Application of mNGS in the Etiological Diagnosis of Thoracic and Abdominal Infection in Patients With End-Stage Liver Disease

BackgroundDespite the obvious advantages of metagenomic next-generation sequencing (mNGS) in etiological diagnosis of various infectious diseases, there are few reports on etiological diagnosis of suspected thoracic and abdominal infections in patients with end-stage liver disease (ESLD).MethodsSeventy-three ESLD patients were enrolled from January 2019 to May 2021 due to suspected complicated thoracic and abdominal infections with poor response to empirical anti-infective treatment. Pleural effusion and ascites samples of these patients were collected for mNGS detection and conventional pathogen culture. The application value of mNGS in etiological diagnosis of thoracic and abdominal infections in ESLD patients was finally evaluated.ResultsA total of 96 pathogens were detected using mNGS method, including 47 bacteria, 32 viruses, 14 fungi, 2 Mycobacterium tuberculosis, and 1 parasite. The positive rate of mNGS reached 42.5%, which was significantly higher than that of conventional culture method (21.9%) (p = 0.008). Considering neutrophil counts, the overall positive rate of bacteria detection of both methods in Polymorphonuclear Neutrophils (PMN) ≥250/mm3 group was 64.3% and in PMN <250/mm3 group was 23.7%. Compared with the final clinical diagnosis, the agreement rate of mNGS in patients with positive bacteria detection and with suspected positive bacteria detection was 78.6% (11/14) and 44.4% (8/18), respectively. In addition, the agreement rate of mNGS was 66.7% (4/6, respectively) in patients with positive and suspected fungal detection. Interestingly, of the 11 patients with fungal detection, 5 had alcoholic liver disease, accounting for 45.5% of all patients with alcoholic liver disease. We also detected 32 strains of viruses using mNGS, mainly cytomegalovirus (62.5%).ConclusionsThe mNGS method is a useful supplement to conventional culture methods, which performs a higher positive rate, higher sensitivity, and broader pathogen spectrum, especially for rare pathogens and those difficult to culture. For ESLD patients, mNGS has great prospects in early etiological diagnosis of thoracic and abdominal infections. In addition, the cutoff values for the diagnosis of bacterial infection (PMN ≥250/mm3) in the thoracic and abdominal cavities may need to be redefined.

Antibiotic resistance profiling of clinical isolates of Salmonella enterica Serovar Paratyphi A in Dhaka, Bangladesh

The study was conducted to detect the antibiotic resistance profile of the clinical isolates of Salmonella enterica Serovar Paratyphi A from 100 blood samples of patients from different age groups suspected to be suffering from enteric fever. The pure cultures of the bacterial isolates were collected from some renowned diagnostic centers of Dhaka and they were further characterized through the conventional culture, microscopy and biochemical examinations. These isolates were cross checked for the antibiogram profile by the Kirby-Bauer disc diffusion method against ten different types of antibiotics. Most of the isolates were found resistant against azithromycin (100%), nalidixic acid (100%) and ceftazidime (75%). However, isolates showed sensitivity to ciprofloxacin (95%), levofloxacin (97%), cotrimoxazole (96%) and chloramphenicol (95%). These findings highlight the need for continuous monitoring of the drug resistance pattern of S. enterica Serovar Paratyphi A for better public health management. Stamford Journal of Microbiology, Vol.11 (1) 2021: 14-16

Performance of Conventional Urine Culture Compared to 16S rRNA Gene Amplicon Sequencing in Children with Suspected Urinary Tract Infection

Concordance between conventional culture and 16S rRNA gene amplicon sequencing appears to be high. In children with equivocal culture results, 16S rRNA gene results may provide information that may help clarify the diagnosis.

Occurrence of Escherichia coli in cloacal samples of broiler chicken from Kollam and Kottayam districts

Foodborne pathogens like E. coli are considered as the major causes of foodborne illness in humans worldwide. The present study was undertaken to determine the occurrence of E. coli in cloacal samples of broiler chicken from Kollam and Kottayam districts. The occurrence of E. coli in cloacal samples from broiler chicken was 76.5 per cent from Kollam and 79 per cent from Kottayam through culture techniques. Out of the total 400 cloacal swab samples collected from broiler chicken, 77.8 per cent were positive for E. coli. The samples which were subjected to conventional culture techniques were further analysed for PCR confirmation. The study revealed that, 56.5 and 67 per cent samples were positive for E. coli from Kollam and Kottayam, respectively. An overall occurrence of 61.8 per cent out of 400 samples were confirmed for E. coli by PCR. One Health approach can be used as a suitable tool to combat the foodborne zoonotic diseases, since it is an integrated, multidisciplinary, holistic approach. Proper implementation of biosecurity measures in farms is mandatory to control foodborne zoonotic diseases.

