A Snapshot of T Cell Subset Cytokines in Pemphigus Vulgaris: A Cross-Sectional Study.

OBJECTIVE: The purpose of this study was to assess the serum levels of cytokines produced by the Th1 (IFN-γ, IL-12), Th2 (IL-4), Th17 (IL-6, IL-17A, IL-23), and Treg (IL-10 and TGF-ß) pathways in individuals with active pemphigus vulgaris (PV) and to determine whether these levels were correlated with the severity of the disease condition. PATIENTS AND METHODS: This study was conducted with 90 individuals, of which 50 were PV patients and 40 healthy individuals (age and gender-matched) as controls. Serum samples were collected and tested for cytokine levels by ELISA (enzyme-linked immunosorbent assay). The cytokine levels in the serum of PV patients and healthy controls were compared statistically using the Mann-Whitney test for nonparametric samples. The strength of the association between the variables was evaluated using the Spearman correlation test. RESULTS: The mean serum levels of IFN- γ (p < 0.001), IL-6 (p < 0.001), IL-10 (p < 0.001), IL-12 (p < 0.05), and IL-17 (p < 0.001) were significantly higher and TGF-ß were significantly low in the PV patients than those observed in the control group. The mean concentration of serum IL-4 in patients with PV did not differ from those in the control group. CONCLUSIONS: In active PV, the Th1 and Th17 pathways are involved in the development and progression of the disease, whereas the Th2 pathway is blocked. Both of these pathways play a significant role in the disease. It is possible that the Treg pathway acts as an antagonist to the Th1 and Th17 pathways, which would cause the disease to become more localised. This study lays the foundation for a better understanding of the aetiology of PV and implies that cytokines could be used as potential therapeutic targets and disease activity biomarkers.

Aetiology of hospital-acquired diarrhoea in under-five children from an urban hospital in East Delhi, India.

Background & objectives: Majority of the studies of hospital-acquired diarrhoea conducted in Western countries have focused on the detection of Clostridium difficile in stool samples. Limited Asian and Indian literature is available on hospital-acquired diarrhoea. This study was aimed to describe the aetiological profile for hospital-acquired diarrhoea in children aged below five years. Methods: One hundred children aged one month to five years who developed diarrhoea (≥3 loose stools for >12 h) after hospitalization for at least 72 h were enrolled. Children who were prescribed purgatives or undergoing procedures such as enema and endoscopy or those with underlying chronic gastrointestinal disorders such as celiac disease and inflammatory bowel disease were excluded from the study. Stool samples from the enrolled children were subjected to routine microscopic examination, modified Ziel-Nielson (ZN) staining for Cryptosporidium and culture for various enteropathogens. Multiplex PCR was used to identify the strains of diarrhoeagenic Escherichia coli. Rotavirus detection was done using rapid antigen kit. Toxins (A and B) of C. difficile were detected using enzyme immunoassay. Results: Of the 100 samples of hospital-acquired diarrhoea analysed, diarrhoeagenic E. coli (DEC) was found to be the most common organism, detected in 37 per cent of cases (enteropathogenic E. coli-18%, enterotoxigenic E. coli-8%, enteroaggregative E. coli-4% and mixed infections-7%). Cryptosporidium was detected in 10 per cent of cases. Rotavirus was detected in six per cent and C. difficile in four per cent of cases. Interpretation & conclusions: The findings of this study suggest that the aetiological profile of hospital-acquired diarrhoea appears to be similar to that of community-acquired diarrhoea, with DEC and Cryptosporidium being the most common causes. The efforts for the prevention and management of hospital-acquired diarrhoea should, thus, be directed towards these organisms.

Antitussive noscapine and antiviral drug conjugates as arsenal against COVID-19: a comprehensive chemoinformatics analysis.

