A training program for improving the capacity of infection high-throughput sequencing and diagnosis in China.

BACKGROUND: Infectious diseases are a serious threat to human especially since the COVID-19 outbreak has proved the importance and urgency of their diagnosis and treatment again. Metagenomic next-generation sequencing (mNGS) has been widely used and recognized in clinical and carried out localized testing in hospitals. Increasing the training of mNGS detection technicians can enhance their professional quality and more effectively realize the application value of the hospital platform. METHODS: Based on the initial theoretical understanding and practice of the mNGS platform for localization construction, we have designed a training program to enhance the ability of technicians to detect pathogens by utilizing mNGS, and hence to conduct training practices nationwide. RESULTS: Until August 30, 2022, the page views of online classes have reached 51,500 times and 6 of offline small-scale training courses have been conducted. A total of 67 trainees from 67 hospitals have participated in the training with a qualified rate of 100%. After the training course, the localization platform of 1 participating hospital has been put into use, 2 have added the mNGS localization platform for admission, among which 3 have expressed strong intention of localization. CONCLUSIONS: This study focuses on the training procedures and practical experience of the project which is the first systematic standardized program of mNGS in the world. It solves the training difficulties in the current industry, and effectively promotes the localization construction and application of mNGS in hospitals. It has great development potential in the future and is worth further promotion.

Novel Biomarker Panel of Let-7d-5p and MiR-140-5p Can Distinguish Latent Tuberculosis Infection from Active Tuberculosis Patients.

Background: Mycobacterium tuberculosis (Mtb) survives inside a human host for a long time in the form of latent tuberculosis infection (LTBI). Latent infection of tuberculosis has the opportunity of developing into active tuberculosis (ATB), which has greatly endangered human health. The existing diagnostic methods cannot effectively distinguish LTBI from ATB. Therefore, more effective diagnostic biomarkers and methods are urgently needed. Methods: Here, we screened the GEO data set, conducted joint differential analysis and target gene enrichment analysis, after filtering the disease-related database, we screened the differential miRNA related to TB. The qPCR was used to verify the miRNAs in 84 serum samples. Different combinations of biomarkers were evaluated by logistic regression to obtain a biomarker panel with good performance for diagnosing LTBI. Results: A panel with two miRNAs (hsa-let-7d-5p, hsa-miR-140-5p) was established to differentiate LTBI from ATB. Receiver operating characteristic (ROC) curve showed that the area under the curve (AUC) are 0.930 (sensitivity = 100%, specificity = 88.5%) and 0.923 (sensitivity = 100%, specificity = 92.3%) with the biomarker panel for the training set and test set respectively. Conclusion: The findings indicated that the logistic regression model built by let-7d-5p and miR-140-5p has the ability to distinguish LTBI from active TB patients.

Clinical Diagnostic Value of Serum 25-Hydroxyvitamin D in Severe Fever with Thrombocytopenia Syndrome.

Purpose: Severe Fever with Thrombocytopenia Syndrome (SFTS) is an infectious disease with rapid onset and high case fatality rate. The study was to explore the clinical value by examining the serum level of 25-hydroxyvitamin D (25 (OH) D) in SFTS patients. Methods: One hundred and five patients and 156 healthy controls were included. Univariate and multivariate regression analyses were performed to identify independent risk factors for disease progression. Subject operating characteristics (ROC) curves were drawn, and the corresponding area under the curve (AUC) was calculated to assess the sensitivity and specificity of the diagnostic disease. Results: The 25 (OH) D level of disease group was lower than that of healthy control group (22.12 (18.43, 25.86) ng/mL vs 27.36 (23.20, 32.71) ng/mL; P<0.05). The 25 (OH) D level of severe disease group was lower than that of mild disease group (20.55(16.30, 24.44) ng/mL vs 24.94(20.89, 31.91) ng/mL; P<0.05). And there was no significant difference of 25 (OH) D level between the survival group and death group in severe disease group. Multivariate Logistic regression analysis showed that the 25 (OH) D level under 19.665 ng/mL was an independent risk factor for the development of SFTS (OR = 0.901, P=0.040). Furthermore, age more than 68.5 years old and lactate dehydrogenase (LDH) more than 1023.5U/L were independent risk factors for death in severe patients with SFTS. Conclusion: Patients with SFTS have reduced 25 (OH) D level, and 25 (OH) D is a risk factor for disease severity in patients with SFTS. Vitamin D supplementation may be an effective measure to reduce the risk of infection and improve the prognosis.

Analysis of Metagenomic Next-Generation Sequencing Results of 25 Pus Samples.

