Molecular characterization of rotavirus indicates predominance of G9P[4] genotype among children with acute gastroenteritis: First report after vaccine introduction in Pakistan.

Globally, Group A rotavirus (RVA) is the leading cause of acute gastroenteritis in children under 5 years old, with Pakistan having the highest rates of RVA-related morbidity and mortality. The current study aims to determine the genetic diversity of rotavirus and evaluate the impact of Rotarix-vaccine introduction on disease epidemiology in Pakistan. A total of 4749 children, hospitalized with acute gastroenteritis between 2018 and 2020, were tested at four hospitals in Lahore and Karachi. Of the total, 19.3% (918/4749) cases were tested positive for RVA antigen, with the positivity rate varying annually (2018 = 22.7%, 2019 = 14.4%, 2020 = 20.9%). Among RVA-positive children, 66.3% were under 1 year of age. Genotyping of 662 enzyme-linked immuno sorbent assay-positive samples revealed the predominant genotype as G9P[4] (21.4%), followed by G1P[8] (18.9%), G3P[8] (11.4%), G12P[6] (8.7%), G2P[4] (5.7%), G2P[6] (4.8%), and 10.8% had mixed genotypes. Among vaccinated children, genotypes G9P[4] and G12P[6] were more frequently detected, whereas a decline in G2P[4] was observed. Phylogenetic analysis confirmed the continued circulation of indigenous genotypes detected earlier in the country except G9 and P[6] strains. Our findings highlight the predominance of G9P[4] genotype after the vaccine introduction thus emphasizing continual surveillance to monitor the disease burden, viral diversity, and their impact on control of rotavirus gastroenteritis in children.

To evaluate the role of interleukin-6 in alopecia areata.

Objective: TTo investigate the level of interleukin-6 in alopecia areata patients. METHODS: The exploratory study was conducted from September to December 2021 at the Sindh Institute of Skin Disease, Karachi, and comprised alopecia areata patients regardless of age and gender in group A, while healthy controls matched for age and gender formed group B. Alopecia areata classification and severity were done using the Severity of Alopecia Tool. Serum interleukin-6 was measured using enzyme-linked immune sorbent assay. Data was analysed using R statistical software v4.2.1. RESULTS: Of the 100 subjects, 50(50%) with mean age 15.52±10.14 years were cases in group A; 26(52%) females with mean age 16.78±10.77 years, and 24(48%) males with mean age 16.44±10.3 years. The remaining 50(50%) were controls in group B. Interleukin-6 concentration was significantly higher in group A (p<0.05). The concentration was not significantly different between the genders (p>0.05). The concentration was the highest in patients aged 11-20 years, followed by 21-30 years, 31-40 years and 1-10 years. Conclusion: The concentration of circulatory pro-inflammatory interleukin-6 was significantly higher in alopecia areata patients than in the healthy controls.

Insights into measles virus: Serological surveillance and molecular characterization.

BACKGROUND: Measles has been a significant public health concern in Pakistan, especially in the Khyber Pakhtunkhwa (KPK) province, where sporadic and silent epidemics continue to challenge existing control measures. This study aimed to estimate the prevalence and investigate the molecular epidemiology of the measles virus (MeV) in KPK and explore the vaccination status among the suspected individuals. METHODS: A cross-sectional study was conducted between February and October 2021. A total of 336 suspected measles cases from the study population were analyzed for IgM antibodies using Enzyme-Linked Immunosorbent Assay (ELISA). Throat swabs were randomly collected from a subset of positive cases for molecular analysis. Phylogenetic analysis of MeV isolates was performed using the neighbor-joining method. The vaccination status of individuals was also recorded. RESULTS: Among the suspected participants, 61.0% (205/336) were ELISA positive for IgM antibodies, with a higher prevalence in males (64.17%) compared to females (57.04%). The majority of cases (36.0%) were observed in infants and toddlers, consistent with previous reports. The majority of IgM-positive cases (71.7%) had not received any dose of measles vaccine, highlighting gaps in vaccine coverage and the need for improved immunization programs. Genetic analysis revealed that all MeV isolates belonged to the B3 genotype, with minor genetic variations from previously reported variants in the region. CONCLUSION: This study provides valuable insights into the genetic epidemiology of the MeV in KPK, Pakistan. The high incidence of measles infection among unvaccinated individuals highlights the urgency of raising awareness about vaccine importance and strengthening routine immunization programs.

