Orientia tsutsugamushi: An Unusual Intracellular Bacteria-Adaptation Strategies, Available Antibiotics, and Alternatives for Treatment.

During evolution Orientia tsutsugamushi became a smarter obligate bacterium to establish as intracellular pathogens. O. tsutsugamushi is a human pathogenic bacterium responsible for 1 billion infections of scrub typhus. Several novel mechanisms make this bacterium unique (cell wall, genetic constitutions, secretion system, etc.). In 2007, O. tsutsugamushi Boryong was pioneer strain for whole-genome sequencing. But the fundamental biology of this bacterial cell is a mystery till date. The unusual biology makes this organism as model for host cell interaction. Only a few antibiotics are effective against this intracellular pathogen but emergence of less susceptibility toward antibiotics make the situation alarming. The review was captivated to highlight the unusual aspects of adaptation, antibiotics, and drugs beyond antibiotics.

Production and testing of mahua oil-based biodiesel synthesized through heterogeneous catalyst using experimental and numerical method.

A two-step treatment  of mahua oil was conducted to synthesize mahua biodiesel using heterogeneous biomass-based catalyst derived from mahua shell. Mahua oil having higher free fatty acid (FFA) content (about 19%) was esterified to reduce the FFA content up to 1%. The esterification process was carried out using 200 mL mahua oil, 5:1 molar ratio (methanol:oil), and 2.25 weight% of H2SO4 at a temperature of 60 °C for 3 h. Post esterification, a set of 16 experiments were created using a Box-Behnken design (BBD)-based response surface methodology (RSM) approach to conduct the transesterification of the esterified oil. Molar ratio, catalyst loading, reaction temperature, and reaction time were the four input variables chosen for the design of experiments. The optimized conditions for maximum biodiesel yield (87.7%) were found to be 14.88 molar ratio, 3.578% catalyst loading, 69.7 °C reaction temperature, and 81.9 min reaction time. The Diesel RK engine simulation tool which was experimentally validated for baseline diesel fuel was used for numerical simulation of mahua biodiesel. The performance, combustion, and emission behavior of mahua biodiesel analyzed using numerical simulation presented the sustainability of mahua biodiesel as an alternate fuel.

Molecular and serological evidence of chikungunya virus infection with high case fatality among pediatric population with acute encephalitis syndrome: first report from Eastern Uttar Pradesh, India.

Acute encephalitis syndrome (AES) outbreaks in children of Eastern Uttar Pradesh (E-UP) region of India have been a longstanding public health issue, with a significant case fatality rate of 20-25%. Since past decade, a rise in chikungunya (CHIK) cases has been occurring, which is a reported etiology of AES. However, the burden of chikungunya virus (CHIKV) among pediatric AES (pAES) is unknown from E-UP. We included 238 hospitalized pAES cases. The presence of IgM antibodies for CHIKV, and Dengue virus (DENV) was tested, and RT-PCR was performed for CHIKV and DENV in serologically confirmed CHIKV and DENV pAES cases. Positive samples were sequenced using Sangers sequencing. Further, to check for co-infection, IgM antibodies for other AES etiologies including Japanese encephalitis virus (JEV), Leptospira and Orientia tsutsugamushi (OT) in serum were also investigated. IgM ELISA demonstrated 5.04% (12) positivity for CHIKV. Among CHIKV IgM positive, 3 (25%, 3/12) pAES patients died. CHIKV genome was detected in 3 pAES specimens. Among which, 2 CHIKV cases were also positive for OT DNA. Partially sequenced CHIKV were genotyped as ECSA. The overall finding indicates evidence of CHIKV infection with high case fatality among pAES patients from E-UP. This study advocates constant serological and molecular surveillance of CHIKV in AES endemic regions of India.

Integration of IgM ELISA and 56 kDa gene PCR in management of pediatric acute encephalitis syndrome associated with scrub typhus.