Coronavirus pandemic has caused a vast number of deaths worldwide. Thus creating an urgent need to develop effective counteragents against novel coronavirus disease (COVID-19). Many antiviral drugs have been repurposed for treatment but implicated minimal recovery, which further advanced the need for clearer insights and innovation to derive effective therapeutics. Strategically, Noscapine, an approved antitussive drug with positive effects on lung linings may show favorable outcomes synergistically with antiviral drugs in trials. Hence, we have theoretically examined the combinatorial drug therapy by culminating the existing experimental results with in silico analyses. We employed the antitussive noscapine in conjugation with antiviral drugs (Chloroquine, Umifenovir, Hydroxychloroquine, Favlplravir and Galidesivir). We found that Noscapine-Hydroxychloroquine (Nos-Hcq) conjugate has strong binding affinity for the main protease (Mpro) of SARS-CoV-2, which performs key biological function in virus infection and progression. Nos-Hcq was analyzed through molecular dynamics simulation. The MD simulation for 100 ns affirmed the stable binding of conjugation unprecedentedly through RMSD and radius of gyration plots along with critical reaction coordinate binding free energy profile. Also, dynamical residue cross-correlation map with principal component analysis depicted the stable binding of Nos-Hcq conjugate to Mpro domains with optimal secondary structure statistics of complex dynamics. Also, we reveal the drugs with stable binding to major domains of Mpro can significantly improve the work profile of reaction coordinates, drug accession and inhibitory regulation of Mpro. The designed combinatorial therapy paves way for further prioritized in vitro and in vivo investigations for drug with robust binding against Mpro of SARS-CoV-2.

Perception and determinants leading to antimicrobial (mis)use: A knowledge, attitude, and practices study in the rural communities of Odisha, India.

Background: Antimicrobial resistance (AMR) has emerged as one of the major public health issues globally. This cross-sectional study determined knowledge, attitudes and practices (KAP) regarding antimicrobial use and AMR among rural communities of Tigiria (Odisha), India. Methods: A semi-structured questionnaire based on socio-demographic characteristics, antibiotics usage, awareness of antimicrobial resistance, healthcare utilization and quality of life were asked to the participants using an electronic device with Open Data Kit. Descriptive statistics, independent t-test and ANOVA were performed to analyze the variables. Results: A total of 1,003 participants were surveyed in the study from 25 villages of Tigiria. About 44.47% (95% CI: 41.36-47.60) of study participants have heard about antimicrobial medicines and 14.75% (95% CI: 12.65-17.13) of participants were involved in buying antibiotics without prescription over the counter. Around 20.14% (95% CI: 17.72-22.78) of participants, stopped taking antibiotics before completing the full course. The physical domain was the most affected with low scores compared to other domains of quality of life (QOL). The QOL scores were found significant (p < 0.05) across age, gender, education and ethnicity. Conclusion: The study documented a significant level of KAP regarding antimicrobial (mis)use in the study. It is essential that antimicrobial stewardship programs for various stakeholders and educational programmes must be initiated to increase awareness of people on antimicrobial resistance.

Prevalence of sibling-species of Anopheles (Cellia) fluviatilis complex in Himachal Pradesh, India.

BACKGROUND & OBJECTIVES: Malaria is one of the most infectious and life-threatening vector borne disease in the tropics. Climate change can significantly influence malaria epidemiology and expansion of malaria vectors to hilly regions of Himachal Pradesh in India, hitherto considered areas of low transmission. Entomological surveillance in Kangra district of Himachal Pradesh revealed high density of a proven efficient vector of malaria, Anopheles fluviatilis, but transmission intensity of malaria was found very low. It was therefore considered prudent to investigate the sibling-species composition of An. fluviatilis complex in Kangra valley to ascertain their role in transmission of malaria. METHODS: The study was undertaken in six villages in Kangra district of Himachal Pradesh, India. A total of 4446 mosquitoes were collected during the one-year study period (2018) and processed in pools of ten for molecular characterization. DNA extraction and multiplex PCR was performed on 900 An. fluviatilis mosquitoes for differentiation of sibling-species. ELISA was used to detect Plasmodium falciparum and Plasmodium vivax circumsporozoite proteins in 3790 An. fluviatilis samples. RESULTS: Among prevalent mosquito species, An. fluviatilis was the predominant species constituting 69.5% of total mosquito collection. Sibling-species U was found in 92.22% and species T in 7.78% samples assayed. ELISA confirmed the absence of evidence of malaria parasite in any of the An. fluviatilis mosquitoes screened. Based on the difference in the sequences of conserved regions of the 28SrDNA, sibling-species U was confirmed as prevalent in the study villages. INTERPRETATION & CONCLUSION: Study revealed that in Kangra district, An. fluviatilis sibling-species U is predominant followed by species T, and both are non-vectors. The absence of malaria parasite and zoophagic nature of An. fluviatilis established through blood meal analysis, confirmed that both U and T are non-vector sibling-species.

