Hepatitis C virus subtype diversity and transmission clusters characteristics among drug users in Zhuhai, South China.

BACKGROUND: Hepatitis C virus (HCV) infection poses a major public health challenge globally, especially among injecting drug users. China has the world's largest burden of HCV infections. However, little is known about the characteristics of transmission networks among drug user populations. This study aims to investigate the molecular epidemiology and transmission characteristics of HCV infections among drug users in Zhuhai, a bustling port city connecting Mainland China and its Special Administrative Regions. METHODS: Participants enrolled in this study were drug users incarcerated at Zhuhai's drug rehabilitation center in 2015. Their sociodemographic and behavioral information, including gender, promiscuity, drug use method, and so forth, was collected using a standardized questionnaire. Plasmas separated from venous blood were analyzed for HCV infection through ELISA and RT-PCR methods to detect anti-HCV antibodies and HCV RNA. The 5'UTR fragment of the HCV genome was amplified and further sequenced for subtype identifications and phylogenetic analysis. The phylogenetic tree was inferred using the Maximum Likelihood method based on the Tamura-Nei model, and the transmission cluster network was constructed using Cytoscape3.8.0 software with a threshold of 0.015. Binary logistic regression models were employed to assess the factors associated with HCV infection. RESULTS: The overall prevalence of HCV infection among drug users was 44.37%, with approximately 19.69% appearing to clear the HCV virus successfully. Binary logistic regression analysis revealed that those aged over 40, engaging in injecting drug use, and being native residents were at heightened risk for HCV infection among drug user cohorts. The predominant HCV subtypes circulating among those drug users were 6a (60.26%), followed by 3b (16.7%), 3a (12.8%), 1b (6.41%) and 1a (3.85%), respectively. Molecular transmission network analysis unveiled the presence of six transmission clusters, with the largest propagation cluster consisting of 41 individuals infected with HCV subtype 6a. Furthermore, distinct transmission clusters involved eight individuals infected with subtype 3b and seven with subtype 3a were also observed. CONCLUSION: The genetic transmission networks revealed a complex transmission pattern among drug users in Zhuhai, emphasizing the imperative for a targeted and effective intervention strategy to mitigate HCV dissemination. These insights are pivotal for shaping future national policies on HCV screening, treatment, and prevention in port cities.

Bioinspired Hierarchical Multi-Protective Membrane for Extreme Environments via Co-Electrospinning-Electrospray Strategy.

Extreme environments can cause severe harm to human health, and even threaten life safety. Lightweight, breathable clothing with multi-protective functions would be of great application value. However, integrating multi-protective functions into nanofibers in a facile way remains a great challenge. Here, a one-step co-electrospinning-electrospray strategy is developed to fabricate a superhydrophobic multi-protective membrane (S-MPM). The water contact angle of S-MPM can reach up to 164.3°. More importantly, S-MPM can resist the skin temperature drop (11.2 °C) or increase (17.2 °C) caused by 0 °C cold or 70 °C hot compared with pure electrospun membrane. In the cold climate (-5 °C), the anti-icing time of the S-MPM is extended by 2.52 times, while the deicing time is only 1.45 s due to the great photothermal effect. In a fire disaster situation, the total heat release and peak heat release rate values of flame retarded S-MPM drop sharply by 24.2% and 69.3%, respectively. The S-MPM will serve as the last line of defense for the human body and has the potential to trigger a revolution in the practical application of next-generation functional clothing.

Electrosprayed Environment-Friendly Dry Triode-Like Facial Masks for Skincare.

The cosmetics industry has a worrying impact on the environment, including the plastics used in products and packaging and environmentally unfriendly additives. In this study, we present an environment-friendly triode-like facial mask (TFM) that utilizes only green and degradable raw materials, nontoxic and harmless solvents, and electric energy to achieve distinct switchable directional water transport properties, avoids a wet storage environment, and reduces excessive packaging. The TFM demonstrates droplet stability when not in contact with the skin while facilitating rapid liquid transfer (15 µL) within durations of 2.8 s (dry skin) and 1.9 s (moist skin) upon contact. We elucidate the underlying mechanism behind this triode-like behavior, emphasizing the synergistic interaction of the wettability gradient, Gibbs pinning, and additional circumferential capillary force. Moreover, the TFM exhibits a reduction in the proportion of aging cells, decreasing from 44.33 to 13.75%, while simultaneously providing antibacterial and skin-beautifying effects. The TFM brings a novel experience while also holding the potential to reduce environmental pollution in the production, packaging, use, and recycling of cosmetics products.