A hidden dilemma; post COVID-19 first detection of Varicella zoster M4 genotype from Pakistan.

OBJECTIVES: During the COVID-19 pandemic, the risk of childhood infectious diseases was increased. Post-COVID-19 escalation of chickenpox cases, becoming an emerging public health concern. Thus, the study was designed to compare chickenpox prevalence and Varicella zoster virus (VZV) genotypes circulating before, during, and post-COVID-19 in Pakistan. METHODS: A total of 267 lesion specimens collected from tertiary care hospitals, and chickenpox outbreaks from Pakistan were analysed by a two-amplicon approach with phylogenetic analysis. RESULTS: Among suspected cases, overall 178/267 were VZV positive. Majority (84.2 %; 150/178) cases were of post-COVID-19 pandemic time. Small outbreaks occurred soon after COVID-19 in Rawalpindi and Islamabad (Pakistan), 40 positive cases out of 178 cases were outbreak cases. There was first time detection of the M4 genotype, which was significantly associated with disease severity (p = 0.0006) and post-COVID-19 chickenpox outbreaks in 2021 (77.9 %; 46/59; p < 0.00001). However, in pre-COVID-19 only M2 genotype was detected. The M2 prevalence varied from 2019 (100 %; 19/19) to 2022 (3.2 %; 3/91). However, the most prevalent strain of 2022 belonged to the M1 genotype (64.8 %; 59/91). CONCLUSION: A significant rise in chickenpox cases detected soon after COVID-19 in Pakistan, and oscillation of different VZV genotypes with first time detection of M4 genotype is an alarming situation. This demands further detailed genotypic studies on transmission dynamics of a rare M4 with other genotypes to protect the local population and restrict spread in other regions.

In silico designing and characterization of outer membrane protein K (OmpK) from Vibrio anguillarum and its expression in Nicotiana tabacum for the development of a plant-based vaccine against fish vibriosis.

Vibriosis is caused by Vibrio anguillarum in various species of aquaculture. A novel, secure, and stable vaccine is needed to eradicate vibriosis. Here, for reverse vaccinology and plant-based expression, the outer membrane protein K (OmpK) of V. anguillarum was chosen due to its conserved nature in all Vibrio species. OmpK, an ideal vaccine candidate against vibriosis, demonstrated immunogenic, non-allergic, and non-toxic behavior by using various bioinformatics tools. Docking showed the interaction of the OmpK model with TLR-5. In comparison to costly platforms, plants can be used as alternative and economic bio-factories to produce vaccine antigens. We expressed OmpK antigen in Nicotiana tabacum using Agrobacterium-mediated transformation. The expression vector was constructed using Gateway® cloning. Transgene integration was verified by polymerase chain reaction (PCR), and the copy number via qRT-PCR, which showed two copies of transgenes. Western blotting detected monomeric form of OmpK protein. The total soluble protein (TSP) fraction of OmpK was equivalent to 0.38% as detected by ELISA. Mice and fish were immunized with plant-derived OmpK antigen, which showed a significantly high level of anti-OmpK antibodies. The present study is the first report of OmpK antigen expression in higher plants for the potential use as vaccine in aquaculture against vibriosis, which could provide protection against multiple Vibrio species due to the conserved nature OmpK antigen.

Vaccinomics Approach for Multi-Epitope Vaccine Design against Group A Rotavirus Using VP4 and VP7 Proteins.

