Identification of circulating miRNA biomarkers in leptospirosis for early detection: A promising diagnostic approach.

Leptospirosis is a zoonotic disease of global significance, contributing to morbidity and mortality worldwide. It is endemic to tropical regions, with outbreaks during monsoons. The disease manifestations are similar to that of other febrile illness such as dengue, malaria hence often misdiagnosed and underreported. The zoonoses if undetected, progresses to cause severe life-threatening complications also known as Weil's disease. Routine diagnostic tests are based on the detection of antibodies in patient serum and are not accurate during the initial phase of the infection. Therefore, it is necessary to detect novel biomarkers that can be used in early detection of leptospirosis. Circulating miRNAs are known to be promising biomarkers for various diseases including cancer, tuberculosis, influenza; hence in this study the potential of miRNAs as biomarkers for leptospirosis was evaluated. A total of 30 leptospirosis cases were screened for the differential expression of 10 miRNA by RT-qPCR assay. The differential expression was calculated by relative quantification using healthy individuals as controls. Among the 10 miRNA,3 miRNA, miR-28-5p, miR-302c-3p and miR-302a-3p were reported to exhibit a significant trend of upregulation. Further their role in immune pathways and biological processes was investigated by KEGG analysis and Gene Ontology. The 3 miRNAs were observed to target various immune response pathways, thus confirming their role in host immune response. Based on the results obtained in this study, miR-28-5p, miR-302c-3p and miR-302a-3p can be considered as potential biomarkers for the detection of leptospirosis.

Non-clinical isolates of Vibrio parahaemolyticus harbouring traits of potential pathogenicity and fitness: A molecular analysis.

Here we investigated the distribution of virulence and fitness attributes V. parahaemolyticus isolated from marine environment (n = 105). We discovered ∼1% of isolates positive for tdh, 8.57% for trh, and 4.76% had tdh and trh genes. More than 50% of the isolates had pathogenicity islands specific to pandemic clones and secretion systems which are detected partially or entirely. VPaI-1 found in 59.04%; VPaI-4 in 60%; VPaI-5 in 34.28%; VPaI-2 in 99.04%; VPaI-3 in 91.42% and VPaI-6 in 99.04% isolates. Also, 34.28% of the isolates harboured T3SS2 encoding VPaI 7; T3SS1 in 98.09%; T6SS2 in 99.04% isolates and T6SS1 in 60.95% isolates. The cytotoxicity analysis showed a significant effect by causing when infected with trh+ environmental isolates. The expression of the trh, VopC, and VopA genes during infection showed a significant upregulation. This suggests the presence of virulence traits among V. parahaemolyticus that could threaten public health.

Isothermal amplification-based assays for rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2: Opportunities and recent developments.

The coronavirus disease 2019 (COVID-19) is a global pandemic caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, the virus has been detected in 219 countries of the world. Therefore, managing the disease becomes the priority, in which detecting the presence of the virus is a crucial step. Presently, real-time RT polymerase chain reaction (RT-qPCR) is considered a gold standard nucleic acid amplification test (NAAT). The test protocol of RT-qPCR is complicated, places high demands on equipment, testing reagents, research personnel skills and is expensive. Therefore, simpler point-of-care (POC) tests are needed to accelerate clinical decision-making and take some of the workload from centralized test laboratories. Various isothermal amplification-based assays have been developed for the sensitive detection of different microorganisms, and recently some of them have been applied for detection of SARS-CoV-2. These do not require any programable thermocycler, can produce the results in a single temperature, and therefore, are considered simple. Unlike RT-qPCR, these methods are highly sensitive, specific, less time-consuming, simple and affordable, and can be used as POC diagnostic kit for COVID-19. In this review, we have discussed the potential of isothermal amplification-based assays as an alternative to RT-qPCR for the detection of SARS-CoV-2.

Integrons as the potential targets for combating multidrug resistance in Enterobacteriaceae using CRISPR- Cas9 technique.