The global transcriptomes of Salmonella enterica serovars Gallinarum, Dublin and Enteritidis in the avian host.

Salmonella enterica serovar Gallinarum causes Fowl Typhoid in poultry, and it is host specific to avian species. The reasons why S. Gallinarum is restricted to avians, and at the same time predominately cause systemic infections in these hosts, are unknown. In the current study, we developed a surgical approach to study gene expression inside the peritoneal cavity of hens to shed light on this. Strains of the host specific S. Gallinarum, the cattle-adapted S. Dublin and the broad host range serovar, S. Enteritidis, were enclosed in semi-permeable tubes and surgically placed for 4 h in the peritoneal cavity of hens and for control in a minimal medium at 41.2 °C. Global gene-expression under these conditions was compared between serovars using tiled-micro arrays with probes representing the genome of S. Typhimurium, S. Dublin and S. Gallinarum. Among other genes, genes of SPI-13, SPI-14 and the macrophage survival gene mig-14 were specifically up-regulated in the host specific serovar, S. Gallinarum, and further studies into the role of these genes in host specific infection are highly indicated. Analysis of pathways and GO-terms, which were enriched in the host specific S. Gallinarum without being enriched in the two other serovars indicated that host specificity was characterized by a metabolic fine-tuning as well as unique expression of virulence associated pathways. The cattle adapted serovar S. Dublin differed from the two other serovars by a lack of up-regulation of genes encoded in the virulence associated pathogenicity island 2, and this may explain the inability of this serovar to cause disease in poultry.

Mycobacterium tuberculosis hijacks host TRIM21- and NCOA4-dependent ferritinophagy to enhance intracellular growth.

Ferritin, a key regulator of iron homeostasis in macrophages, has been reported to confer host defenses against Mycobacterium tuberculosis (Mtb) infection. Nuclear receptor coactivator 4 (NCOA4) was recently identified as a cargo receptor in ferritin degradation. Here, we show that Mtb infection enhanced NCOA4-mediated ferritin degradation in macrophages, which in turn increased the bioavailability of iron to intracellular Mtb and therefore promoted bacterial growth. Of clinical relevance, the upregulation of FTH1 in macrophages was associated with tuberculosis (TB) disease progression in humans. Mechanistically, Mtb infection enhanced NCOA4-mediated ferritin degradation through p38/AKT1- and TRIM21-mediated proteasomal degradation of HERC2, an E3 ligase of NCOA4. Finally, we confirmed that NCOA4 deficiency in myeloid cells expedites the clearance of Mtb infection in a murine model. Together, our findings revealed a strategy by which Mtb hijacks host ferritin metabolism for its own intracellular survival. Therefore, this represents a potential target for host-directed therapy against tuberculosis.

Construction of a risk model and deep learning network based on patients with active pulmonary tuberculosis and pulmonary inflammation.

Most patients with active pulmonary tuberculosis (TB) are difficult to be differentiated from pneumonia (PN), especially those with acid-fast bacillus smear-negative (AFB-) and interferon-γ release assay-positive (IGRA+) results. Thus, the aim of the present study was to develop a risk model of low-cost and rapid test for the diagnosis of AFB- IGRA+ TB from PN. A total of 41 laboratory variables of 204 AFB- IGRA+ TB and 156 PN participants were retrospectively analyzed. Candidate variables were identified by t-statistic test and univariate logistic model. The logistic regression analysis was used to construct the multivariate risk model and nomogram with internal and external validation. A total of 13 statistically differential variables were compared between AFB- IGRA+ TB and PN by false discovery rate (FDR) and odds ratio (OR). By integrating five variables, including age, uric acid (UA), albumin (ALB), hemoglobin (Hb) and white blood cell counts (WBC), a multivariate risk model with a concordance index (C-index) of 0.7 (95% CI: 0.61, 0.8) was constructed. The nomogram showed that UA and Hb acted as protective factors with an OR <1, while age, WBC and ALB were risk factors for TB occurrence. Internal and external validation revealed that nomogram prediction was consistent with the actual observations. Collectively, it was revealed that an integration of five biomarkers (age, UA, ALB, Hb and WBC) may be used to quickly predict TB in AFB- IGRA+ clinical samples from PN.

