Challenges and future solutions for detection of Clostridioides difficile in adults.

There are no established standards for the diagnosis of Clostridioides difficile infection (CDI), even though the importance of this infection in humans is well known. The effectiveness of the commercially available techniques, which are all standardized for use with human feces, is also limited in terms of the accuracy of the tests. Furthermore, the current approach lacks a point-of-care diagnosis with an acceptable range of sensitivity and specificity. This article reviews the challenges and possible future solutions for the detection of CDI in adults. Existing diagnostic methods, such as enzyme-linked immunoassays and microbial culturing for the detection of toxins A and B, appear to work poorly in samples but exhibit great sensitivity for glutamate dehydrogenase. Real-time polymerase chain reaction and nucleic acid amplification tests have been investigated in a few studies on human samples, but so far have shown poor turnaround times. Thus, developing a multiplex point-of-care test assay with high sensitivity and specificity is required as a bedside approach for diagnosing this emerging infection.

RNA-Seq of untreated wastewater to assess COVID-19 and emerging and endemic viruses for public health surveillance.

Background: The COVID-19 pandemic showcased the power of genomic sequencing to tackle the emergence and spread of infectious diseases. However, metagenomic sequencing of total microbial RNAs in wastewater has the potential to assess multiple infectious diseases simultaneously and has yet to be explored. Methods: A retrospective RNA-Seq epidemiological survey of 140 untreated composite wastewater samples was performed across urban (n = 112) and rural (n = 28) areas of Nagpur, Central India. Composite wastewater samples were prepared by pooling 422 individual grab samples collected prospectively from sewer lines of urban municipality zones and open drains of rural areas from 3rd February to 3rd April 2021, during the second COVID-19 wave in India. Samples were pre-processed and total RNA was extracted prior to genomic sequencing. Findings: This is the first study that has utilised culture and/or probe-independent unbiased RNA-Seq to examine Indian wastewater samples. Our findings reveal the detection of zoonotic viruses including chikungunya, Jingmen tick and rabies viruses, which have not previously been reported in wastewater. SARS-CoV-2 was detectable in 83 locations (59%), with stark abundance variations observed between sampling sites. Hepatitis C virus was the most frequently detected infectious virus, identified in 113 locations and co-occurring 77 times with SARS-CoV-2; and both were more abundantly detected in rural areas than urban zones. Concurrent identification of segmented virus genomic fragments of influenza A virus, norovirus, and rotavirus was observed. Geographical differences were also observed for astrovirus, saffold virus, husavirus, and aichi virus that were more prevalent in urban samples, while the zoonotic viruses chikungunya and rabies, were more abundant in rural environments. Interpretation: RNA-Seq can effectively detect multiple infectious diseases simultaneously, facilitating geographical and epidemiological surveys of endemic viruses that could help direct healthcare interventions against emergent and pre-existent infectious diseases as well as cost-effectively and qualitatively characterising the health status of the population over time. Funding: UK Research and Innovation (UKRI) Global Challenges Research Fund (GCRF) grant number H54810, as supported by Research England.

Development of multiple reaction monitoring (MRM) assays to identify Brucella abortus proteins in the serum of humans and livestock.

In the present study, a targeted multiple reaction monitoring-mass spectrometry (MRM-MS) approach was developed to screen and identify protein biomarkers for brucellosis in humans and livestock. The selection of proteotypic peptides was carried out by generating in silico tryptic peptides of the Brucella proteome. Using bioinformatics analysis, 30 synthetic peptides corresponding to 10 immunodominant Brucella abortus proteins were generated. MRM-MS assays for the accurate detection of these peptides were optimized using 117 serum samples of human and livestock stratified as clinically confirmed (45), suspected (62), and control (10). Using high throughput MRM assays, transitions for four peptides were identified in several clinically confirmed and suspected human and livestock serum samples. Of these, peptide NAIYDVVTR corresponding to B. abortus proteins: BruAb2_0537 was consistently detected in the clinically confirmed serum samples of both humans and livestock with 100% specificity. To conclude, a high throughput MRM-MS-based protocol for detecting endogenous B. abortus peptides in serum samples of humans and livestock was developed. The developed protocol will help design sensitive assays to accurately diagnose brucellosis in humans and livestock. The data associated with this study are deposited in Panorama Public (https://panoramaweb.org/rNOZCy.url with ProteomeXchange ID: PXD034407).