The emergence of multi-drug resistance (MDR) to pan-drug resistance (PDR) in Enterobacteriaceae has made treatment extremely challenging. Genetic mutations and horizontal gene transfer (HGT) through mobile genetic elements (MGEs) were frequently associated mechanisms of drug resistance in pathogens. However, transposons, plasmids, and integrons transfer MDR genes in bacterium via HGT much faster. Integrons are dsDNA segment that plays a crucial role in the adaptation and evolution of bacteria. They contain multiple gene cassettes that code for antibiotic resistance determinants that are expressed by a single promoter (Pc). Integrons are the cause of drug resistance in Enterobacteriaceae. Although alternatives to antibiotics such as bacteriophages, phage proteins, antimicrobial peptides, and natural compounds have been widely used to treat MDR infections, there have been limited efforts to reverse the antibiotic resistance ability of bacteria. Thus, silencing the genes harboured on MGEs achieved by Gene Editing Techniques (GETs) might prevent the spread of MDR. One such GETs, which has a simple design, good repeatability, low cost, and high efficiency, is CRISPR- Cas9 system. Thus, this review is a first of the kind that focuses on utilizing the structure of an integron to make it an ideal target for GETs like CRISPR- Cas9 systems.

Role of oral microbiota in irreversible pulpitis - Current strategies and future perspectives.

Irreversible pulpitis is an inflammation of the tooth pulp caused by an opportunity-driven invasion of the pulp space by oral microbiota typically prevalent in the oral cavity. Microbial organisms are extensively recognised to be the fundamental cause of endodontic infections and treatment failures. Previously, bacterial species responsible for these infections were largely recognised using conventional microbial culture techniques, lending credence to the widely held belief that anaerobic Gram-negative bacteria frequently enter the pulp space and trigger endodontic infections. The advent of novel technologies grants the advantage of detecting and studying microbial populations via an amalgamation of the modern "Omics" techniques and meticulous bioinformatics analysis, additionally detecting the metatranscriptome, metaproteome and metabolome along with the metagenome. Amongst these analytical strategies, metagenomic analyses are essentially pragmatic for investigating the oral microbiome. Metagenomics favor not only assessment of microbial composition in diseased conditions, but also contributes to detection of novel, potentially pathogenic species inclusive of non-viable bacteria. The present review describes current knowledge of root canal microbiome, including its composition and functional attributes, the novel strategies available for detection of microbiome as well as challenges associated and provides some crucial pointers for areas of future research.

Characterisation of broad-spectrum phiKZ like jumbo phage and its utilisation in controlling multidrug-resistant Pseudomonas aeruginosa isolates.

The emergence of highly virulent multidrug-resistant P. aeruginosa has become increasingly evident among hospital-acquired infections and has raised the need for alternative therapies. Phage therapy can be one such alternative to antibiotic therapy to combat multidrug-resistant pathogenic bacteria, but this requires the availability of phages with a broad host range. In this study, isolation and molecular characterisation of P. aeruginosa specific phages were carried out. A total of 17 phages isolated showed different spectra of activity and efficiency of lysis against 82 isolates of P. aeruginosa obtained from clinical samples (n = 13), hospital effluent (n = 46) and fish processing plant effluent (n = 23). Antibiotic susceptibility test results revealed multi-drug resistance in 61 of the total 82 isolates. Three new jumbo lytic P. aeruginosa specific broad host range phages were isolated and characterised in this present study belonged to the family Myoviridae (order Caudovirales). The genetic analysis of ɸU5 revealed that phage has a genome size of 282.6 kbp with 373 putative open reading frames (ORFs), and its genetic architecture is similar to phiKZ like jumbo phages infecting P. aeruginosa. The bacteriophages isolated in this study had lytic ability against biofilm-forming and multidrug-resistant P. aeruginosa and could be candidates for further studies towards phage therapy.

Effect of NaCl, high iron, iron chelator and antibiotics on growth, virulence gene expression and drug susceptibility in non-typhoidal Salmonella: an in vitro fitness study.

