a certain extent by geographical factors, the salivary flora of each sample was still dominated by Streptococcus, Neisseria, and Rothia. In addition, the microbial community in the saliva samples in this study was significantly different from that in the vaginal secretions, semen, and skin samples reported in our previous studies. Accordingly, saliva can be distinguished from the other three body fluids and tissues. Moreover, we established a prediction model based on the random forest algorithm that could distinguish saliva between different regions at the genus level even though the model has a certain probability of misjudgment which needs more in-depth research. Overall, the microbial community information in saliva stains might have prospects for potential application in body fluid identification and biogeographic inference.

Response of salivary microbiome to temporal, environmental, and surface characteristics under in vitro exposure.

The microbiome of saliva stains deposited at crime scenes and in everyday settings is valuable for forensic investigations and environmental ecology. However, the dynamics and applications of microbial communities in these saliva stains have not been fully explored. In this study, we analyzed saliva samples that were exposed to indoor conditions for up to 1 year and to different carriers (cotton, sterile absorbent cotton swab, woolen, dacron) in both indoor and outdoor environments for 1 month using high-throughput sequencing. The analysis of microbial composition and Mfuzz clustering showed that the salivary flora, specifically Streptococcus (cluster7), which was associated with microbial contamination, remained stable over short periods of time. However, prolonged exposure led to significant differences due to the invasion of environmental bacteria such as Pseudomonas and Achromobacter. The growth and colonization of environmental flora were promoted by humidity. The neutral model predictions indicated that the assembly of salivary microbial communities in outdoor environments was significantly influenced by stochastic processes, with environmental characteristics having a greater impact on community change compared to surface characteristics. By incorporating data from previous studies on fecal and vaginal secretion microbiology, we developed RF and XGBoost classification models that achieved high accuracy (>98 %) and AUC (>0.8). Additionally, a RF regression model was created to determine the time since deposition (TsD) of the stains. Time inference models yielded a mean absolute error (MAE) of 7.1 days for stains exposed for 1 year and 14.2 h for stains exposed for 14 days. These findings enhance our understanding of the changes in the microbiome of saliva stains over time, in different environments, and on different surfaces. They also have potential applications in assessing potential microbial contamination, identifying body fluids, and inferring the time of deposition.

Changes in the microbial community after vaginal fluid exposure in different simulated forensic situations.

If vaginal fluid is found on clothing or on the body of the suspect, it may indicate the occurrence of sexual assault. Therefore, it is important to collect the victim's vaginal fluid at different sites from the suspect. Previous studies have revealed that fresh vaginal fluids can be identified based on 16S rRNA gene sequencing data. However, the influence of environmental factors on the stability of microbial markers must be investigated before being used in forensic practice. We collected vaginal fluid from nine unrelated individuals and placed each individual of vaginal swab on five different substrates. A total of 54 vaginal swabs were analyzed using 16S rRNA on the V3-V4 regions. Then, we constructed a random forest model including the samples of all vaginal fluids in this study and the other four types of body fluids in our previous studies. The alpha diversity of vaginal samples increased after exposure to the substrate environment for 30 days. The dominant vaginal bacteria were Lactobacillus and Gardnerella, which remained relatively stable after exposure, with Lactobacillus being the most abundant in all substrates, while Gardnerella was more abundant in other substrates than in the polyester fiber substrate. Except for bed sheets, Bifidobacterium significantly declined when placed on other substrates. Rhodococcus and Delftia from the substrate environment migrated to the vaginal samples. Rhodococcus was abundant in polyester fibers, and Delftia was abundant in wool substrates, while those environmental bacteria were all in low abundance in bed sheets. Overall, the bed sheet substrates showed a good retention capacity for the dominant flora and could reduce the number of taxa migrated by the environment compared with the other substrates. Both fresh and exposed vaginal samples of the same individuals could mostly be clustered and clearly distinguished from different individuals, showing the potential of individual identification, and the confusion matrix value of body fluid identification for vaginal samples was 1. In summary, vaginal samples placed on the surface of different substrates retained their stability and demonstrated good application potential for individual and body fluid identification.

Herbal Granules of Heat-Clearing and Detoxifying for Children with Mild Hand, Foot, and Mouth Disease: A Bayesian Network Meta-Analysis.

Background: Regarding ethical considerations of randomized controlled trials (RCTs) in children, limited evidence for mild hand, foot, and mouth disease (HFMD) is available. Recently, with the increasing but result-conflicting RCTs published around herbal granules of heat-clearing and detoxifying (HGs-HD), a head-to-head comparison is urgently needed to choose a suitable therapy for clinical practice. Materials and Methods: This study was conducted according to the preferred reporting items for systematic review and meta-analysis (PRISMA) extension statement for network meta-analysis (NMA). Eight databases (Medline, Embase, and so on) and two trial registry platforms (https://www.clinicaltrials.gov and https://www.chictr.org.cn) were searched from inception to May 26, 2021. The NMA was performed using a random-effect model. The treatment hierarchy was summarized and reported as the surface under the cumulative ranking curve (SUCRA) probability values. The rankings of each HGs-HD at primary outcomes were estimated by the inverse probability weighting (IPW) approach and averaged, which presents the comprehensive improvement effect. Results: Forty-five RCTs involving 18 interventions were included that studied 5,652 children with mild HFMD. The best performance probability for improving symptoms were respectively presented in terms of fever (Xiao'er Resuqing granules, XRGs, 94.9%), rash (Xiao'er Jinqiao granules, 83.9%), hospitalization (Xiao'er Chiqiao Qingre granules, XCQGs, 92.7%), vesicles (Jinlianhua granules, 91.0%), appetite (Xiao'er Chiqiao Qingre granules, XCQGs, 86.7%), and ulcers (Kouyanqing granules, KouGs, 88.8%). Furthermore, the top 5 rankings for comprehensive improvement effect were Yanning granules (YNGs, 2.256), XCQGs (2.858), XRGs (3.270), KouGs (7.223), and Houerhuan Xiaoyan granules (HXGs, 7.597). Conclusions: This is the first NMA of HGs-HD head-to-head comparisons for children with mild HFMD. Of those, YNGs, XCQGs, XRGs, KouGs, and HXGs could be recommended as potential choices for clinical practice. Of course, the results should be interpreted with caution due to the limited high-quality RCTs.