The Influence of Gut Dysbiosis in the Pathogenesis and Management of Ischemic Stroke.

Recent research on the gut microbiome has revealed the influence of gut microbiota (GM) on ischemic stroke pathogenesis and treatment outcomes. Alterations in the diversity, abundance, and functions of the gut microbiome, termed gut dysbiosis, results in dysregulated gut-brain signaling, which induces intestinal barrier changes, endotoxemia, systemic inflammation, and infection, affecting post-stroke outcomes. Gut-brain interactions are bidirectional, and the signals from the gut to the brain are mediated by microbially derived metabolites, such as trimethylamine N-oxide (TMAO) and short-chain fatty acids (SCFAs); bacterial components, such as lipopolysaccharide (LPS); immune cells, such as T helper cells; and bacterial translocation via hormonal, immune, and neural pathways. Ischemic stroke affects gut microbial composition via neural and hypothalamic-pituitary-adrenal (HPA) pathways, which can contribute to post-stroke outcomes. Experimental and clinical studies have demonstrated that the restoration of the gut microbiome usually improves stroke treatment outcomes by regulating metabolic, immune, and inflammatory responses via the gut-brain axis (GBA). Therefore, restoring healthy microbial ecology in the gut may be a key therapeutic target for the effective management and treatment of ischemic stroke.

Clostridioides difficile epidemiology in India.

Clostridioides difficile infection (CDI) continues to affect hospitalized patients and community populations worldwide. In contrast to the substantial resources invested in the diagnosis and prevention of CDI in high-income countries, this anaerobic toxigenic bacterium has been largely overlooked in low-and-middle-income countries (LMICs) such as India, where there remains a paucity of epidemiologic data evaluating the burden of CDI. Extensive multi-institutional studies describing C. difficile epidemiology in India have not yet been performed. Given recent economic growth in many Asian countries, with aging populations, increased access to healthcare and widespread inappropriate use of antimicrobials, C. difficile is likely to be highly prevalent and causing significant disease burden. Greater efforts are required to enhance awareness of this neglected pathogen, through educating healthcare practitioners to test for CDI. There is also an urgent need to strengthen laboratory capacity, and ideally establish a national reference laboratory, to help facilitate a greater understanding of the molecular epidemiology of CDI in India and other LMICs. This mini-review aims to summarize the existing research evaluating the burden of CDI in humans and the environment in India.

Gut dysbiosis, defective autophagy and altered immune responses in neurodegenerative diseases: Tales of a vicious cycle.

The human microbiota comprises trillions of symbiotic microorganisms and is involved in regulating gastrointestinal (GI), immune, nervous system and metabolic homeostasis. Recent observations suggest a bidirectional communication between the gut microbiota and the brain via immune, circulatory and neural pathways, termed the Gut-Brain Axis (GBA). Alterations in gut microbiota composition, such as seen with an increased number of pathobionts and a decreased number of symbionts, termed gut dysbiosis or microbial intestinal dysbiosis, plays a prominent role in the pathogenesis of central nervous system (CNS)-related disorders. Clinical reports confirm that GI symptoms often precede neurological symptoms several years before the development of neurodegenerative diseases (NDDs). Pathologically, gut dysbiosis disrupts the integrity of the intestinal barrier leading to ingress of pathobionts and toxic metabolites into the systemic circulation causing GBA dysregulation. Subsequently, chronic neuroinflammation via dysregulated immune activation triggers the accumulation of neurotoxic misfolded proteins in and around CNS cells resulting in neuronal death. Emerging evidence links gut dysbiosis to the aggravation and/or spread of proteinopathies from the peripheral nervous system to the CNS and defective autophagy-mediated proteinopathies. This review summarizes the current understanding of the role of gut microbiota in NDDs, and highlights a vicious cycle of gut dysbiosis, immune-mediated chronic neuroinflammation, impaired autophagy and proteinopathies, which contributes to the development of neurodegeneration in Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We also discuss novel therapeutic strategies targeting the modulation of gut dysbiosis through prebiotics, probiotics, synbiotics or dietary interventions, and faecal microbial transplantation (FMT) in the management of NDDs.