A Digital Microfluidic RT-qPCR Platform for Multiple Detections of Respiratory Pathogens.

The coronavirus disease 2019 pandemic has spread worldwide and caused more than six million deaths globally. Therefore, a timely and accurate diagnosis method is of pivotal importance for controlling the dissemination and expansions. Nucleic acid detection by the reverse transcription-polymerase chain reaction (RT-PCR) method generally requires centralized diagnosis laboratories and skilled operators, significantly restricting its use in rural areas and field settings. The digital microfluidic (DMF) technique provides a better option for simultaneous detections of multiple pathogens with fewer specimens and easy operation. In this study, we developed a novel digital microfluidic RT-qPCR platform for multiple detections of respiratory pathogens. This method can simultaneously detect eleven respiratory pathogens, namely, mycoplasma pneumoniae (MP), chlamydophila pneumoniae (CP), streptococcus pneumoniae (SP), human respiratory syncytial virus A (RSVA), human adenovirus (ADV), human coronavirus (HKU1), human coronavirus 229E (HCoV-229E), human metapneumovirus (HMPV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (FLUA) and influenza B virus (FLUB). The diagnostic performance was evaluated using positive plasmids samples and clinical specimens compared with off-chip individual RT-PCR testing. The results showed that the limit of detections was around 12 to 150 copies per test. The true positive rate, true negative rate, positive predictive value, negative predictive value, and accuracy of DMF on-chip method were 93.33%, 100%, 100%, 99.56%, and 99.85%, respectively, as validated by the off-chip RT-qPCR counterpart. Collectively, this study reported a cost-effective, high sensitivity and specificity on-chip DMF RT-qPCR system for detecting multiple respiratory pathogens, which will greatly contribute to timely and effective clinical management of respiratory infections in medical resource-limited settings.

Evaluations of the serological test in the diagnosis of 2019 novel coronavirus (SARS-CoV-2) infections during the COVID-19 outbreak.

We developed a chemiluminescence immunoassay method based on the recombinant nucleocapsid antigen and assessed its performance for the clinical diagnosis of severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infections by detecting SARS-CoV-2-specific IgM and IgG antibodies in patients. Full-length recombinant nucleocapsid antigen and tosyl magnetic beads were used to develop the chemiluminescence immunoassay approach. Plasmas from 29 healthy cohorts, 51 tuberculosis patients, and 79 confirmed SARS-CoV-2 patients were employed to evaluate the chemiluminescence immunoassay method performance for the clinical diagnosis of SARS-CoV-2 infections. A commercial ELISA kit (Darui Biotech, China) using the same nucleocapsid antigen was used for the in-parallel comparison with our chemiluminescence immunoassay method. The IgM and IgG manner of testing in the chemiluminescence immunoassay method showed a sensitivity and specificity of 60.76% (95% CI 49.1 to 71.6) and 92.25% (95% CI 83.4 to 97.2) and 82.28% (95% CI 72.1 to 90.0) and 97.5% (95% CI 91.3 to 99.7), respectively. Higher sensitivity and specificity were observed in the chemiluminescence immunoassay method compared with the Darui Biotech ELISA kit. The developed high sensitivity and specificity chemiluminescence immunoassay IgG testing method combined with the RT-PCR approach can improve the clinical diagnosis for SARS-CoV-2 infections and thus contribute to the control of COVID-19 expansion.