In Situ Functionalization of Cellulose with Zinc Pyrithione for Antimicrobial Applications.

Considering the public health demands for stronger and effective personal protective clothing, herein, antimicrobial fabrics using a known bacteriostatic and fungistatic drug zinc pyrithione (ZPT) have been reported. ZPT was synthesized in situ on cellulosic fabric, viscose (VC), using a zinc metal precursor and 2-mercaptopyridine-N-oxide as a ligand (VC-ZPT). For comparison, viscose was also phosphorylated (VP) before in situ functionalization with ZPT (VP-ZPT). Both approaches provided adequate protection from microbes; however, functionalization of cellulose with phosphate (VP) resulted in the formation of a linking group between cellulose and ZPT, which exhibited better uniformity of ZPT over the fabric surface and higher durability to washing. The functionalization was confirmed by inductively coupled plasma mass spectroscopy (ICP-MS), scanning electron microscopy (SEM), and Raman spectroscopy. Further, the bonding of phosphate with ZPT was confirmed by 31P solid-state NMR. The physical properties, such as appearance, bending length, and mechanical strength, of the treated fabrics remained unchanged. The antimicrobial activities of VP-ZPT with VC-ZPT were studied against Escherichia coli, Staphylococcus aureus, and Candida albicans, which were found to be effective until 20 laundry cycles in VP-ZPT. Additionally, VP-ZPT samples exhibited poor adherence of bacteria on the fabric surface. The functionalized fabrics may find applications for topical skin diseases in reducing the necessity of repeated use of antibiotic ointments.

Typical and atypical enteropathogenic Escherichia coli in children with acute diarrhoea: Changing trend in East Delhi.

BACKGROUND: Worldwide around 2 million deaths occur every year due to diarrhoeal illnesses among children less than 5 years of age. Among diarrhoeagenic Escherichia coli, Enteropathogenic E. coli (EPEC) is highly prevalent in both community and hospital settings and is one of the main causes of persistent diarrhea in children in developing countries. EPEC remains underdiagnosed in India due to lack of conventional tools for identification. METHODS: We in this study investigated the prevalence and regional variation of EPEC in paediatric population suffering from diarrhoea in East Delhi, India. Two hundred stool samples were collected from children, aged between 0.5 and 5 years, with acute diarrhoea. E. coli were identified by conventional tests and PCR. RESULTS: We observed 7% atypical EPEC (aEPEC) and 2.5% typical EPEC (tEPEC), with an overall 9.5% EPEC prevalence amongst total samples. E. coli phylogenetic group A was the predominant. The most common age group affected was 6-23 months with common symptoms being vomiting, watery diarrhoea and severe dehydration. High drug resistance pattern was observed in EPEC isolates. CONCLUSION: The study depicts a changing trend of aEPEC over tEPEC in children less than 5 years with diarrhoea, an emerging drug resistant enteropathogen and a public health concern demanding monitoring and surveillance.

Integron mediated antimicrobial resistance in diarrheagenic Escherichia coli in children: in vitro and in silico analysis.

The exchange of genes between bacterial chromosome and plasmid(s) and their integration into integrons are mainly responsible for acquisition and dissemination of antibiotic resistance. We investigated the role of integrons and their underlying molecular mechanisms leading to development of adaptability in E. coli and eventual resistance to antimicrobials. Escherichia coli isolates (n = 120); including 40 diarrheagenic isolates, an even number of isolates from cases other than diarrhea, and equal number of isolates from healthy children recovered from fresh stool samples were used for identification of integron genes and gene cassettes. The association of integrons with antibiotic resistance was assayed before phylogenetic analysis. DNA sequence analysis revealed class 1 and 2 integrons in 55.83% and 21.66% isolates, respectively. The integron presence was found significantly associated with the probability of antibiotic resistance in E. coli; the association being highest with class 1 integron. Modelling and molecular docking along with molecular dynamics simulation analyses found ceftriaxone and amoxicillin as potential inhibitors of dihydrofolate reductase (DHFR). The class 1 integrons of these pathogenic isolates can serve as prospective therapeutic targets using specific silencing strategies and combinational antimicrobial therapy. The findings may be useful for the development of a potent and versatile drug for DHFR inhibition.