Rotavirus A is the most common cause of Acute Gastroenteritis globally among children <5 years of age. Due to a segmented genome, there is a high frequency of genetic reassortment and interspecies transmission which has resulted in the emergence of novel genotypes. There are concerns that monovalent (Rotarix: GlaxoSmithKline Biologicals, Rixensart, Belgium) and pentavalent (RotaTeq: MERCK & Co., Inc., Kenilworth, NJ, USA) vaccines may be less effective against non-vaccine strains, which clearly shows the demand for the design of a vaccine that is equally effective against all circulating genotypes. In the present study, a multivalent vaccine was designed from VP4 and VP7 proteins of RVA. Epitopes were screened for antigenicity, allergenicity, homology with humans and anti-inflammatory properties. The vaccine contains four B-cell, three CTL and three HTL epitopes joined via linkers and an N-terminal RGD motif adjuvant. The 3D structure was predicted and refined preceding its docking with integrin. Immune simulation displayed promising results both in Asia and worldwide. In the MD simulation, the RMSD value varied from 0.2 to 1.6 nm while the minimum integrin amino acid fluctuation (0.05-0.1 nm) was observed with its respective ligand. Codon optimization was performed with an adenovirus vector in a mammalian expression system. The population coverage analysis showed 99.0% and 98.47% in South Asia and worldwide, respectively. These computational findings show potential against all RVA genotypes; however, in-vitro/in-vivo screening is essential to devise a meticulous conclusion.

Fabrication of Ternary MoS2/CdS/Bi2S3-Based Nano Composites for Photocatalytic Dye Degradation.

The synthesis and design of low-cost visible-light-active catalysts for the photodegradation of organic dyes have been regarded as an efficient way to use solar energy in addressing environmental issues. We report the fabrication of MoS2/CdS nanoparticles functionalized with Bi2S3 nanoflakes. The ternary composites of "MoS2/CdS/Bi2S3" were synthesized in situ by a hydrothermal method at different temperatures. The changes in structural, optical, and morphological properties of the synthesized CdS/MoS2/Bi2S3 were explored. The effects of Bi2S3 on CdS/MoS2 were thoroughly studied by performing an X-ray diffractometer (XRD), a scanning electron microscope (SEM), an ultra-violet-visible spectrometer (Uv-vis), and Fourier transform infrared spectroscopic (FT-IR) studies of the nanoparticles. XRD confirms the cubical crystal structure of the nanoparticles. SEM studies possess the modulation in the surface morphology with the tenability in volume ratios of "MoS2/CdS/Bi2S3" composites. It was observed that the bandgaps calculated using absorption measurements could be manipulated from 2.40 eV to 0.97 eV with varying Bi2S3 in the MoS2/CdS nanostructures. FT-IR confirmed the synthesis of "MoS2/CdS/Bi2S3" nanoparticles. On allowing the visible light to fall for 120 min, it was observed that "MoS2/CdS/Bi2S3" degrades the methylene blue up to 90%. The calculated results of "MoS2/CdS/Bi2S3" suggest that the synthesized material could be a strong candidate for photodegradation applications. This research work explains the synthesis of MoS2/CdS/Bi2S3-based nanocomposites for the degradation of dye using a photocatalytic process. The final results show that this catalyst effectively degrades the dye.

First report of coinfection and whole-genome sequencing of norovirus and sapovirus in an acute gastroenteritis patient from Pakistan.

Acute gastroenteritis is one of the most common diseases in infants and children in developing countries including Pakistan. In Pakistan, rotavirus (RVA) is known to contribute significantly to pediatric diarrheal illness, but the contribution of other viruses is still unclear. In the current study we have identified a case of mixed infection of norovirus (NoV) and sapovirus (SaV) in a 2-year-old child with acute gastroenteritis. The sample was initially processed for the detection of group A RVA through ELISA followed by NoV using RT-PCR assay. The sample tested positive for NoV RNA and was later subjected to whole-genome sequencing using meta-genome approach on Miseq (Illumina) platform. Sequencing results revealed GII.15 genotype of NoV that clustered with viruses from China and USA from 2017 to 2021. We also retrieved the complete genome of SaV (GI.1 genotype) from the same sample and phylogenetic analysis showed clustering with strains reported from Japan, South Korea, US, and Taiwan during 2012-2016. This is the first report from Pakistan that confirms coinfection of NoV and SaV and elucidates their whole genomes. We recommend initiation of NoV and SaV surveillance program to ascertain disease burden and explore genetic diversity, especially as RVA vaccines have been included in national immunization program.