Salmonella is one among the most versatile and resilient enteric pathogens that is known to have developed various survival strategies within the host system. The ability of the bacteria to circumvent the physiological parameters as well as dodge the antimicrobial stress environment within the host is one of the most crucial steps in establishing an infection. With an alarming rise in multi-drug resistant serovars of non-typhoidal Salmonella and lack of vaccine for combatting the infections, behaviour of the bacteria in the presence of host physiological conditions (NaCl, high and low iron) and antibiotics will help in understanding the survival strategies as well as mechanisms of resistance. Two multi-drug resistant and two sensitive serovars of Salmonella Weltevreden and Salmonella Newport isolated from poultry and seafood were used for growth kinetics and virulence gene expression study. The results obtained revealed that despite similar resistance pattern, effect of individual class of antibiotics on the growth of serovars varied. On the contrary, no significant difference was observed in growth pattern on exposure to these in vitro experimental conditions. Nevertheless, coupling these conditions with antibiotics drastically reduced the minimum inhibitory concentration (MIC) of antibiotics in resistant strains. A first of its kind study that draws attention on the significant effect of antibiotics and physiological conditions on MIC between resistant and sensitive non-typhoidal Salmonella serovars and expression of virulence genes from Salmonella pathogenicity island (SPI) 1 and 2 (invA, hilC, fliC2, sseA and ssrB).

Identification and evaluation of an appropriate housekeeping gene for real time gene profiling of hepatocellular carcinoma cells cultured in three dimensional scaffold.

BACKGROUND: Assessing an optimal reference gene as an internal control for target gene normalization is important during quantitative real time polymerase chain reaction (RT-qPCR) of three dimensional (3D) cell culture. Especially, gene profiling of cancer cells under a complex 3D microenvironment in a polymer scaffold provides a deeper understanding of tumor functioning in vivo. METHODS AND RESULTS: Expression of six housekeeping genes (HKG's): Glyceraldehyde-3-phosphodehydrogenase (GAPDH), ß-actin (ACTB), beta-2-microglobulin (B2M), 18S ribosomal RNA (18S rRNA), peptidyl-propyl-isomerase A (PPIA), and ribosomal protein L13 (RPL-13) during two dimensional (2D) culture, and alginate-carboxymethylcellulose scaffold based 3D culture conditioned up to 21 days was analysed for hepatocellular carcinoma (Huh-7) cells. The gene expression studies were performed by determining primer efficiency, melting curve and threshold cycle analysis. Further, RT-qPCR data was validated statistically using geNorm and NormFinder softwares. The study indicated RPL-13, 18S rRNA and B2M to be stable among selected referral HKG candidates. CONCLUSION: An exploration of a reliable HKG is necessary for normalization of gene expression in RT-qPCR during varying cell culture conditions.

Synthesis and inhibition studies towards the discovery of benzodiazepines as potential antimalarial compounds.

The target-based discovery of therapeutics against apicoplast, an all-important organelle is an overriding perspective. MEP pathway, an accredited drug target provides an insight into the importance of apicoplast in the survival of the parasite. In this study, we present the rational design strategy employing sustainable catalysis for the synthesis of benzodiazepine (BDZ) conformers followed by their biological evaluation as prospective inhibitors against the potential target of the IPP pathway, 1-deoxy-D-xylulose-5-phosphatereductoisomerase (DXR). The study reported the inhibitory profile of 8c and 6d against the quintessential step of the only drug target in the erythrocytic stages of parasite development. The potential compounds were identified to represent a novel class of inhibitors that serve as the lead molecules to impede the pathway and further affect the survival of the parasite.

Phenotypic characterization of auxotrophic mutant of nontyphoidal Salmonella and determination of its cytotoxicity, tumor inhibiting cytokine gene expression in cell line models.