OBJECTIVES: To identify the potential target genes for detection of Orientia tsutsugamushi (OT) in pediatric acute encephalitis syndrome (pAES). METHODS: DNA was extracted from whole blood of 100 pAES cases having tested positive (n = 41) and negative (n = 59) for scrub typhus (ST) by IgM ELISA. These samples were subjected to standard PCR for 56 kDa, 47 kDa, 16 s rRNA, groEL, traD genes and the newly identified 27 kDa gene. RESULTS: Among the selected gene targets, 56 kDa demonstrated its superiority for OT detection over the other tested genes. The presence of OT was confirmed via PCR targeting 56 kDa gene in 17 out of the 41 (41.4 %) IgM-positive ST AES cases and 38 out of the 59 (64.4 %) ST IgM negative cases. None of the other gene targets were amplified. CONCLUSION: Integration of serological diagnosis with molecular diagnostics targeting the 56 kDa gene for routine testing of AES patients would facilitate detection of OT in AES endemic regions.

Synergistic Antibacterial Activity of Gallic Acid Based Chalcone Indl 2 by Inhibiting Efflux Pump Transporters.

As a part of novel discovery of drugs from natural resources, present study was undertaken to explore the antibacterial potential of chalcone Indl-2 in combination with different group of antibiotics. MIC of antibiotics was reduced up to eight folds against the different cultures of E. coli by both chalcones. Among the two compounds, the i. e. 1-(3', 4,'5'-trimethoxyphenyl)-3-(3-Indyl)-prop-2-enone (6, Indl-2), a chalcone derivative of gallic acid (Indl-2) was better along with tetracycline (TET) worked synergistically and was found to inhibit efflux transporters as obvious by ethidium bromide efflux confirmed by ATPase assays and docking studies. In combination, Indl-2 kills the MDREC-KG4 cells, post-antibiotic effect (PAE) of TET was prolonged and mutant prevention concentration (MPC) of TET was also decreased. In-vivo studies revealed that Indl-2 reduces the concentration of TNF-α. In acute oral toxicity study, Indl-2 was non-toxic and well tolerated up-to dose of 2000 mg/kg. Perhaps, the study is going to report gallic acid derived chalcone as synergistic agent acting via inhibiting the primary efflux pumps.

CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub Typhus.

Orientia tsutsugamushi is responsible for causing scrub typhus (ST) and is the leading cause of acute encephalitis syndrome (AES) in AES patients. A rapid and sensitive method to detect scrub typhus on-site is essential for the timely deployment of control measures. In the current study, we developed a rapid, sensitive, and instrument-free lateral flow assay (LFA) detection method based on CRISPR/Cas12a technology for diagnosing ST (named LoCIST). The method is completed in three steps: first, harnessing the ability of recombinase polymerase for isothermal amplification of the target gene; second, CRISPR/Cas12a-based recognition of the target; and third, end-point detection by LFA. The detection limit of LoCIST was found to be one gene copy of ST genomic DNA per reaction, and the process was complete within an hour. In 81 clinical samples, the assay showed no cross-reactivity with other rickettsial DNA and was 100% consistent with PCR detection of ST. LoCIST demonstrated 97.6% sensitivity and 100% specificity. Overall, the LoCIST offers a novel alternative for the portable, simple, sensitive, and specific detection of ST, and it may help prevent and control AES outbreaks due to ST. In conclusion, LoCIST does not require specialized equipment and poses a potential for future applications as a point-of-care diagnostic.

Prevalence and factors associated with tuberculosis infection in India.

BACKGROUND: The risk of tuberculosis (TB) disease is higher in individuals with TB infection. In a TB endemic country like India, it is essential to understand the current burden of TB infection at the population level. The objective of the present analysis is to estimate the prevalence of TB infection in India and to explore the factors associated with TB infection. METHODS: Individuals aged > 15 years in the recently completed National TB prevalence survey in India who were tested for TB infection by QuantiFERON-TB Gold Plus (QFT-Plus) assay were considered for this sub-analysis. TB infection was defined as positive by QFT-Plus (value >0.35 IU/ml). The estimates for prevalence, prevalence ratio (PR) and adjusted risk ratio (aRR) estimates with 95% confidence intervals (CIs) were calculated. RESULTS: Of the 16864 individuals analysed, the prevalence of TB infection was 22.6% (95% CI:19.4 -25.8). Factors more likely to be associated with TB infection include age > 30 years (aRR:1.49;95% CI:1.29-1.73), being male (aRR:1.26; 95%CI: 1.18-1.34), residing in urban location (aRR:1.58; 95%CI: 1.03-2.43) and past history of TB (aRR:1.49; 95%CI: 1.26-1.76). CONCLUSION: About one fourth (22.6%) of the individuals were infected with TB in India. Individuals aged > 30 years, males, residing in urban location, and those with past history of TB were more likely to have TB infection. Targeted interventions for prevention of TB and close monitoring are essential to reduce the burden of TB in India.