Changing paradigm of antibiotic resistance amongst Escherichia coli isolates in Indian pediatric population.

Antimicrobial resistance happens when microorganisms mutates in manners that render the drugs like antibacterial, antiviral, antiparasitic and antifungal, ineffective. The normal mutation process is encouraged by the improper use of antibiotics. Mutations leading to quinolone resistance occur in a highly conserved region of the quinolone resistance-determining region (QRDR) of DNA gyrAse and topoisomerase IV gene. We analyzed antibiotic resistant genes and single nucleotide polymorphism (SNP) in gyrA and parC genes in QRDR in 120 E. coli isolates (both diarrheagenic and non-pathogenic) recovered from fresh stool samples collected from children aged less than 5 years from Delhi, India. Antibiotic susceptibility testing was performed according to standard clinical and laboratory standards institute (CLSI) guidelines. Phylogenetic analysis showed the clonal diversity and phylogenetic relationships among the E. coli isolates. The SNP analysis depicted mutations in gyrA and parC genes in QRDR. The sul1 gene, responsible for sulfonamide resistance, was present in almost half (47.5%) of the isolates across the diseased and healthy samples. The presence of antibiotic resistance genes in E. coli isolates from healthy children indicate the development, dissemination and carriage of antibiotic resistance in their gut. Our observations suggest the implementation of active surveillance and stewardship programs to promote appropriate antibiotic use and minimizing further danger.

Transcriptome analysis of beta-lactamase genes in diarrheagenic Escherichia coli.

Beta (ß)-lactamases are the most important agents that confer drug resistance among gram-negative bacteria. Continuous mutations in ß-lactamases make them remarkably diverse. We carried out the transcriptome analysis of 10 ß-lactamase genes of Extended-Spectrum ß-lactamases (ESBL), Metallo ß-lactamases (MBL), and AmpC ß-lactamases (ABL) in drug-resistant and sensitive diarrheagenic E. coli (DEC) isolates obtained from children up to 5 years of age. Out of the 10 ß-lactamase genes, four belonged to ESBL (TEM, SHV, CTX, and OXA); three to MBL (NDM-1, IMP, and VIM); and three to ABL (ACT, DHA and CMY) class of genes. The different categories of DEC were estimated for ß-lactamases production using a set of conventional phenotypic tests, followed by detection of their messenger RNA (mRNA) expression. The study revealed a direct correlation between mRNA expression of these genes and the presence of antibiotic resistance; also corroborated by mutation analysis of the AmpC promoter region. All the 10 ß-lactamase genes showed a significant increase in their expression levels in resistant isolates, compared to those of the sensitive isolates, indicating their possible role in the disease pathogenesis. Increase in mRNA expression of ß-lactamase genes, and thereby virulence, may be due to multifactorial parameters causing phenotypic as well as genotypic changes. Our study highlights the necessity of instantaneous detection of ß-lactamase gene expression to curb the overwhelming threat posed by emergence of drug resistance amongst the commensal E. coli strains in children from developing countries for larger public health interest.

Typical & atypical enteropathogenic Escherichia coli in diarrhoea & their role as carrier in children under five.