ESX-5-targeted export of ESAT-6 in BCG combines enhanced immunogenicity & efficacy against murine tuberculosis with low virulence and reduced persistence.

Tuberculosis (TB) is the leading infectious cause of death globally. The only licensed TB vaccine, Bacille Calmette-Guérin (BCG), has low efficacy against TB in adults and is not recommended in people with impaired immunity. The incorporation of the Mycobacterium tuberculosis (Mtb) secretion system ESX-1 into BCG improves immunogenicity and protection against TB in animal models, which is associated with the secretion of the ESX-1-dependent protein ESAT-6. However, the resulting strain, BCG::ESX1Mtb, has been deemed unsafe as a human vaccine, due to prolonged persistence and increased virulence in immunocompromised mice. In this study, we describe a new recombinant BCG strain that uncouples the beneficial aspects of ESAT-6 secretion from the detrimental ESX-1effects on virulence and persistence. The strain was constructed by fusing the ESAT-6-encoding gene esxA to the general secretion signal for the mycobacterial type VII secretion pathway protein PE25. This new strain, BCG::ESAT6-PE25SS, secretes full-length ESAT-6 via the ESX-5 secretion system, which in contrast to ESX-1 is also present in BCG. In vivo testing revealed that ESX-5-targeted ESAT-6 export, induces cytosolic contact, generates ESAT-6-specific T cells and enhances the protective efficacy against TB disease, but is associated with low virulence and reduced persistence in immunocompetent and immunocompromised mice. Additionally, compared to BCG::ESX1Mtb and parental BCG, mucosal administration of BCG::ESAT6-PE25SS is associated with more rapid clearance from the lung. These results warrant further studies to evaluate BCG::ESAT6-PE25SS as a potential live attenuated vaccine candidate for TB.

Multiomics Profiling Reveals Signatures of Dysmetabolism in Urban Populations in Central India.

BACKGROUND: Non-communicable diseases (NCDs) have become a major cause of morbidity and mortality in India. Perturbation of host-microbiome interactions may be a key mechanism by which lifestyle-related risk factors such as tobacco use, alcohol consumption, and physical inactivity may influence metabolic health. There is an urgent need to identify relevant dysmetabolic traits for predicting risk of metabolic disorders, such as diabetes, among susceptible Asian Indians where NCDs are a growing epidemic. METHODS: Here, we report the first in-depth phenotypic study in which we prospectively enrolled 218 adults from urban and rural areas of Central India and used multiomic profiling to identify relationships between microbial taxa and circulating biomarkers of cardiometabolic risk. Assays included fecal microbiota analysis by 16S ribosomal RNA gene amplicon sequencing, quantification of serum short chain fatty acids by gas chromatography-mass spectrometry, and multiplex assaying of serum diabetic proteins, cytokines, chemokines, and multi-isotype antibodies. Sera was also analysed for N-glycans and immunoglobulin G Fc N-glycopeptides. RESULTS: Multiple hallmarks of dysmetabolism were identified in urbanites and young overweight adults, the majority of whom did not have a known diagnosis of diabetes. Association analyses revealed several host-microbe and metabolic associations. CONCLUSIONS: Host-microbe and metabolic interactions are differentially shaped by body weight and geographic status in Central Indians. Further exploration of these links may help create a molecular-level map for estimating risk of developing metabolic disorders and designing early interventions.