Optimization of nutritional strategies using a mechanistic computational model in prediabetes: Application to the J-DOIT1 study data.

Lifestyle interventions have been shown to prevent or delay the onset of diabetes; however, inter-individual variability in responses to such interventions makes lifestyle recommendations challenging. We analyzed the Japan Diabetes Outcome Intervention Trial-1 (J-DOIT1) study data using a previously published mechanistic simulation model of type 2 diabetes onset and progression to understand the causes of inter-individual variability and to optimize dietary intervention strategies at an individual level. J-DOIT1, a large-scale lifestyle intervention study, involved 2607 subjects with a 4.2-year median follow-up period. We selected 112 individuals from the J-DOIT1 study and calibrated the mechanistic model to each participant's body weight and HbA1c time courses. We evaluated the relationship of physiological (e.g., insulin sensitivity) and lifestyle (e.g., dietary intake) parameters with variability in outcome. Finally, we used simulation analyses to predict individually optimized diets for weight reduction. The model predicted individual body weight and HbA1c time courses with a mean (±SD) prediction error of 1.0 kg (±1.2) and 0.14% (±0.18), respectively. Individuals with the most and least improved biomarkers showed no significant differences in model-estimated energy balance. A wide range of weight changes was observed for similar model-estimated caloric changes, indicating that caloric balance alone may not be a good predictor of body weight. The model suggests that a set of optimal diets exists to achieve a defined weight reduction, and this set of diets is unique to each individual. Our diabetes model can simulate changes in body weight and glycemic control as a result of lifestyle interventions. Moreover, this model could help dieticians and physicians to optimize personalized nutritional strategies according to their patients' goals.

Thermo-hydraulic performance of solar air heater having discrete D-shaped ribs as artificial roughness.

In this paper, the thermo-hydraulic performance of a solar air heater (SAH) duct roughened with discrete D-shaped ribs is numerically investigated using ANSYS Fluent 2020 R2. The numerical investigation is carried out at rib radius to transverse pitch ratio (r/Pt) from 0.1 to 0.35 and longitudinal pitch to rib radius ratio (Pl /r) from 4 to 10 under various operating conditions with Reynolds number (Re) varied from 10,200 to 20,200. The numerical results are validated with previous experimental results for the Nusselt number (Nu) values, and good agreement is found with mean absolute percentage error (MAPE) of 3.6%. Based on the results of the numerical investigation, it was found that the value of Nu and the friction factor (f) decreases with the increase of the value of Pl/r, while the ratio r/Pt is kept constant. From the overall analysis, it is concluded that the optimum results are obtained for r/Pt of 0.25 and Pl/r = 4, and the maximum thermo-hydraulic performance parameter is 1.12. Further correlations are developed for the value of Nu and f for the whole range of r/Pt as 0.10-0.35 and Pl/r as 4-10. According to the developed correlations, the values of Nu are within ± 2% of the results of CFD, while the values of f are within ± 2.7% of the results of CFD.

Clinical Pharmacology Applications of Real-World Data and Real-World Evidence in Drug Development and Approval-An Industry Perspective.