Personal protective equipment (PPE) disposal during COVID-19: An emerging source of microplastic and microfiber pollution in the environment.

Waste generated by healthcare facilities during the COVID-19 pandemic has become a new source of pollution, particularly with the widespread use of single-use personal protective equipment (PPE). Releasing microplastics (MPs) and microfibers (MFs) from discarded PPE becomes an emerging threat to environmental sustainability. MPs/MFs have recently been reported in a variety of aquatic and terrestrial ecosystems, including water, deep-sea sediments, air, and soil. As COVID-19 spreads, the use of plastic-made PPE in healthcare facilities has increased significantly worldwide, resulting in massive amounts of plastic waste entering the terrestrial and marine environments. High loads of MPs/MFs emitted into the environment due to excessive PPE consumption are easily consumed by aquatic organisms, disrupting the food chain, and potentially causing chronic health problems in humans. Thus, proper management of PPE waste is critical for ensuring a post-COVID sustainable environment, which has recently attracted the attention of the scientific community. The current study aims to review the global consumption and sustainable management of discarded PPE in the context of COVID-19. The severe impacts of PPE-emitted MPs/MFs on human health and other environmental segments are briefly addressed. Despite extensive research progress in the area, many questions about MP/MF contamination in the context of COVID-19 remain unanswered. Therefore, in response to the post-COVID environmental remediation concerns, future research directions and recommendations are highlighted considering the current MP/MF research progress from COVID-related PPE waste.

Interactive salinity and water stress severely reduced the growth, stress tolerance, and physiological responses of guava (Psidium Guajava L.).

Salinity and water stress are serious environmental issues that reduced crop production worldwide. The current research was initiated (2012) in the wirehouse of the Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad, Pakistan to investigate the growth, stress tolerance, and physiological responses of guava to salinity and water shortage. Guava was grown for one year in pots containing soil with Eight treatments (control, 10 dS m-1, 20 dS m-1, 40 dS m-1, control + water stress (WS), 10 dS m-1 + WS, 20 dS m-1 + WS, 40 dS m-1 + WS) in a completely randomized design. The results indicated that plant growth, stress tolerance, and physiological parameters declined at higher salinity and water stress and could not survive at 40 dS m-1. The 20 dS m-1 + WS caused a > 70% decline in dry weights of shoot and root regarding control. Similarly, the highest decrease in stress tolerance was noticed in 20 dS m-1 + WS followed by the 20 dS m-1 treatment than control. Our findings validated that guava can be cultivated on soils having salinity ≤ 10 dS m-1 but it could not be cultivated on soils having salinity ≥ 20 dS m-1 with limited water supply.

Breakthrough cases of Omicron and Delta variants of SARS-CoV-2 during the fifth wave in Pakistan.