Interaction Differences of the Avian Host-Specific Salmonella enterica Serovar Gallinarum, the Host-Generalist S. Typhimurium, and the Cattle Host-Adapted S. Dublin with Chicken Primary Macrophage.

Most Salmonella serovars cause disease in many host species, while a few serovars have evolved to be host specific. Very little is known about the mechanisms that contribute to Salmonella host specificity. We compared the interactions between chicken primary macrophages (CDPM) and host-generalist serovar Salmonella enterica serovar Typhimurium, host-adapted Salmonella enterica serovar Dublin, and avian host-specific Salmonella enterica serovar Gallinarum. S Gallinarum was taken up in lower numbers by CDPM than S Typhimurium and S Dublin; however, a higher survival rate was observed for this serovar. In addition, S Typhimurium and S Dublin caused substantially higher levels of cell death to the CDPM, while significantly higher concentrations of NO were produced by S Gallinarum-infected cells. Global transcriptome analysis performed 2 h postinfection showed that S Gallinarum infection triggered a more comprehensive response in CDPM with 1,114 differentially expressed genes (DEGs) compared to the responses of S Typhimurium (625 DEGs) and S Dublin (656 DEGs). Comparable levels of proinflammation responses were observed in CDPM infected by these three different serovars at the initial infection phase, but a substantially quicker reduction in levels of interleukin-1ß (IL-1ß), CXCLi1, and CXCLi2 gene expression was detected in the S Gallinarum-infected macrophages than that of two other groups as infections proceeded. KEGG cluster analysis for unique DEGs after S Gallinarum infection showed that the JAK-STAT signaling pathway was top enriched, indicating a specific role for this pathway in response to S Gallinarum infection of CDPM. Together, these findings provide new insights into the interaction between Salmonella and the host and increase our understanding of S Gallinarum host specificity.

The SPI-19 encoded type-six secretion-systems (T6SS) of Salmonella enterica serovars Gallinarum and Dublin play different roles during infection.

Salmonella Pathogenicity Islands 19 (SPI19) encodes a type VI secretion system (T6SS). SPI19 is only present in few serovars of S. enterica, including the host-adapted serovar S. Dublin and the host-specific serovar S. Gallinarum. The role of the SPI19 encoded T6SS in virulence in these serovar is not fully understood. Here we show that during infection of mice, a SPI19/T6SS deleted strain of S. Dublin 2229 was less virulent than the wild type strain after oral challenge, but not after IP challenge. The mutant strain also competed significantly poorer than the wild type strain when co-cultured with strains of E. coli, suggesting that this T6SS plays a role in pathogenicity by killing competing bacteria in the intestine. No significant difference was found between wild type S. Gallinarum G9 and its ΔSPI19/T6SS mutant in infection, whether chicken were challenged orally or by the IP route, and the S. Gallinarum G9 ΔSPI19/T6SS strain competed equally well as the wild type strain against strains of E. coli. However, contrary to what was observed with S. Dublin, the wild type G9 strains was significantly more cytotoxic to monocyte derived primary macrophages from hens than the mutant, suggesting that SPI19/T6SS in S. Gallinarum mediates killing of eukaryotic cells. The lack of significant importance of SPI19/T6SS after oral and systemic challenge of chicken was confirmed by knocking out SPI19 in a second strain, J91. Together the results suggest that the T6SS encoded from SPI19 have different roles in the two serovars and that it is a virulence-factor after oral challenge of mice in S. Dublin, while we cannot confirm previous results that SPI19/T6SS influence virulence significantly in S. Gallinarum.

Dynamics and Outcome of Macrophage Interaction Between Salmonella Gallinarum, Salmonella Typhimurium, and Salmonella Dublin and Macrophages From Chicken and Cattle.