Metagenomics reveals impact of geography and acute diarrheal disease on the Central Indian human gut microbiome.

BACKGROUND: The Central Indian gut microbiome remains grossly understudied. Herein, we sought to investigate the burden of antimicrobial resistance and diarrheal diseases, particularly Clostridioides difficile, in rural-agricultural and urban populations in Central India, where there is widespread unregulated antibiotic use. We utilized shotgun metagenomics to comprehensively characterize the bacterial and viral fractions of the gut microbiome and their encoded functions in 105 participants. RESULTS: We observed distinct rural-urban differences in bacterial and viral populations, with geography exhibiting a greater influence than diarrheal status. Clostridioides difficile disease was more commonly observed in urban subjects, and their microbiomes were enriched in metabolic pathways relating to the metabolism of industrial compounds and genes encoding resistance to 3rd generation cephalosporins and carbapenems. By linking phages present in the microbiome to their bacterial hosts through CRISPR spacers, phage variation could be directly related to shifts in bacterial populations, with the auxiliary metabolic potential of rural-associated phages enriched for carbon and amino acid energy metabolism. CONCLUSIONS: We report distinct differences in antimicrobial resistance gene profiles, enrichment of metabolic pathways and phage composition between rural and urban populations, as well as a higher burden of Clostridioides difficile disease in the urban population. Our results reveal that geography is the key driver of variation in urban and rural Indian microbiomes, with acute diarrheal disease, including C. difficile disease exerting a lesser impact. Future studies will be required to understand the potential role of dietary, cultural, and genetic factors in contributing to microbiome differences between rural and urban populations.

Modelling of cerebral tuberculosis in BALB/c mice using clinical strain from patients with CNS tuberculosis infection.

BACKGROUND & OBJECTIVES: Central nervous system (CNS) infection caused by Mycobacterium tuberculosis (MTB) is the most severe form of extrapulmonary tuberculosis (EPTB) due to a high level of mortality and morbidity. Limited studies are available on CNS-TB animal model development. The present study describes the development of a murine model of CNS-TB using a clinical strain (C3) isolated from the cerebrospinal fluid (CSF) of CNS-TB patients. METHODS: Groups of mice were infected by the intravenous route with MTB C3 strain isolated from the CSF of CNS-TB patients. Brain and lung tissue were evaluated for bacterial burden, histopathology and surrogate markers of TB infection at 30 and 50 days post-infection. RESULTS: Mice infected intravenously with MTB C3 strains showed progressive development of CNS disease with high bacillary burden in lungs at the initial stage (30 days), which eventually disseminated to the brain at a later stage (50 days). Similarly, high mortality (60%) was associated in mice infected with C3 strain compared to control. INTERPRETATION & CONCLUSIONS: The study showed development of a novel murine model of CNS-TB using the C3 strain of MTB that replicated events of extrapulmonary dissemination. The developed model would be helpful in understanding the pathogenesis of CNS-TB infection for the development of improved therapeutic interventions in future.

Determination of Toll-like receptor-induced cytokine profiles in the blood and cerebrospinal fluid of Chikungunya patients.