Molecular Methods for Pathogenic Bacteria Detection and Recent Advances in Wastewater Analysis

With increasing concerns about public health and the development of molecular techniques, new detection tools and the combination of existing approaches have increased the abilities of pathogenic bacteria monitoring by exploring new biomarkers, increasing the sensitivity and accuracy of detection, quantification, and analyzing various genes such as functional genes and antimicrobial resistance genes (ARG). Molecular methods are gradually emerging as the most popular detection approach for pathogens, in addition to the conventional culture-based plate enumeration methods. The analysis of pathogens in wastewater and the back-estimation of infections in the community, also known as wastewater-based epidemiology (WBE), is an emerging methodology and has a great potential to supplement current surveillance systems for the monitoring of infectious diseases and the early warning of outbreaks. However, as a complex matrix, wastewater largely challenges the analytical performance of molecular methods. This review synthesized the literature of typical pathogenic bacteria in wastewater, types of biomarkers, molecular methods for bacterial analysis, and their recent advances in wastewater analysis. The advantages and limitation of these molecular methods were evaluated, and their prospects in WBE were discussed to provide insight for future development.

Quorum Sensing Mediated Pathogenicity, Virulence Genes and Class 1 Integron in Carbapenem-Resistant Clinical Pseudomonas Aeruginosa Isolates

Carbapenems are the most effective agents for treating carbapenem-resistant clinical P. aeruginosa (CRPsA) infections. During an infection, a quorum-sensing (QS) system and its regulating virulence genes have a great role. The aim of the study was to detect the presence of a las and rhl QS system and related virulence genes and a class 1 (Cls1) integron. A total of 52 CRPsA isolates obtained from Kastamonu, Turkey was analyzed. A conventional culture method, an oprL gene-based molecular assay for P. aeruginosa isolates, and an automated VITEK-2 compact system were applied for the isolation and identification of CRPsA isolates. The oprL gene was detected in all of the isolates tested. At least one of the las or rhl system genes was detected in 98.07% of the isolates, and the percentage of las system genes (98.07%) were higher than that of rhl system genes (90.38%). algD, lasB, toxA and aprA genes were detected in between 46.15% and 88.46% of the isolates, and co-existence of four genes were detected in 40.38% of the isolates. Cls1 integron and slime production using Congo Red Agar (CRA) were present in 51.92% and 67.30%, respectively, of the isolates. There was a significant positive correlation (p <.10) between the las system and the rhl system and a strongly positive correlation (p<.01 or p <.05) between the rhl system-four virulence genes and slime production-and among some virulence genes. In conclusion, the CRPsA isolates tested in the study are highly virulent and QS systems have a significant role in pathogenesis.

It Takes Two to Tango: Combining Conventional Culture with Molecular Diagnostics Enhances Accuracy of Streptococcus pneumoniae Detection and Pneumococcal Serogroup/Serotype determination in Carriage

Background: The specificity of molecular methods for the detection of Streptococcus pneumoniae carriage is under debate. We propose a procedure that increases the accuracy of molecular detection of live pneumococci in polymicrobial respiratory samples. Methods: Culture and qPCR methods were applied to detect S. pneumoniae and pneumococcal serotypes in 1549 nasopharyngeal samples collected in the Netherlands (n=972) and England (n=577) from 946 toddlers and 603 adults, and in paired oropharyngeal samples collected exclusively from 319 Dutch adults. Samples with no live pneumococci isolated at primary diagnostic culture yet generating pneumococcus-specific signal in qPCRs were re-examined with a second, qPCR-guided culture. Optimal Cq cut-offs for positivity in qPCRs were determined via receiver operating characteristic (ROC) curve analysis using isolation of live pneumococci from the primary and qPCR-guided cultures as reference. Results: Detection of S. pneumoniae and pneumococcal serotypes with qPCRs in cultured (culture-enriched) nasopharyngeal samples exhibited near-perfect agreement with conventional culture (Cohens kappa: 0.95). Molecular methods also displayed increased sensitivity of detection for multiple serotype carriage. Among paired samples from adults, the sensitivity of S. pneumoniae detection in primary nasopharyngeal plus oropharyngeal cultures was significantly lower compared with molecular detection in both culture-enriched samples together (p<0.0001) and also in culture-enriched oropharyngeal samples alone (p<0.05). Conclusions: The sensitivity of S. pneumoniae carriage surveillance can be greatly improved by complementing conventional culture with qPCR and vice versa. The specificity of molecular methods for the detection of live pneumococci can be enhanced by incorporating statistical procedures based on ROC curve analysis. The procedure we propose improves detection of S. pneumoniae carriage in adults in particular and enhances specificity of serotype carriage detection.