Purpose: To explore the clinical value of detecting pathogens in pus samples by metagenomic next-generation sequencing (mNGS). Methods: The 25 pus samples from infected patients were collected in this research. The positive rate and consistency of pathogenic bacteria detected by mNGS and conventional methods were compared. The pathogen types detected by the two methods were analyzed. Furthermore, the modifications of antibiotic treatment therapy were also evaluated based on mNGS results. Results: The sensitivity of mNGS method in detecting pathogenic bacteria in pus samples was better than that of conventional method (96% vs 40%; P < 0.01). Only 10 samples were detected pathogens by conventional methods, but 24 samples were detected by mNGS method. In specific, the results of conventional methods showed 10 samples had 11 kinds of pathogenic bacteria, of which 9 samples were single pathogen and 1 sample had two kinds of pathogenic bacteria. The results of mNGS method showed 24 samples were detected with 54 kinds of pathogenic bacteria, of which 15 samples were detected with single pathogen, and 9 samples were detected with two or more kinds of pathogenic bacteria. The two methods had 9(36%) consistent results, 14 (56%) completely different results, and 2 (8%) partially consistent results, and the kappa value was 0.19. Notably, mNGS could detect viruses, anaerobic bacteria, and other uncommon pathogens simultaneously. Conclusion: The application of mNGS in the detection of pus specimens from different parts not only have high accuracy rate and also reduce the turnaround time of diagnosis. In addition, the performance of mNGS detection of anaerobic bacteria and caustic bacteria is better than conventional methods. The mNGS diagnosis in pus sample may play an important role in clinical diagnosis and treatment strategy decisions.

Understanding etiology of community-acquired central nervous system infections using metagenomic next-generation sequencing.

Background: Community-acquired central nervous system infections (CA-CNS infections) have the characteristics of acute onset and rapid progression, and are associated with high levels of morbidity and mortality worldwide. However, there have been only limited studies on the etiology of this infections. Here, metagenomic next-generation sequencing (mNGS), a comprehensive diagnosis method, facilitated us to better understand the etiology of CA-CNS infections. Methods: We conducted a single-center retrospective study between September 2018 and July 2021 in which 606 cerebrospinal fluid (CSF) samples were collected from suspected CNS infectious patients for mNGS testing, and all positive samples were included in this analysis. Results: After the exclusion criteria, a total of 131 mNGS-positive samples were finally enrolled. Bacterial, viral, fungal, parasitic, specific pathogen and mixed infections were accounted for 32.82% (43/131), 13.74% (18/131), 0.76% (1/131), 2.29% (3/131) and 6.87% (9/131), respectively. A total of 41 different pathogens were identified, including 16 bacteria, 12 viruses, 10 fungi, and 1 parasite and 3 specific pathogens. The most frequent infecting pathogens are Epstein-Barr virus (n = 14), Herpes simplex virus 1 (n = 14), Mycobacterium tuberculosis (n = 13), Streptococcus pneumoniae (n = 13), and Cryptococcus neoformans (n = 8). Some difficult-to-diagnose pathogen infections were also detected by mNGS, such as Streptococcus suis, Pseudorabies virus, Bunyavirus, Orientia tsutsugamushi and Toxoplasma gondii. Conclusion: In this study, mNGS identified a wide variety of pathogens of CA-CNS infections and many of which could not be detected by conventional methods. Our data provide a better understanding of the etiology of CA-CNS infections and show that mNGS represents a comparative screening of CSF in an unbiased manner for a broad range of human pathogens.

Gclc overexpression inhibits apoptosis of bone marrow mesenchymal stem cells through the PI3K/AKT/Foxo1 pathway to alleviate inflammation in acute lung injury.

BACKGROUND: Acute lung injury (ALI) represents a serious heterogenous pulmonary disorder with high mortality. Bone marrow mesenchymal stem cells (BMSCs) have a good therapeutic effect on ALI, but their survival rate in vivo is not high. GCLc has all the activities of Glutamate cysteine ligase (GCL) and can reduce reactive oxygen species, antioxidant stress response and improve cell survival. Therefore, in our study, overexpressing GCLc BMSCs were constructed by lentiviral transduction and intratracheally transplanted into ALI mice to evaluate their therapeutic effects, and we explored the mechanism of anti-apoptosis of GCLc in BMSCs. METHODS: Overexpressing GCLc hBMSCs were constructed using lentiviral vectors. The cell viability of MSCs was detected by CCK-8 assay. GSH, MDA, SOD and ROS were detected by the manufacturer's kit. Western blot and RT-qPCR were used to detect the expression of GCLc, bax, bcl2, cleaved-caspase 3, caspase 3, cleaved-caspase 9, caspase 9 and Foxo1 in BMSCs stimulated by H2O2. Apoptosis of BMSCs was analyzed by flow cytometry, JC-1 and TUNEL method. Confocal microscopy was to observe the nuclear extracellular migration of Foxo1. We then examined the expression levels of the pathway proteins by Western blot. In ALI animal model, we evaluated the therapeutic effect of the overexpressing GCLc BMSCs by H&E staining, in vitro imaging, wet/dry weight ratio of lung tissue, and extraction of bronchoalveolar lavage fluid from mice to analyze protein concentrations, neutrophil, leukocyte and macrophage counts and ELISA for inflammatory factors. RESULTS: We demonstrated that overexpression of GCLc reduced MDA and ROS and increased GSH and SOD, while GCLc reduced the expression of pro-apoptotic proteins (bax, cleaved-caspase 3, caspase 3, cleaved-caspase 9, caspase 9) and elevated the expression of anti-apoptotic proteins (bcl-2) in BMSCs. We verified that it acts through the PI3K/AKT/Foxo1 pathway. In ALI vivo, overexpression of GCLc BMSCs had a longer retention time in the lung compared to vector BMSC and improved pulmonary edema, decreased alveolar protein concentration and reduced TNF-α, IL-1ß, IL-6 levels and increased IL-10 levels in the lung. CONCLUSIONS: These results show that GCLc overexpressing BMSCs with anti-apoptotic effects significantly improve acute lung injury.