An auxotrophic mutant of nontyphoidal Salmonella (NTS) strain (Salmonella Oslo) was phenotypically characterized in this study. The characterization was based on phenotype, morphology, motility, biofilm forming ability, growth kinetics, etc. The phenotypic results from the above experiments determined that the mutant showed variation in phenotypic characters from that of wild-type strain. Subsequently, mutant and wild-type NTS were subjected to epithelial cell invasion and intracellular replication assays. The real-time PCR analysis was also performed to analyse expression of tumor inhibiting cytokine genes and virulence genes post-bacterial infection in cell lines. The mutant showed highest invasion potential than wild-type NTS whereas the replication of mutant was slower in both the cell lines. Similar to the wild-type strain, the mutant also retained the cytotoxic potential when analysed in vitro. Furthermore, the expression of proinflammatory cytokine genes such as TNF-α and IL-1ß was upsurged with the downregulation of anti-inflammatory cytokine genes like TGF-ß, IL-6 and IL-10 post-infection of the mutant strain in cell lines. In addition, virulence genes of Salmonella pathogenicity island one and two of mutant were downregulated in vitro except invA in HeLa cell line. Therefore, the auxotrophic mutant showed positive attributes of a potential antitumor agent in terms of expressing tumor inhibiting cytokine genes when assessed in vitro. Though the study did not check the tumor inhibitory effect of NTS strain directly, findings of the study emphasizes on the development of a novel strain of NTS with less virulence and more immunogenic traits to inhibit tumor cells.

Detection technologies and recent developments in the diagnosis of COVID-19 infection.

COVID-19 is a disease caused by SARS-CoV-2 capable of causing mild to severe infections in humans. Since its first appearance in China in December 2019, the pandemic has spread rapidly throughout the world. Despite considerable efforts made to contain the disease, the virus has continued its prevalence in many countries with varying degrees of clinical manifestations. To contain this pandemic, collaborative approach involving accurate diagnosis, epidemiology, surveillance, and prophylaxis is essential. However, proper diagnosis using rapid technologies plays a crucial role. With increasing incidence of COVID-19 cases, the accurate and early detection of the SARS-CoV-2 is need of the hour for effective prevention and management of COVID-19 cases as well as to curb its spread. RT-qPCR assay is considered to be the gold standard for the early detection of virus, but this protocol has limited application to use as bedside test because of its technical complexity. To address these challenges, several POC assays have been developed to facilitate the COVID-19 diagnosis outside the centralized testing laboratories as well to accelerate the clinical decision making with a least turnaround time. Hence, in this report, we review different nucleic acid-based and serological techniques available for the diagnosis and effective prevention of COVID-19. KEY POINTS : • Provides comprehensive information on the different diagnostic tools available for COVID-19 • Nucleic acid based tests or antigen detection tests are used for diagnostic purpose • Accurate diagnosis is essential for the efficient management of COVID-19.

Recombinant viral proteins delivered orally through inactivated bacterial cells induce protection in Macrobrachium rosenbergii (de Man) against White Tail Disease.

White tail disease (WTD) is a disease of Macrobrachium rosenbergii caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) with the potential to devastate the aquaculture industry. The present study aimed to explore the possible protection of M. rosenbergii against the disease by oral administration of bacterially expressed recombinant capsid proteins of MrNV and XSV. Juvenile M. rosenbergii were fed with the feed coated with inactivated bacteria encapsulated expressed recombinant viral proteins either individually or in combination for 7 days. Challenge studies using WTD causing agents were carried out after 3 (group I), 10 (group II) and 20 (group III) days post-feeding of viral proteins. Recombinant capsid protein of MrNV showed better protection when compared to other treatments with relative per cent survival of 62.5% (group I), 57.9% (group II) and 39.5% (group III). Treatment controls of groups I, II and III showed 100%, 95% and 95% mortality, respectively. The study demonstrates that oral administration of recombinant capsid proteins of MrNV and XSV provides effective protection against WTD in freshwater prawn.

Revolutionizing the diagnosis of irreversible pulpitis - Current strategies and future directions.