Salmonella Gallinarum only infects avian species, where it causes a severe systemic infection in birds of all ages. It is generally accepted that interaction with phagocytic cells plays an important role in the development of systemic, host-specific Salmonella infections. The current study detailed the interaction of S. Gallinarum with macrophages derived from chicken (HD11) and cattle (Bomac) compared to interaction of the broad host range serovar, Salmonella Typhimurium and the cattle adapted serovar Salmonella Dublin. Results showed a weaker invading ability of S. Gallinarum in both kinds of macrophages, regardless whether the bacteria were opsonized or not before infections. However, opsonization of S. Gallinarum by chicken serum increased its intracellular survival rate in chicken macrophages. No significant induction of nitrogen oxide was observed in the infected HD11 cells within the first 6 h, and levels of reactive oxygen species (ROS) were similar among the three serovars. S. Gallinarum infection was associated with low cell deaths in both chicken and cattle macrophages, whereas S. Dublin only induced a comparable high level of cell death in chicken macrophages, but not in macrophages of its preferred host species (Bomac) compared to host generalist S. Typhimurium. S. Gallinarum-infected HD11 macrophages exhibited low induction of pro-inflammation genes [interleukin (IL)1ß, CXCLi1, and CXCLi2] compared to the two other serovars, and contrary to the other serovars, it did not induce significant downregulation of Toll-like receptor (TLR)2, TLR4, and TLR5. In in vivo infection of 1-week-old chicken, a significant upregulation of the TLR4 and TLR5 genes in the spleen was observed in S. Gallinarum-infected chickens, but not in S. Typhimurium-infected chicken at 5 days post-infections. Taken together, results show that S. Gallinarum infection of macrophages was characterized by low uptake and low cytotoxicity, possibly allowing long-term persistence in the intracellular environment, and it caused a low induction of pro-inflammatory responses.

Streptococcus suis 2 Transcriptional Regulator TstS Stimulates Cytokine Production and Bacteremia to Promote Streptococcal Toxic Shock-Like Syndrome.

Two large-scale outbreaks of streptococcal toxic shock-like syndrome (STSLS) have revealed Streptococcus suis 2 to be a severe and evolving human pathogen. We investigated the mechanism by which S. suis 2 causes STSLS. The transcript abundance of the transcriptional regulator gene tstS was found to be upregulated during experimental infection. Compared with the wild-type 05ZY strain, a tstS deletion mutant (ΔtstS) elicited reduced cytokine secretion in macrophages. In a murine infection model, tstS deletion resulted in decreased virulence and bacterial load, and affected cytokine production. Moreover, TstS expression in the P1/7 strain of S. suis led to the induction of STSLS in the infected mice. This is noteworthy because, although it is virulent, the P1/7 strain does not normally induce STSLS. Through a microarray-based comparative transcriptomics analysis, we found that TstS regulates multiple metabolism-related genes and several virulence-related genes associated with immune evasion.

Investigation of the Role of Genes Encoding Zinc Exporters zntA, zitB, and fieF during Salmonella Typhimurium Infection.

The transition metal zinc is involved in crucial biological processes in all living organisms and is essential for survival of Salmonella in the host. However, little is known about the role of genes encoding zinc efflux transporters during Salmonella infection. In this study, we constructed deletion mutants for genes encoding zinc exporters (zntA, zitB, and fieF) in the wild-type (WT) strain Salmonella enterica serovar Typhimurium (S. Typhimurium) 4/74. The mutants 4/74ΔzntA and 4/74ΔzntA/zitB exhibited a dramatic growth delay and abrogated growth ability, respectively, in Luria Bertani medium supplemented with 0.25 mM ZnCl2 or 1.5 mM CuSO4 compared to the WT strain. In order to investigate the role of genes encoding zinc exporters on survival of S. Typhimurium inside cells, amoeba and macrophage infection models were used. No significant differences in uptake or survival were detected for any of the mutants compared to the WT during infection of amoebae. In natural resistance-associated macrophage protein 1 (Nramp1)-negative J774.1 murine macrophages, significantly higher bacterial counts were observed for the mutant strains 4/74ΔzntA and 4/74ΔzntA/zitB compared to the WT at 4 h post-infection although the fold net replication was similar between all the strains. All four tested mutants (4/74ΔzntA, 4/74ΔzitB, 4/74ΔfieF, and 4/74ΔzntA/zitB) showed enhanced intracellular survival capacity within the modified Nramp1-positive murine RAW264.7 macrophages at 20 h post-infection. The fold net replication was also significantly higher for 4/74ΔzntA, 4/74ΔzitB, and 4/74ΔzntA/zitB mutants compared to the WT. Intriguingly, the ability to survive and cause infection was significantly impaired in all the three mutants tested (4/74ΔzntA, 4/74ΔzitB, and 4/74ΔzntA/zitB) in C3H/HeN mice, particularly the double mutant 4/74ΔzntA/zitB was severely attenuated compared to the WT in all the three organs analyzed. These findings suggest that these genes encoding zinc exporters, especially zntA, contribute to the resistance of S. Typhimurium to zinc and copper stresses during infection.