Detection of Bartonella vinsonii subsp. berkhoffii in an HIV patient using metagenomic Next-Generation Sequencing.

Bartonella species are fastidious, aerobic bacteria that are transmitted by blood-sucking arthropods. Bartonella spp. are responsible for cat scratch disease, Carrion's disease, bacillary angiomatosis and trench fever. On the other hand, Bartonella vinsonii is rarely reported in the literature and there exist a few reports of systemic infection caused by Bartonella vinsonii in patients with acquired immunodeficiency syndrome. A 31-year-old male (diagnosed with AIDS six years ago) had persistent fever and ulceration in the right knee. The elevated levels of inflammatory markers suggested an infectious aetiology. Despite the negative findings of blood culture, metagenomic Next-Generation Sequencing of plasma detected Bartonella vinsonii. The polymerase chain reaction of whole blood and Sanger sequencing confirmed the mNGS findings. Immunohistochemical staining had later suggested bacillary angiomatosis, which was consistent with Bartonella infection. Following antibiotic treatment, the ulcers subsided significantly, but a high fever persisted. The patient died due to sudden respiratory failure.

Fermentation Blues: Analyzing the Microbiota of Traditional Indigo Vat Dyeing in Hunan, China.

Traditional indigo dyeing through anaerobic fermentation has recently gained worldwide attention in efforts to address concerns regarding the sustainability of industrial indigo dyeing and the impact of toxic reducing agents such as sodium dithionite (Na2S2O4) on human health and the ecological environment. Intriguingly, changes in the microbiota during indigo fermentation are known to potently affect the onset of indigo reduction, and thus elucidation of the microbial community transitions could help develop methods to control the initiation of indigo reduction. Here, we investigated the microbiota associated with the traditional indigo dyeing practiced in Hunan, China. Specifically, we identified the bacterial and fungal components of the microbiota at distinct stages in the indigo fermentation process by analyzing 16S rRNA gene and internal transcribed spacer sequences. Our analyses revealed two substantial changes in the microbiota during the traditional indigo fermentation process. The first change, which was probably caused by the introduction of Chinese liquor (featuring a high alcohol concentration), resulted in decreased bacterial diversity and increased proportions of Pseudomonas, Stenotrophomonas, and Bacillaceae family members. The second change, which could be attributed to the addition of specific plant species, led to an increase in the abundance of Alkalibacterium, Amphibacillus, the obligate anaerobe Turicibacter, the facultative anaerobe Enterococcus, and ZOR0006, as well as to a decrease in the pH and redox potential values. Our results indicate that the specific plant mixture included in the procedure here could be used as an effective additive to accelerate the initiation of indigo reduction during the fermentation process. To the best of our knowledge, this is the first report revealing the fungal diversity during the indigo fermentation process and, furthermore, showing that the fungal diversity has remained in transition despite the relatively stable bacterial diversity in the proper indigo fermentation process. Although traditional indigo fermentation in China is challenging to manage, we can benefit from local knowledge of the fermentation process, and understanding the scientific bases of traditional indigo fermentation will facilitate the development of environmentally friendly procedures. IMPORTANCE Chemical reducing agents included in modern indigo dyeing to initiate indigo reduction can be harmful to both human health and the environment. Given that traditional indigo dyeing involves natural fermentation in a dye vat using natural organic additives without the use of toxic chemicals and that changes in the microbiota during traditional indigo fermentation potently affect the onset of indigo reduction, elucidation of these microbial community transitions could help develop methods to control the initiation of indigo reduction. This study on the microbiota associated with the traditional indigo dyeing practiced in Hunan, China, has identified the bacterial and fungal communities at distinct stages of the indigo fermentation process. Notably, the addition of specific plant species might yield the desired microbial communities and appropriate fermentation conditions, which could be used as an effective additive to accelerate the initiation of indigo reduction. This study has also revealed the fungal diversity during the indigo fermentation process for the first time and shown that the fungal diversity has remained in transition despite the relatively stable bacterial diversity. Thus, this work provides new insights into the traditional indigo fermentation process used in China and substantially enhances current efforts devoted to designing environmentally friendly methods for industrial indigo dyeing.