COVID-19 pandemic has severely affected Pakistan with 1,557,134 cases as of August 4, 2022. However, the data regarding breakthrough infections in Pakistan is scant. Hence, the objective was to analyze SARS-CoV-2 breakthrough infections with respect to vaccines and variants during the fifth wave in Pakistan. Therefore, the Department of Virology (NIH, Pakistan) genotyped 2,467 randomly selected individuals between November 2021 and February 2022 using the SNPsig® SARS-CoV-2 (EscapePLEX) kit (PrimerDesign, UK). P681R and K417N mutations were used to distinguish delta and omicron. Data on the patient's age, gender, date of collection, variant, and vaccination status were analyzed using Statistical Package for Social Sciences (SPSS) software. Among 2,467 genotyped samples, Omicron was detected in 58.6% (n = 1445), Delta in 40.4% (n = 998) and undetermined/wildtype variant in 24 samples. The vaccination status of omicron-positive patients showed (49.7%; n = 718/1445) and Delta-positive patients (39.67%; n = 396/998) to be fully vaccinated. Of note, a high percentage 85% of breakthrough cases (n = 947) were identified among fully vaccinated individuals (n = 1114). Among them, 85.9% (n = 617/718) belonged to omicron and 83.3% (n = 330/396) to delta. Moreover, 76.7% (n = 855) of vaccinated individuals (n = 1114) received Sinopharm (n = 432) and Sinovac (n = 423) vaccines. The majority of breakthrough subjects who contracted Omicron were vaccinated with Sinopharm (93.0%, n = 256) and delta with Cansino (100%, n = 44). Individuals vaccinated with Sinovac showed the most frequent breakthrough cases for both Omicron and Delta variant between the 4th and 6th months (n = 278) after primary vaccination as compared to the 7th to 9th months (n = 24) category. While in case of Sinopharm, maximum breakthrough cases occurred between 7th to 9th months (n = 234) as compared to the 4th to 6th months (n = 120) after primary vaccination. Omicron and Delta breakthrough cases in men (n = 364 and 193) are more frequently seen than women (n = 253 and 138) respectively and breakthrough majority cases (n = 392) occurred in individuals aged 18-33 years. Breakthrough cases limiting monitoring in Pakistan impose a substantial constraint on policymakers' ability to take timely effective decisions. Since the current study consists of only a 2,467-genotyped sample, comprehensive data should be analyzed.

SeDeM tool-driven full factorial design for osmotic drug delivery of tramadol HCl: Formulation development, physicochemical evaluation, and in-silico PBPK modeling for predictive pharmacokinetic evaluation using GastroPlus™.

The study is based on using SeDeM expert system in developing controlled-release tramadol HCl osmotic tablets and its in-silico physiologically based pharmacokinetic (PBPK) modeling for in-vivo pharmacokinetic evaluation. A Quality by Design (QbD) based approach in developing SeDEM-driven full factorial osmotic drug delivery was applied. A 24 Full-factorial design was used to make the trial formulations of tramadol HCl osmotic tablets using NaCl as osmogen, Methocel K4M as rate controlling polymer, and avicel pH 101 as diluent. The preformulation characteristics of formulations (F1-F16) were determined by applying SeDeM Expert Tool. The formulation was optimized followed by in-vivo predictive pharmacokinetic assessment using PBPK "ACAT" model of GastroPlus™. The FTIR results showed no interaction among the ingredients. The index of good compressibility (ICG) values of all trial formulation blends were ≥5, suggesting direct compression is the best-suited method. Formulation F3 and F4 were optimized based on drug release at 2, 10, and 16 h with a zero-order kinetic release (r 2 = 0.992 and 0.994). The SEM images confirmed micropores formation on the surface of the osmotic tablet after complete drug release. F3 and F4 were also stable (shelf life 29.41 and 23.46 months). The in vivo simulation of the pharmacokinetics of the PBPK in-silico model revealed excellent relative bioavailability of F3 and F4 with reference to tramadol HCl 50 mg IR formulations. The SeDeM expert tool was best utilized to evaluate the compression characteristics of selected formulation excipients and their blends for direct compression method in designing once-daily osmotically controlled-release tramadol HCl tablets. The in-silico GastroPlus™ PBPK modeling provided a thorough pharmacokinetic assessment of the optimized formulation as an alternative to tramadol HCl in vivo studies.

Nanoclay-Based Composite Films for Transdermal Drug Delivery: Development, Characterization, and in silico Modeling and Simulation.