Since the 21st Century Cures Act was signed into law in 2016, real-world data (RWD) and real-world evidence (RWE) have attracted great interest from the healthcare ecosystem globally. The potential and capability of RWD/RWE to inform regulatory decisions and clinical drug development have been extensively reviewed and discussed in the literature. However, a comprehensive review of current applications of RWD/RWE in clinical pharmacology, particularly from an industry perspective, is needed to inspire new insights and identify potential future opportunities for clinical pharmacologists to utilize RWD/RWE to address key drug development questions. In this paper, we review the RWD/RWE applications relevant to clinical pharmacology based on recent publications from member companies in the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) RWD Working Group, and discuss the future direction of RWE utilization from a clinical pharmacology perspective. A comprehensive review of RWD/RWE use cases is provided and discussed in the following categories of application: drug-drug interaction assessments, dose recommendation for patients with organ impairment, pediatric plan development and study design, model-informed drug development (e.g., disease progression modeling), prognostic and predictive biomarkers/factors identification, regulatory decisions support (e.g., label expansion), and synthetic/external control generation for rare diseases. Additionally, we describe and discuss common sources of RWD to help guide appropriate data selection to address questions pertaining to clinical pharmacology in drug development and regulatory decision making.

Optimization of performance characteristics in diesel engine utilizing Chlorella vulgaris fuel-a green approach towards sustainable development.

Sustainable renewable energy fuel is used to reduce fossil fuel consumption and mitigate global warming pollution. The effect of diesel and biodiesel blends on engine combustion, performance, and emissions were studied at various engine loads, compression ratios, and engine speed. Chlorella vulgaris biodiesel is derived through a transesterification process and diesel and biodiesel blends are prepared at 20% incremental volume up to CVB100. The performance such as brake thermal efficiency reduced by 1.49%, specific fuel consumption increased by 2.78%, and exhaust gas temperature increased by 0.43% for CVB20 as compared to diesel. Similarly, emissions were reduced such as smoke, particulate matters. CVB20 shows close performance and lower emission than diesel at a 15.5 compression ratio and 1500 rpm engine speed. The increasing compression ratio has a positive impact on engine performance and emission except for NOx. Similarly, increasing engine speed has a positive impact on engine performance and emission except for exhaust gas temperature. The performance of a diesel engine fueled with a blend of diesel and Chlorella vulgaris biodiesel is optimized by varying compression ratio, engine speed, load, and blend. It was found that at 8 compression ratio, 1835 rpm speed, 88% engine load, and 20% biodiesel blend the maximum BTE obtained 34% while minimum SFC 0.158 kg/kWh is obtained employing research surface methodology tool.

Achieving big with small: quantitative clinical pharmacology tools for drug development in pediatric rare diseases.

Pediatric populations represent a major fraction of rare diseases and compound the intrinsic challenges of pediatric drug development and drug development for rare diseases. The intertwined complexities of pediatric and rare disease populations impose unique challenges to clinical pharmacologists and require integration of novel clinical pharmacology and quantitative tools to overcome multiple hurdles during the discovery and development of new therapies. Drug development strategies for pediatric rare diseases continue to evolve to meet the inherent challenges and produce new medicines. Advances in quantitative clinical pharmacology research have been a key component in advancing pediatric rare disease research to accelerate drug development and inform regulatory decisions. This article will discuss the evolution of the regulatory landscape in pediatric rare diseases, the challenges encountered during the design of rare disease drug development programs and will highlight the use of innovative tools and potential solutions for future development programs.

Phylogenetic and immunological investigations of complete TSA56 ORF of Orientia tsutsugamushi present in acute encephalitis syndrome cases from eastern Uttar Pradesh, India.

Scrub typhus (ST) caused by Orientia tsutsugamushi (OT), has long been known to cause acute encephalitis syndrome (AES) and acute febrile illness (AFI). The immunodominant 56 kDa protein of OT, which is encoded by the 56 kDa gene (1600 bp encoding 516-541 amino acids) is a commonly studied antigen for genotype and serotype assignment. Previous studies from India have utilized partial type specific antigen (TSA) 56 kDa sequences for OT strain characterisation. On the other hand, understanding the antigenic diversity of current OT strains, is critical for developing specific diagnostic tests and vaccines against ST. As a result, the current study analyses antigenic variants using the entire TSA56 ORF of OT from AES cases. Phylogenetic investigation using complete TSA56 ORF sequences revealed Karp and Gilliam were the circulating predominant strains of OT. Furthermore, Immuno-informatical analysis demonstrated that the majority of high-binding affinity CD4 TCEs against the most prevalent Indian human leukocyte antigen alleles were present in the S-VDIII/IV and S-VDIV spacer regions of TSA56 ORF. TSA56 conserved spacer is crucial for OT immunological response investigations. Further, the pathophysiological effects of spacer domains in ST require further investigation. Furthermore, the characterization of the TSA56 spacer region of the OT from different parts of India is critical for developing region-specific ST diagnostic assays and vaccines.