BACKGROUND & OBJECTIVES: Multidrug-resistant enteropathogenic Escherichia coli (EPEC) is responsible for a large number of cases of infantile diarrhoea in developing countries, causing failure in treatment with consequent health burden and resulting in a large number of deaths every year. This study was undertaken to determine the proportion of typical and atypical EPEC in under five children with diarrhoea and controls, their function as a carriage and to identify virulent genes associated with them. METHODS: During the study period, 120 stool samples including 80 from controls children were collected and analyzed for the presence of EPEC using standard bacteriological methods. Isolates were subjected to antimicrobial testing by disc diffusion method. Isolates confirmed as E. coli by phenotypic method were further tested for the presence of attaching and effacing (eae) and bundle-forming pilus (bfpA) genes by real-time SYBR Green-based polymerase chain reaction. RESULTS: All isolates were tested for the presence of EPEC. The frequency of typical EPEC was 20 and 16.25 per cent whereas the frequency of atypical EPEC strains was 5 and 23.75 per cent in patients and controls, respectively (PbfpA was seen in 45 and 18.75 per cent isolates of diarrhoeal patients and controls, respectively. INTERPRETATION & CONCLUSIONS: Our results showed that typical EPEC was a common cause of diarrhoea, but at the same time, atypical EPEC was emerging as colonizers in the intestine of children with and without diarrhoea in and around Delhi. Children can be considered asymptomatic carriers of these pathogens and can transmit them to other susceptible children. Adequate steps need to be taken to stop these strains from developing and spreading further.

Spectrum of diarrhoeagenic Escherichia coli in paediatric population suffering from diarrhoea and as commensals in healthy children.

BACKGROUND: Diarrhoeagenic Escherichia coli (DEC) is associated with early death of children in developing countries and are being identified now as an important evolving pathogen. The objective of this study was to perform multiplex polymerase chain reaction (PCR) for simultaneous detection of six categories of DEC in two sets of PCR reactions using 11 virulent genes. MATERIALS AND METHODS: During 1-year study period, forty isolates each from outpatient, inpatient and healthy groups were collected from children. E. coli was identified using conventional biochemical methods. DNA extraction was done using kit, and the extracted DNA was used as a template for multiplex PCR. RESULTS: Virulent genes of DEC were detected in 106 (88.33%) samples. Overall, elt and est were detected in 8.33% and 30.83% of specimens; typical, atypical enteropathogenic E. coli and bfp were detected in 13.33%, 29.16% and 19.16% specimens; eagg was detected in 39.16% and east in 13.33% specimens and stx and hyla were isolated in 1.66% specimens each. While diffusely adherent E. coli and enteroinvasive E. coli genes were not isolated. CONCLUSION: Multiplex PCR is a rapid method for the simultaneous detection of 11 virulent genes of DEC at a time and it will provide a platform in understanding the diarrheal diseases in a more improved manner.

Distribution of Integrons and Phylogenetic Groups among Enteropathogenic Escherichia coli Isolates from Children <5 Years of Age in Delhi, India.

Integrons by means of horizontal gene transfer carry multidrug resistance genes (MDR) among bacteria, including E. coli. The aim of this study was to determine the antibiotic resistance profiles and the genes associated with them, to gain insights in the distribution of phylogroups, prevalence, and characterization of class 1, 2 and 3 integrons among Enteropathogenic E. coli (EPEC) isolates, from children upto 5 years of age from Delhi and National Capital Region (NCR), India. A total of 120 E. coli isolates, including 80 from diarrheagenic E. coli (cases) and 40 from healthy isolates (controls) were recruited in this study. After isolation of E. coli, screening for EPEC was done by conventional multiplex PCR. Antibiotic suseptibility test was performed using disk diffusion method and further confirmed by minimum inhibitory concentration (MICs) by E-test. The presence and characterization of integrons and antimicrobial resistance genes were performed by PCR and DNA sequencing. Phylogeny determination was carried out by quadruplex PCR. EPEC strains were found in 64 of the 80 diarrheagenic cases, out of which 38 were MDR. In the 40 healthy controls, 23 were found to be EPEC strain, out of which only 2 were MDR. Amongst 80 diarrheagenic cases, class 1 integron were observed in 43 isolates, class 2 integron in 12 isolates and 9 isolates were found with co-existence of both. Similarly, in healthy controls; class 1 integron in 9 and class 2 integron in 7 isolates were observed with co-existence in 3 isolates. None of the isolates included class 3 integron. The dfr was the most commonly identified gene cassette within the integron-positive isolates. Phylogenetic studies showed considerable representation of phylogroup B2 in both diarrheagenic cases and healthy controls. This study reiterates the importance of class 1 integron predominantly for acquisition of antibiotic resistance genes among EPEC isolates. Furthermore, it also ascertains the possible association between multidrug resistance and presence of integrons. Approximately 91% of isolates were easily assigned to their respective phylogroups. Assessment of the relationship between antibiotic resistance and dominant phylogroups detected was also attempted. This study also highlights the increased burden of antimicrobial resistance in healthy controls.