BACKGROUND: Pulpitis primarily arises from the pulp space infection by oral microbiota. Vital pulp therapy is a minimally invasive approach that relies on assessing the severity of pulpal inflammation to facilitate repair. However, the current evaluation methods prescribed by the American Association of Endodontics are subjective, leading to ambiguity in assessment. Therefore, this review aims to explore molecular strategies for evaluating the severity of pulpal inflammation to accurately predict the success of pulp vitality preservation in clinical settings. METHODOLOGY: This review was conducted by searching relevant keywords, such as irreversible pulpitis, pulpitis biomarkers, molecular diagnosis, inflammation, and genomic strategies, in databases such as PubMed, Web of Science, and Scopus to address the subjective nature of diagnosis. The data included in this review were collected up to April 2023. The literature search revealed well-documented limitations in clinically assessing the pulp inflammatory. Molecular approaches that aid in clinical differentiation between irreversible and reversible pulpitis may potentially enhance favorable outcomes in vital pulp therapy. Non-invasive diagnostic methods for pulpal assessment would also be valuable for determining whether the inflamed pulp is reversible, irreversible, or necrotic. CONCLUSION: The present review examines the various molecular diagnostic approaches that have revolutionized the medical field and are considered the most promising empirical methodologies for the proactive detection of pulpal diseases. It also provides comprehensive insights into the current diagnostic methods, associated challenges, next-generation strategies, and future directions for diagnosing the severity of pulp inflammation.

Astaxanthin and DHA Supplementation Modulates the Maternal Undernutrition-induced Impairment of Cognitive Behavior and Synaptic Plasticity in Adult Life of Offspring's -Exploring the Molecular Mechanism.

Maternal nutrition was recognized as a significant part of brain growth and maturation in most mammalian species. Timely intervention with suitable nutraceuticals would provide long-term health benefits. We aim to unravel the molecular mechanisms of perinatal undernutrition-induced impairments in cognition and synaptic plasticity, employing animal model based on dietary nutraceutical supplementation. We treated undernourished dams at their gestational, lactational, and at both the time point with Astaxanthin (AsX) and Docosahexaenoic acid (DHA), and their pups were used as experimental animals. We evaluated the cognitive function by subjecting the pups to behavioral tests in their adult life. In addition, we assessed the expression of genes in the hippocampus related to cognitive function and synaptic plasticity. Our results showed downregulation of Brain-derived neurotrophic factor (BDNF), Neurotrophin-3 (NT-3), cAMP response-element-binding protein (CREB), and uncoupling protein-2 (UCP2) gene expression in pups born to undernourished dams in their adult life, which AsX and DHA modulated. Maternal AsX and DHA supplementation ameliorated the undernutrition-induced learning impairment in novel object recognition (NOR) tests and partially baited radial arm maze (RAM) tasks in offspring's. The expressions of Synapsin-1 and PSD-95 decreased in perinatally undernourished groups compared to control and AsX-DHA treated groups at CA1, CA2, CA3, and DG. AsX and DHA supplementation upregulated BDNF, NT-3, CREB, and UCP2 gene expressions in perinatally undernourished rats, which are involved in intracellular signaling cascades like Ras, PI3K, and PLC. The results of our study give new insights into neuronal differentiation, survival, and plasticity, indicating that the perinatal period is the critical time for reversing maternal undernutrition-induced cognitive impairment in offspring's.

Identification of Dominant Leptospira Serogroups among Leptospirosis Cases and Their Clinical Outcomes: A Prospective Hospital-Based Study in Mangaluru, India.