A Pulmonary Vascular Model From Endothelialized Whole Organ Scaffolds

The development of an in vitro system for the study of lung vascular disease is critical to understanding human pathologies. Conventional culture systems fail to fully recapitulate native microenvironmental conditions and are typically limited in their ability to represent human pathophysiology for the study of disease and drug mechanisms. Whole organ decellularization provides a means to developing a construct that recapitulates structural, mechanical, and biological features of a complete vascular structure. Here, we developed a culture protocol to improve endothelial cell coverage in whole lung scaffolds and used single-cell RNA-sequencing analysis to explore the impact of decellularized whole lung scaffolds on endothelial phenotypes and functions in a biomimetic bioreactor system. Intriguingly, we found that the phenotype and functional signals of primary pulmonary microvascular revert back—at least partially—toward native lung endothelium. Additionally, human induced pluripotent stem cell-derived endothelium cultured in decellularized lung systems start to gain various native human endothelial phenotypes. Vascular barrier function was partially restored, while small capillaries remained patent in endothelial cell-repopulated lungs. To evaluate the ability of the engineered endothelium to modulate permeability in response to exogenous stimuli, lipopolysaccharide (LPS) was introduced into repopulated lungs to simulate acute lung injury. After LPS treatment, proinflammatory signals were significantly increased and the vascular barrier was impaired. Taken together, these results demonstrate a novel platform that recapitulates some pulmonary microvascular functions and phenotypes at a whole organ level. This development may help pave the way for using the whole organ engineering approach to model vascular diseases.

Wnt/β-catenin Signaling Pathway Promotes Transdifferentiation from Fetal Skin Fibroblasts to Keratinocyte-like Cells

Background: Wound healing is a dynamic, sequential,and complex physiological process, including a variety of cellular events, such as proliferation, adhesion, chemotaxis, and apoptosis. Skin fibroblasts and keratinocytes are the two most important cells involved in wound repair, and Relying on the proliferation and differentiation of keratinocytes to form epithelium to completely cover the wound is the most ideal result for wound repair, so expanding the source of keratinocytes is a huge challenge. In this study, we examined the phenomenon that fetal skin fibroblasts spontaneously transdifferentiated into keratinocyte-like cells in conventional culture, and evaluated the characteristics of KLCs and the potential mechanisms of the transdifferentiation process.Methods: HFF-1 were routinely cultured in ordinary DMEM medium for more than 40 days,and observed the cell morphology. The cytological properties of KLCs at the cellular and molecular levels were detected by RT-PCR, Western-blot, immunofluorescence, Transwell, and cell scratch experiments.The functionality and safety of KLCs were determined through wound healing and tumorigenicity experiments. And high-throughput transcriptome sequencing (RNA-seq) was performed to explore the mechanism underlying HFF-1 transdifferentiation.Results: The transdifferentiation process started on the 25th day and was completed by the 40th day. KLCs and KCs had similar expressions at the molecular and protein levels, both functioned similarly in wound healing and were non-tumorigenic.RNA-seq revealed that the transdifferentiation process was regulated by the activation of the classical Wnt/β-catenin signaling pathway, which could shorten the process to 10 days.Conclusion: This study demonstrates that HFF-1 can spontaneously transdifferentiate into KLCs with conventional culture conditions, and the Wnt/β-catenin signaling pathway regulates the transdifferentiation process.

Universal Enzyme-Based Field Workflow for Rapid and Sensitive Quantification of Water Pathogens

A universal filtration and enzyme-based workflow has been established to allow for the rapid and sensitive quantification of leading pathogens Cryptosporidium parvum, Giardia gamblia, Campylobacter jejuni, and Escherichia coli from tap water samples with volumes up to 100 mL, and the potential to scale up to larger volumes. qPCR limits of quantification as low as four oocysts for Cryptosporidium, twelve cysts for Giardia, two cells for C. jejuni, and nineteen cells for E. coli per reaction were achieved. A polycarbonate filter-based sampling method coupled with the prepGEM enzyme-based DNA extraction system created a single-step transfer workflow that required as little as 20 min of incubation time and a 100 µL reaction mix. The quantification via qPCR was performed directly on the prepGEM extract, bypassing time-consuming, labour-intensive conventional culture-based methods. The tap water samples were shown to contain insoluble particles that inhibited detection by reducing the quantification efficiency of a representative pathogen (C. jejuni) to 30–60%. This sample inhibition was effectively removed by an on-filter treatment of 20% (v/v) phosphoric acid wash. Overall, the established workflow was able to achieve quantification efficiencies of 92% and higher for all four leading water pathogens, forming the basis of a rapid, portable, and low-cost solution to water monitoring.