BACKGROUND: Chikungunya infection caused by Chikungunya virus (CHIKV) is an inflammatory disease affecting the joints and may also lead to neurological complications. We investigated a panel of human Toll-like receptor (TLR)-induced cytokines in Chikungunya patients with and without neurological complications. METHODS: In a case-control study, a panel of 12 cytokines and chemokines, TNF-α, IFN-α, IL-1ß, IL-6, IL-12, IL-17A, IL-8, monocyte chemotactic protein (MCP)-1, RANTES, interferon (IFN)-γ-induced protein (IP)-10, monokine induced by IFN-γ (MIG) and thymus and activation-regulated chemokine (TARC), was analysed using a conventional ELISA protocol in the serum samples of Chikungunya patients without neurological complications and in the cerebrospinal fluid (CSF) and paired serum samples of Chikungunya patients with neurological complications. RESULTS: The levels of 3 cytokines, IL-1ß, IL-17A and IL-8, and 4 chemokines, MCP-1, RANTES, IP-10 and TARC, were raised in serum samples of Chikungunya patients without neurological complications, whereas, 4 cytokines, TNF-α, IFN-α, IL-6 and IL-8, and 4 chemokines, MCP-1, RANTES, MIG and TARC, were elevated in CSF samples of Chikungunya patients with neurological complications. Moreover, the levels of IL-6 and IL-8 cytokines were significantly elevated in the CSF compared to paired serum samples in Chikungunya patients with neurological complications. CONCLUSIONS: In CHIKV infection, multiple cytokines are elevated in serum and CSF. The elevation in IL-6 and IL-8 cytokines in CSF correlates with neurological involvement.

Effect of aqueous extract and fractions of Fagonia arabica on in vitro anticoagulant activity.

Fagonia arabica (FA) is a deobstruent and blood purifier, which possesses thrombolytic and antioxidant activities. In this study, the anticoagulant effects of FA and its derived fractions were evaluated. Plasma recalcification was performed with multisolvent extracts of FA and then with extracts prepared successively with increasing polarity of the solvents. Aqueous extract was the most potent anticoagulant extract, which was fractionated by thin-layer chromatography and column chromatography. Five fractions collected were checked for their anticoagulation effect. The most potent fraction was screened for phytoconstituents. Aqueous extract of FA is the most active anticoagulant (31 minutes). Results were statistically significant when compared to heparin (38 minutes) and saline (4.04 minutes; P > .001). The Fifth fraction (FA5), the most potent fraction (27 minutes), was found positive for flavonoids, saponin, tannin, triterpenoids, carbohydrates, reducing sugar, and monosaccharides. Aqueous FA and fraction FA5 were most active in in vitro anticoagulation, and any of the phytochemicals identified could be considered the active component.

In-vitro clot lytic potential of Fagonia arabica: a comparative study of two methods.

The tube method developed in our laboratory is a simple, inexpensive and a classical whole blood clot lytic procedure through which clot lytic potential of Fagonia arabica was found to be significant. Microtiter plate clot lysis (MPCL) assay is a rapid and precise turbidimetric clot lysis method which includes measurements of maximum absorbance (Max Abs), area under the curve (AUC) along with the standard clot lysis time. In the present study we have compared and validated clot lytic potential of F. arabica extract by tube method and MPCL assay. Percentage of clot lysis was calculated by measuring the difference of the absorbance taken at 0 and 240 min in the case of MPCL assay, whereas with the tube method according to the weight difference. Fagonia arabica (50 ug/ml) was capable of clot lysis by MPCL assay and showed clot lysis pattern similar to 60 U/ml streptokinase (positive control). The clot lysis times were significantly different from one another (P value ≤0.001). When Max Abs and AUC were compared to the clot lysis time the correlation coefficient (r value) was significant too (P value ≤0.001). Moreover, we have also found that both the methods showed almost the same clot lysis percentage by streptokinase as well as F. arabica. The correlation coefficient between streptokinase, and F. arabica done by tube method and MPCL assay was found to be statistically significant (P < 0.05). Fagonia arabica had the clot lytic potential checked by in-vitro methods, namely MPCL assay and the method.

Diagnosis of Chikungunya fever in an Indian population by an indirect enzyme-linked immunosorbent assay protocol based on an antigen detection assay: a prospective cohort study.