Purpose: Halloysite nanotubes (HNTs) are a versatile and highly investigated clay mineral due to their natural availability, low cost, strong mechanical strength, biocompatibility, and binding properties. The present work explores its role for retarding and controlling the drug release from the composite polymer matrix material. Methods: For this purpose, nanocomposite films comprising propranolol HCl and different concentrations of HNTs were formulated using the "solution casting method". The menthol in a concentration of 1% w/v was used as a permeation enhancer, and its effect on release and permeation was also determined. Quality characteristics of the nanocomposite were determined, and in vitro release and permeation studies were performed using the Franz diffusion system. The data was analyzed using various mathematical models and permeation parameters. Optimized formulation was also subjected to skin irritation test, FTIR, DSC, and SEM study. Systemic absorption and disposition of propranolol HCl from the nanocomposites were predicted using the GastroPlus TCAT® model. Results: The control in drug release rate was associated with the higher concentration of HNTs. F8 released 50% of propranolol within 8 hours (drug, HNTs ratio, 1:2). The optimized formulation (F6) with drug: HNTs (2:1), exhibited drug release 80% in 4 hours, with maximum flux of 145.812 µg/cm2hr. The optimized formulation was found to be a non-irritant for skin with a shelf life of 35.46 months (28-30 ℃). The in silico model predicted Cmax, Tmax, AUCt , and AUCinf as 32.113 ng/mL, 16.58 h, 942.34 ng/mL×h, and 1102.9 ng/mL×h, respectively. Conclusion: The study demonstrated that HNTs could be effectively used as rate controlling agent in matrix type transdermal formulations.

Comparative study of ozonation and ozonation catalyzed by Fe-loaded biochar as catalyst to remove methylene blue from aqueous solution.

Ozone-based processes gained much attention in recent years. However, due to low oxidative stability and utilization rate, single ozonation process (SOP) is insufficient for complete mineralization of pollutants. As a result, the single ozonation process is performed in the presence of a catalyst, a process known as catalytic ozonation process (COP). A promising catalyst (Fe/BC) was prepared by impregnating iron on biochar surface to remove methylene blue from aqueous solution via heterogeneous catalytic ozonation process (HCOP). The prepared Fe/BC features were characterized using Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and Brunauer-Emmett-Teller method (BET) before and after HCOP. Furthermore, the effect of various operating parameters such as ozone dose, catalyst dose, initial dye concentration, initial pH on the efficiency of SOP and HCOP were compared. In comparison to single ozonation process, the experimental study found that heterogeneous catalytic ozonation process has the highest efficiency. At pH 7.0, approximately 76% of methylene blue is removed during single ozonation process in 60 min. Heterogeneous catalytic ozonation process showed 95% methylene blue elimination from aqueous solution. The efficiency of heterogeneous catalytic ozonation process was decreased by 52% in the presence of hydroxyl radical (●OH) scavenger, indicating that hydroxyl is the major oxidant during heterogeneous catalytic ozonation process for the removal of methylene blue from aqueous solution. Fe/BC catalyst appears to have a lot of industrial promise, as well as the ability to remove methylene blue from aqueous solution via heterogeneous catalytic ozonation process.

Impairment of Respiratory Chain Function and Involvement of Alternative Respiratory Pathway in Mitochondria of Potato Tubers Infected by Pectobacteriumcarotovorum subsp. carotovorum.

The significance of alternative respiratory pathway (AOXs) during the interaction between soft rot bacteria (Pectobacterium carotovorum subsp. carotovorum, (Pcc.)) and potato tubers is well-defined. However, the role of the AOXs in impaired mitochondrial respiratory chain function during the Pcc. infection is yet to be studied. In this study, the results show that with the aggravation of infection of Pcc., the capacity for alternative respiration in mitochondria of potato tubers increased gradually. The mitochondrial membrane potential increased more significantly after infection with Pcc. when the AOXs in potato tubers was partially blocked using salicylhydroxamic acid (SHAM) beforehand. In addition, the activity of complex III decreased more drastically while the activity of complex IV increased more significantly in the partial absence of the AOXs in the mitochondria. Furthermore, the mitochondrial endogenous respiration, mitochondrial respiratory state 3 and respiratory control rate (RCR) decreased more significantly and the value of RCR reached around 1.0 with the aggravation of infection of Pcc. in the partially absence of AOXs in the mitochondria.