Leptospirosis is a reemerging zoonotic disease of worldwide significance, endemic to the southern region of India, with clinical manifestations similar to other febrile illnesses; hence, it is often misdiagnosed and underreported. Inadequate information about the disease burden and the regional circulating serogroups contributes to its neglected disease status. This study aimed to identify the infecting Leptospira serogroup in the coastal region of Mangaluru and study the clinical symptoms and outcome among leptospirosis patients. Serum samples were collected from 30 patients with confirmed leptospirosis admitted to a tertiary care center in Mangaluru and screened by microscopic agglutination test (MAT) for the infecting serogroup. The clinical profile of these cases was reviewed, and data regarding epidemiological factors such as age, sex, complications, and mortality were recorded. The MAT identified a higher occurrence of serogroup Bataviae (n = 7, 43.75%) and serogroup Australis (n = 5, 31.25%) compared with other serogroups screened in this study population. Patients were aged 16 to 65 years, with a predominance of males. The clinical presentation of leptospirosis ranged from a mild febrile illness to multiorgan failure. Fever (n = 29, 96%) was the common clinical presentation, followed by myalgia, nausea, and abdominal pain. Acute kidney injury, acute respiratory distress syndrome, and multiple organ dysfunction syndrome were the common complications observed. Determining the circulating serogroup is necessary to understand the epidemiology and diversity of Leptospira serogroups among animals and humans to strategize appropriate preventive measures.

Astaxanthin and DHA supplementation ameliorates the proteomic profile of perinatal undernutrition-induced adipose tissue dysfunction in adult life.

Maternal diet is an essential factor that directly and indirectly regulates fetal growth. Exposure to certain environmental conditions substantially impacts an individual's short- and long-term health. Adipose tissue dysfunction is a worldwide chronic disease caused by improper lipid build-up in adipose tissue leading to obesity. Therefore, it is the need of the hour to invent anti-obesity agents. As a keto-carotenoid, Astaxanthin (AsX) has been shown to have preventive effects against problems associated with obesity. A crucial role in the pathogenesis of obesity has been attributed to dietary polyunsaturated fatty acids. Adipose tissue plays a vital role in maintaining overall body homeostasis. Metabolic dysfunction of white adipocytes forms a critical step in the emergence of insulin resistance and related diseases. Here we aim to investigate the effect of AsX and Docosahexaenoic acid (DHA) supplementation on the proteomic profile of perinatal undernutrition-induced adipose tissue dysfunction in adult life using a rat model. The LC-MS/MS quantitative proteomics enabled us to identify differentially expressed proteins in perinatal undernourished but AsX and DHA-supplemented animal models. Data are available via ProteomeXchange with identifier PXD041772.This study explored biological roles, molecular functions of differentially expressed proteins, and pathways related to adipose tissue dysfunction induced by undernutrition and its effective modulation by AsX and DHA.

An insight into clinical and laboratory detections for screening and diagnosis of cervical cancer.

INTRODUCTION: Cervical cancer is the most frequent malignancy among women caused by an unresolved long-term infection with distinct human papillomavirus (HPV) genotypes. It is the fourth most common form of cancer among women worldwide. The two oncogenic genotypes, HPV 16 and 18, are responsible for >70% of all cervical cancers worldwide. Cervical cancer is one of the most successfully preventable and treatable forms of cancer if detected early. AREAS COVERED: In this review article, we have summarizedsummarised the different approaches used in clinical diagnosis and research laboratories to detect HPV-related changes associated with cervical cancer for a better understanding of the advantages and limitations of these tests. EXPERT OPINION: Despite the well-known screening strategies for cervical cancer, developing nations lack effective implementation due to various factors. With the current rate of cervical cancer cases, precise and timely identification of HPV can significantly impact the prevention and efficient management of cervical cancer. Cervical cancer is the most common gynecological cancer in developing countries. The primary screening test with cytology and molecular testing of HPV is important for preventing cervical cancer. To address these issues, several point-of-care assays have been developed to facilitate rapid screening of HPV with the least turnaround time.

Genome analysis of clinical genotype Vibrio vulnificus isolated from seafood in Mangaluru Coast, India provides insights into its pathogenicity.