Quality blues: traditional knowledge used for natural indigo identification in southern China.

BACKGROUND: As one of the oldest traditional dyes, people worldwide have used natural indigo for centuries. Local people have unique knowledge about indigo identification, which is crucial for indigo quality control and determining the dyeing effects. However, such traditional knowledge is rarely documented and explained. Therefore, the aims of this study were to document and assess the traditional knowledge used by local people when identifying natural indigo paste as well as quantitatively explore the characteristics and material basis of such traditional knowledge. METHOD: Three field surveys were conducted between 2019 and 2020. A total of 283 traditional indigo-paste artisans were interviewed in Guizhou, Yunnan, and Fujian Provinces. The frequency of citation, mention index, and fidelity level of each indigo-paste quality criterion were calculated to determine the most commonly used, recognized, and important quality criteria. To explore the characteristics and material basis of the traditional knowledge, we analyzed 21 indigo-paste samples using high-performance liquid chromatography with diode-array detection (HPLC-DAD), pH, and particle size analyses. RESULTS: Local people possess unique knowledge to identify natural indigo. Based on this knowledge accumulated over thousands of years, four criteria (color, taste, touch, and dyeing ability) were chosen by local people, and using these criteria, nature indigo was divided into five quality grades. The best quality indigo paste was judged according to the following folk criteria: dark blue in color with a purple-red luster; smooth and difficult to wipe off; having a sweet, bitter or spicy taste; and easy cloth dyeing. Additionally, the higher the contents of indigo and indirubin-especially indirubin-the better is the quality of the indigo paste. Within the pH range of 9-12, high-quality indigo-paste was more acidic. There was no significant relationship between particle size and quality. CONCLUSION: The ancient methods used by local people for identifying natural indigo are comprehensive and unique. By documenting the various folk quality criteria and conducting quantitative analyses, this study revealed the importance of indirubin and pH for assessing the quality of indigo paste. These findings differ from existing quality standards for synthetic indigo. Amid rapid modernization, traditional knowledge remains invaluable as a world heritage of humanity that warrants preservation.

Island blues: indigenous knowledge of indigo-yielding plant species used by Hainan Miao and Li dyers on Hainan Island, China.

BACKGROUND: Historically, indigo-yielding plant species were important cash crops from Central Asia to the southern United States and Central America. Indigo-dyed textiles were widely traded along the legendary Silk Road that linked China to Europe. Today, due to the labor-intensive nature of indigo extraction at the household level, lifestyle changes and the widespread availability of commercially produced indigo paste, traditional indigo extraction methods have declined in villages. Yet Li textile weavers on Hainan Island are internationally recognized as producers of indigo-dyed textile using warp ikat techniques. In contrast, Hainan Miao weavers produce indigo-dyed textiles using batik (wax resist) techniques. The aim of this study was to document the indigenous knowledge on indigo-yielding plant species used by both Hainan Miao and Li people on Hainan Island, China. METHOD: Ethnic uses were documented during three field surveys, through a questionnaire survey of 193 respondents, comprising 144 Hainan Miao and 49 Li traditional dyers. Mention index (QI), Availability index (AI), and Preference ranking (PR) of each indigo-yielding plant species were calculated to screen out plant resources with potential development value. RESULTS: Five indigo-yielding plant species (from four plant families and four genera) were historically used by Hainan Miao and Li dyers. However, just four species are still in use. Strobilanthes cusia was the main indigo source for Hainan Miao dyers. Li dyers also commonly use Indigofera species (I. tinctoria and I. suffruticosa) for indigo extraction. Wrightia laevis is less commonly used as a contemporary indigo source. Indigo extraction by steeping in water to which lime is added to increase the pH is sharing by the five indigo-yielding plant species. Strobilanthes cusia had the highest QI, AI and PR values in Hainan Miao villages. Indigofera tinctoria had the highest QI and AI values, but Indigofera suffruticosa was preferred by Li dyers. CONCLUSION: In the process of modernization and urbanization, some Hainan Miao and Li dyers retain the traditional indigo extraction methods. We found that Strobilanthes cusia and Indigofera tinctoria have the most potential for sustainable indigo production in the future. Furthermore, this study documents the details of extraction method from Wrightia laevis for the first time and the use of Ricinus communis seeds in that process. As one of the last places globally where Wrightia laevis is still used for indigo production, the may also be a nice market among textile collectors and museums that keeps the tradition of Wrightia laevis production and use for indigo extraction alive.