Interleukin-6-174G > C (rs1800795) polymorphism distribution and its association with rheumatoid arthritis: A case-control study and meta-analysis.

The association of interleukin-6 (IL-6)-174G > C (rs1800795) single nucleotide polymorphism (SNP) with the risk of acquiring rheumatoid arthritis (RA) is a relevant issue because of conflicting and consensus lacking reports published in literature. We investigated IL-6-174G > C promoter polymorphism in 34 RA patients, attending a tertiary care hospital in north India. We also performed a meta-analysis, of the previously published studies reporting this genetic relationship, in overall population, and independently in Asian and Caucasian ethnicities to further elucidate this association. A total of 13 studies, including the current one, involving 3291 RA cases and 3812 controls were analyzed. Out of the 13 studies, 6 were from Asian, 6 from Caucasian and 1 from a mixed population. Our case-control study showed significant association of IL-6-174G > C SNP with increased RA risk: allelic (OR = 3.750, 95% CI = 1.800-7.813, p < 0.001); dominant (OR = 2.800, 95% CI = 1.167-6.721, p = 0.021); and recessive (OR = 36.72, 95% CI = 2.004-672.7, p = 0.015). The meta-analysis revealed the increased RA risk associated with IL-6-174G > C SNP in overall population: allelic (OR = 1.650, 95% CI = 1.169-2.329, p = 0.004); homozygous (OR = 1.380, 95% CI = 0.906-2.101, p = 0.133); heterozygous (OR = 1.559, 95% CI = 1.001-2.428, p = 0.049); dominant (OR = 1.663, 95% CI = 1.078-2.567, p = 0.022); and recessive (OR = 1.366, 95% CI = 0.964-1.935, p = 0.079). Subgroup analysis also showed this polymorphism to be associated with increased RA risk in Asian population: allelic (OR = 3.724, 95% CI = 1.361-10.190, p = 0.010); dominant (OR = 3.823, 95% CI = 1.320-11.074, p = 0.013); and recessive (OR = 4.357, 95% CI = 1.634-11.623, p = 0.003), but not in Caucasian population. This meta-analysis shows that IL-6-174G > C SNP is significantly associated with increased RA risk in overall, and specifically in Asian population.

Tumor necrosis factor (TNF)-α -308G/A (rs1800629) polymorphism distribution in North India and its association with pemphigus: Case-control study and meta-analysis.

Pemphigus is an autoimmune blistering disorder of skin and/or mucosal surfaces characterized by intraepithelial lesions and immunoglobulin-G autoantibodies against desmogleins (proteins critical in cell-to-cell adhesion). Genetic, immunological, hormonal, and environmental factors are known to contribute to its etiology. Tumor necrosis factor-alpha (TNF-α) which plays a key role in pathogenesis of many infectious and inflammatory diseases has been found in high levels in lesional skin and sera of pemphigus patients. However, studies on association of single nucleotide polymorphism (SNP) in promoter region of TNF-α at position -308 affecting G to A transition with pemphigus has been scarce. This study was conducted to evaluate the TNF-α -308G/A SNP distribution in North Indian cohort, and to define the association between the TNF-α -308G/A SNP distribution and pemphigus, globally, by means of meta-analysis. TNF-α -308G/A SNP in pemphigus patients was investigated by cytokine genotyping using genomic DNA by PCR with sequence-specific primers. Meta-analysis of the data, including four previously published studies from other populations, was performed to generate a meaningful relationship. The results of our case-control study indicate non-significant differences between patients and controls in TNF-α -308G/A SNP. The meta-analysis also revealed that TNF-α -308G/A SNP is not associated with pemphigus risk in population at large; however, it may be contributing towards autoimmune phenomenon in pemphigus by being a part of its multi-factorial etiology. This study provides evidence that the TNF-α -308G/A polymorphism is not associated with overall pemphigus susceptibility. Nevertheless, further studies on specific ethnicity and pemphigus variants are necessary to validate the findings.