Characterisation of a novel integrative and conjugative element ICESsD9 carrying erm(B) and tet(O) resistance determinants in Streptococcus suis, and the distribution of ICESsD9-like elements in clinical isolates.

This study identified a novel integrative and conjugative element (ICESsD9) carrying erm(B) and tet(O) resistance determinants in Streptococcus suis D9 and determined its prevalence in clinical isolates. Comparative genome analysis was performed using Mauve and Artemis Comparison Tool visualisation programs. Inverse PCR was utilised to detect its circular intermediate. The transfer capacity of ICESsD9 was evaluated by mating assays using S. suis A7 and Enterococcus faecalis JH2-2 as recipients. A genome walking approach was employed to analyse the characteristics of integration sites in transconjugants. A total of 118 clinical S. suis isolates were tested by PCR mapping assays to detect ICESsD9-like elements. MLST was performed on isolates containing ICESsD9 variants to determine their clonal relatedness. This 55 683-bp element can actively excise from the chromosome. Additionally, it was capable of transferring both into S. suis and E. faecalis with frequencies of 1.2×10-4 and 5.8×10-6 per donor, respectively. When investigating integration site features, it was found that ICESsD9 can enter S. suis and E. faecalis chromosomes by different sites, generating 15-bp and 3-bp direct repeat sequences, respectively. Twelve isolates mainly belonging to sequence types ST1, ST7 and ST28 were confirmed to harbour ICESsD9-like elements. In conclusion, this study provides the first description of an ICE in S. suis that is capable of transferring both into S. suis and E. faecalis. The presence of different ICESsD9 variants in clinical isolates suggests already wide dissemination of this family element in S. suis in China.

Characterization of Spectinomycin Resistance in Streptococcus suis Leads to Two Novel Insights into Drug Resistance Formation and Dissemination Mechanism.

Spectinomycin is an aminocyclitol antibiotic used clinically to treat a variety of infections in animals. Here, we characterized drug resistance prevalence in clinical Streptococcus suis isolates and discovered a novel resistance mechanism in which the s5 mutation (Gly26Asp) results in high spectinomycin resistance. Additionally, a novel integrative and conjugative element encompassing a multidrug resistance spw_like-aadE-lnu(B)-lsa(E) cluster and a cadmium resistance operon were identified, suggesting a possible cause for the wide dissemination of spectinomycin resistance in S. suis.

Identification and characterisation a surface-associated arginine peptidase in Streptococcus suis serotype 2.

Streptococcus suis is an important zoonotic pathogen worldwide and is responsible for disease in swine and humans. In the present study, we identified and characterised a surface-associated peptidase (abpb, amylase-binding protein B) in Streptococcus suis serotype 2 (S. suis 2) that has high hydrolytic activity towards H-Arg-pNa, with maximum activity at pH 7.0. Stimulation of RAW 264.7 macrophages with purified recombinant abpb protein triggered the release of pro-inflammatory cytokines. An abpb-deficient mutant ΔAbpb was constructed by homologous recombination to determine the role of abpb in S. suis 2. The mutant ΔAbpb showed decreased adherence to Hep-2 cells and attenuated virulence in a mouse model compared to the wild type strains. The results of the infection showed impaired bacterial growth in vivo and poor colonisation of the organs. In a protection assay, the recombinant abpb provided excellent protection against a lethal challenge of S. suis 2. Together, these findings suggest that abpb contributes to the pathogenicity of S. suis 2 and may be another target for S. suis prevention and control.