Challenges in Accessing and Delivering Maternal and Child Health Services during the COVID-19 Pandemic: A Cross-Sectional Rapid Survey from Six States of India.

BACKGROUND/OBJECTIVES: Globally, the COVID-19 pandemic and its prevention and control policies have impacted maternal and child health (MCH) services. This study documents the challenges faced by patients in accessing MCH services, and the experiences of health care providers in delivering those services during the COVID-19 outbreak, explicitly focusing on the lockdown period in India. METHODS: A cross-sectional study (rapid survey) was conducted in 18 districts from 6 states of India during March to June, 2020. The sample size included 540 MCH patients, 18 gynaecologists, 18 paediatricians, 18 district immunisation officers and 108 frontline health workers. Bivariate analysis and multivariable analysis were used to assess the association between sociodemographic characteristics, and challenges faced by the patients. RESULTS: More than one-third of patients (n = 212; 39%) reported that accessing MCH services was a challenge during the lockdown period, with major challenges being transportation-related difficulties (n = 99; 46%) unavailability of hospital-based services (n = 54; 23%) and interrupted outreach health services (n = 39; 18.4%). The supply-side challenges mainly included lack of infrastructural preparedness for outbreak situations, and a shortage of human resources. CONCLUSIONS/RECOMMENDATIONS: A holistic approach is required that focuses on both preparedness and response to the outbreak, as well reassignment and reinforcement of health care professionals to continue catering to and maintaining essential MCH services during the pandemic.

Current Treatment Strategies Against Multidrug-Resistant Bacteria: A Review.

There are several bacteria called superbugs that are resistant to multiple antibiotics which can be life threatening specially for critically ill and hospitalized patients. This article provides up-to-date treatment strategies employed against some major superbugs, like methicillin-resistant Staphylococcus aureus, carbapenem-resistant Enterobacteriaceae, vancomycin-resistant Enterococcus, multidrug-resistant Pseudomonas aeruginosa, and multidrug-resistant Escherichia coli. The pathogen-directed therapeutics decrease the toxicity of bacteria by altering their virulence factors by specific processes. On the other hand, the host-directed therapeutics limits these superbugs by modulating immune cells, enhancing host cell functions, and modifying disease pathology. Several new antibiotics against the global priority superbugs are coming to the market or are in the clinical development phase. Medicinal plants possessing potent secondary metabolites can play a key role in the treatment against these superbugs. Nanotechnology has also emerged as a promising option for combatting them. There is urgent need to continuously figure out the best possible treatment strategy against these superbugs as resistance can also be developed against the new and upcoming antibiotics in future. Rational use of antibiotics and maintenance of proper hygiene must be practiced among patients.

Omicron BA.2 lineage predominance in severe acute respiratory syndrome coronavirus 2 positive cases during the third wave in North India.