Vibrio vulnificus an opportunistic human pathogen native to marine/estuarine environment, is one of the leading causes of death due to seafood consumption and exposure of wounds to seawater worldwide. The present study involves the whole genome sequence analysis of an environmental strain of V. vulnificus (clinical genotype) isolated from seafood along the Mangaluru coast of India. The sequenced genome data was subjected to in-silico analysis of phylogeny, virulence genes, antimicrobial resistance determinants, and secretary proteins using suitable bioinformatics tools. The sequenced isolate had an overall genome length of 4.8 Mb and GC content of 46% with 4400 coding DNA sequences. The sequenced strain belongs to a new sequence type (Multilocus sequence typing) and was also found to branch with a phylogenetic lineage that groups the most infectious strains of V. vulnificus. The seafood isolate had complete genes involved in conferring serum resistance yet showed limited serum resistance. The study identified several genes against the antibiotics that are commonly used in their treatment, highlighting the need for alternative treatments. Also, the secretory protein analysis revealed genes associated with major pathways like ABC transporters, two-component systems, quorum sensing, biofilm formation, cationic antimicrobial peptide (CAMP) resistance, and others that play a critical role in the pathogenesis of the V. vulnificus. To the best of our knowledge, this is the first report of a detailed analysis of the genomic information of a V. vulnificus isolated from the Indian subcontinent and provides evidence that raises public health concerns about the safety of seafood.

Sequence-Specific Gene Silencing of acrA in the Multi-drug Efflux System AcrAB Induces Sensitivity in Drug-Resistant Klebsiella pneumoniae.

Multi-drug efflux is one of the resistant determinants in Klebsiella pneumoniae that are encountered in a broad range of clinically relevant antimicrobial agents. An alternative method to strategically induce sensitivity in drug-resistant K. pneumoniae and improve the efficacy of the existing antibiotics is the need of the hour. Hence, an antisense RNA was designed against the acrA gene of the AcrAB-TolC efflux system in a drug-resistant isolate of K. pneumoniae obtained from a blood culture. Minimum inhibitory concentration by E test demonstrated that the antisense RNA could significantly increase the susceptibility of previously resistant K. pneumoniae toward ciprofloxacin (CIP) and co-trimoxazole. Real-time PCR determined the ability of the antisense RNA to inhibit the expression of the acrA-mRNA. The wild-type K. pneumoniae showed increased growth in the presence of CIP, while, under the same condition, the growth of the antisense RNA-treated K. pneumoniae was inhibited up till 12 h. In the presence of co-trimoxazole, delayed growth rate of the antisense RNA-treated K. pneumoniae was seen, in comparison to that of the wild-type K. pneumoniae and also a fourfold reduction was noted in the expression of the efflux gene acrA. Our results underscore the potential of the acrA antisense RNA as an alternative therapeutic against multi-drug-resistant K. pneumoniae.

Evaluation of reverse transcriptase-polymerase spiral reaction assay for rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2.

BACKGROUND AND AIM: Existing real-time reverse transcriptase PCR (RT-qPCR) has certain limitations for the point-of-care detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since it requires sophisticated instruments, reagents and skilled laboratory personnel. In this study, we evaluated an assay termed the reverse transcriptase-polymerase spiral reaction (RT-PSR) for rapid and visual detection of SARS-CoV-2. METHODS: The RT-PSR assay was optimized using RdRp gene and evaluated for the detection of SARS-CoV-2. The time of 60min and a temperature of 63°C was optimized for targeting the RNA-dependent RNA polymerase gene of SARS-CoV-2. The sensitivity of the assay was evaluated by diluting the in-vitro transcribed RNA, which amplifies as low as ten copies. RESULTS: The specific primers designed for this assay showed 100% specificity and did not react when tested with other lung infection-causing viruses and bacteria. The optimized assay was validated with 190 clinical samples in two phases, using automated RTPCR based TrueNat test, and the results were comparable. CONCLUSIONS: The RT-PSR assay can be considered for rapid and sensitive detection of SARS-CoV-2, particularly in resource-limited settings. To our knowledge, there is as yet no RT-PSR-based kit developed for SARS-CoV-2.