A Chikungunya virus (CHIKV) outbreak continues in India. Monitoring of the clinical features of CHIKV infection is an important component of assessing the disease process. Diagnosis is usually made by an immunoglobulin M (IgM)/IgG enzyme-linked immunosorbent assay (ELISA). However, these assays have extremely low sensitivities for the detection of infection in the majority of CHIKV patients during the acute stage of infection (during the 1 to 4 days after infection). In our laboratory, a sensitive ELISA protocol for antigen detection has been developed for the detection of CHIKV infection in the acute stage, and in the present study we assessed the usefulness of this ELISA-based system for the detection of CHIKV infection. We performed a prospective, double-blinded study of 205 Indian patients with suspected CHIKV infection in the Nagpur District. All patients underwent a full clinical assessment, and their serum samples were analyzed for the presence of antigens and of IgM and IgG by an ELISA protocol. In patients with CHIKV infection, the sensitivity of antigen detection was 85%, which was significantly higher (P < 0.001) than that of IgM (17%) or IgG (45%) detection. The sensitivity of IgM (20%) or IgG (25%) detection was significantly lower than that of the antigen assay (95%) for patients with acute infections (i.e., from day 1 to day 5 after infection). Antigen detection not only gives a positive confirmatory result in the early phase of the disease, but it is also useful in the prodromal and subclinical stage and may be useful for field applications for the rapid detection of CHIKV infection.

Effect of Fagonia Arabica (Dhamasa) on in vitro thrombolysis.

BACKGROUND: Atherothrombotic diseases such as myocardial or cerebral infarction are serious consequences of the thrombus formed in blood vessels. Thrombolytic agents are used to dissolve the already formed clots in the blood vessels; however, these drugs have certain limitations which cause serious and sometimes fatal consequences. Herbal preparations have been used since ancient times for the treatment of several diseases. Herbs and their components possessing antithrombotic activity have been reported before; however, herbs that could be used for thrombolysis has not been reported so far. This study's aim was to investigate whether herbal preparations (aqueous extract) possess thrombolytic activity or not. METHODS: An in vitro thrombolytic model was used to check the clot lysis effect of six aqueous herbal extracts viz., Tinospora cordifolia, Rubia cordifolia, Hemidesmus indicus, Glycyrrhiza glabra Linn, Fagonia Arabica and Bacopa monnieri Linn along with Streptokinase as a positive control and water as a negative control. RESULTS: Using an in vitro thrombolytic model, Tinospora cordifolia, Rubia cordifolia, Hemidesmus indicus, Glycyrrhiza glabra Linn, Fagonia Arabica and Bacopa monnieri Linn showed 19.3%, 14.5%, 20.3%, 17.8%, 75.6% and 41.8% clot lysis respectively . Among the herbs studied Fagonia arabica showed significant % of clot lysis (75.6%) with reference to Streptokinase (86.2%). CONCLUSION: Through our study it was found that Dhamasa possesses thrombolytic properties that could lyse blood clots in vitro; however, in vivo clot dissolving properties and active component(s) of Dhamasa for clot lysis are yet to be discovered. Once found Dhamasa could be incorporated as a thrombolytic agent for the improvement of patients suffering from Atherothrombotic diseases.

Evaluation of adenosine deaminase assay for analyzing T-lymphocyte density in vitro.

The proliferative capacity of T cells in response to various stimuli is commonly determined by radioactive assay based on incorporation of [3H]thymidine ([3H]TdR) into newly synthesized DNA. In order to assess techniques for application in laboratories where radioactive facilities are not present, an alternative method was tested. As an alternative, T-cell proliferation was measured by spectrophotometrically analyzing the presence of an enzyme adenosine deaminase in lymphocytes and also using a standard XTT assay. Jurkat (human) T-cell line (clone E6.1) was used for lymphocyte population. The Jurkat cell concentration was adjusted according to different cell densities and enzyme activity was determined. Cells were also seeded in complete medium up to 72 h and harvested for estimation of enzyme activity. A significant correlation between the standard cell-proliferation assay and adenosine deaminase assay was observed. The present study indicates that the assay of adenosine deaminase is a reliable and accurate method for measuring proliferation of T lymphocytes.