Background: Recent studies on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reveal that Omicron variant BA.1 and sub-lineages have revived the concern over resistance to antiviral drugs and vaccine-induced immunity. The present study aims to analyze the clinical profile and genome characterization of the SARS-CoV-2 variant in eastern Uttar Pradesh (UP), North India. Methods: Whole-genome sequencing (WGS) was conducted for 146 SARS-CoV-2 samples obtained from individuals who tested coronavirus disease 2019 (COVID-19) positive between the period of 1 January 2022 and 24 February 2022, from three districts of eastern UP. The details regarding clinical and hospitalized status were captured through telephonic interviews after obtaining verbal informed consent. A maximum-likelihood phylogenetic tree was created for evolutionary analysis using MEGA7. Results: The mean age of study participants was 33.9 ± 13.1 years, with 73.5% accounting for male patients. Of the 98 cases contacted by telephone, 30 (30.6%) had a travel history (domestic/international), 16 (16.3%) reported having been infected with COVID-19 in past, 79 (80.6%) had symptoms, and seven had at least one comorbidity. Most of the sequences belonged to the Omicron variant, with BA.1 (6.2%), BA.1.1 (2.7%), BA.1.1.1 (0.7%), BA.1.1.7 (5.5%), BA.1.17.2 (0.7%), BA.1.18 (0.7%), BA.2 (30.8%), BA.2.10 (50.7%), BA.2.12 (0.7%), and B.1.617.2 (1.3%) lineages. BA.1 and BA.1.1 strains possess signature spike mutations S:A67V, S:T95I, S:R346K, S:S371L, S:G446S, S:G496S, S:T547K, S:N856K, and S:L981F, and BA.2 contains S:V213G, S:T376A, and S:D405N. Notably, ins214EPE (S1- N-Terminal domain) mutation was found in a significant number of Omicron BA.1 and sub-lineages. The overall Omicron BA.2 lineage was observed in 79.5% of women and 83.2% of men. Conclusion: The current study showed a predominance of the Omicron BA.2 variant outcompeting the BA.1 over a period in eastern UP. Most of the cases had a breakthrough infection following the recommended two doses of vaccine with four in five cases being symptomatic. There is a need to further explore the immune evasion properties of the Omicron variant.

Next-Generation Diagnostic with CRISPR/Cas: Beyond Nucleic Acid Detection.

The early management, diagnosis, and treatment of emerging and re-emerging infections and the rising burden of non-communicable diseases (NCDs) are necessary. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system has recently acquired popularity as a diagnostic tool due to its ability to target specific genes. It uses Cas enzymes and a guide RNA (gRNA) to cleave target DNA or RNA. The discovery of collateral cleavage in CRISPR-Cas effectors such as Cas12a and Cas13a was intensively repurposed for the development of instrument-free, sensitive, precise and rapid point-of-care diagnostics. CRISPR/Cas demonstrated proficiency in detecting non-nucleic acid targets including protein, analyte, and hormones other than nucleic acid. CRISPR/Cas effectors can provide multiple detections simultaneously. The present review highlights the technical challenges of integrating CRISPR/Cas technology into the onsite assessment of clinical and other specimens, along with current improvements in CRISPR bio-sensing for nucleic acid and non-nucleic acid targets. It also highlights the current applications of CRISPR/Cas technologies.

A Computational Platform Integrating a Mechanistic Model of Crohn's Disease for Predicting Temporal Progression of Mucosal Damage and Healing.

INTRODUCTION: Physicians are often required to make treatment decisions for patients with Crohn's disease on the basis of limited objective information about the state of the patient's gastrointestinal tissue while aiming to achieve mucosal healing. Tools to predict changes in mucosal health with treatment are needed. We evaluated a computational approach integrating a mechanistic model of Crohn's disease with a responder classifier to predict temporal changes in mucosal health. METHODS: A hybrid mechanistic-statistical platform was developed to predict biomarker and tissue health time courses in patients with Crohn's disease. Eligible patients from the VERSIFY study (n = 69) were classified into archetypical response cohorts using a decision tree based on early treatment data and baseline characteristics. A virtual patient matching algorithm assigned a digital twin to each patient from their corresponding response cohort. The digital twin was used to forecast response to treatment using the mechanistic model. RESULTS: The responder classifier predicted endoscopic remission and mucosal healing for treatment with vedolizumab over 26 weeks, with overall sensitivities of 80% and 75% and overall specificities of 69% and 70%, respectively. Predictions for changes in tissue damage over time in the validation set (n = 31), a measure of the overall performance of the platform, were considered good (at least 70% of data points matched), fair (at least 50%), and poor (less than 50%) for 71%, 23%, and 6% of patients, respectively. CONCLUSION: Hybrid computational tools including mechanistic components represent a promising form of decision support that can predict outcomes and patient progress in Crohn's disease.