Increasing usage of rapid diagnostics for Dengue virus detection in Pakistan.

OBJECTIVE: To evaluate the trends in usage of dengue virus diagnostics in Pakistan. METHODS: This retrospective study was conducted at the Aga Khan University Hospital, Karachi, and comprised data for specimens tested for dengue virus from January 2012 to December 2015. Test for dengue virus ribonucleic acid by reverse transcription polymerase chain reaction, dengue virus antigen by immunochromatic assay and for human immunoglobulin M against dengue virus by enzyme-linked immunosorbent assay were reviewed. SPSS 17 was used for data analysis. RESULTS: Overall, 33,577 specimens tested for dengue virus. Of them, 11,995 (35.7%) were positive. among them, 1,039(8.66%) were reported in 2012; 5,791(48.28%) in 2013; 1,027(8.56%) in 2014; and 4,138(34.49%) in 2015. In 2012, 966(93%) of the positive samples were diagnosed by immunoglobulin M-based method and 73(7%) by non-structural protein-1 antigen. In 2013, 4,401(76%) samples were tested positive by immunoglobulin M, 1,332(23%) by antigen and 58(1%) by polymerase chain reaction. The trend continued in 2014, but in 2015, 2,111(51%) of all dengue positive tests were determined by antigen testing, 1,969(47.6%) by immunoglobulin M and 58(1.4%) by polymerase chain reaction. CONCLUSIONS: There was a shift in usage of direct virus identification for rapid diagnosis of dengue virus compared with host immunoglobulin M testing.

In-silico formulation of a next-generation polyvalent vaccine against multiple strains of monkeypox virus and other related poxviruses.

Mpox (formerly known as monkeypox) virus and some related poxviruses including smallpox virus pose a significant threat to public health, and effective prevention and treatment strategies are needed. This study utilized a reverse vaccinology approach to retrieve conserved epitopes for monkeypox virus and construct a vaccine that could provide cross-protection against related viruses with similar antigenic properties. The selected virulent proteins of monkeypox virus, MPXVgp165, and Virion core protein P4a, were subjected to epitope mapping for vaccine construction. Two vaccines were constructed using selected T cell epitopes and B cell epitopes with PADRE and human beta-defensins adjuvants conjugated in the vaccine sequence. Both constructs were found to be highly antigenic, non-allergenic, nontoxic, and soluble, suggesting their potential to generate an adequate immune response and be safe for humans. Vaccine construct 1 was selected for molecular dynamic simulation studies. The simulation studies revealed that the TLR8-vaccine complex was more stable than the TLR3-vaccine complex. The lower RMSD and RMSF values of the TLR8 bound vaccine compared to the TLR3 bound vaccine suggested better stability and consistency of hydrogen bonds. The Rg values of the vaccine chain bound to TLR8 indicated overall stability, whereas the vaccine chain bound to TLR3 showed deviations throughout the simulation. These results suggest that the constructed vaccine could be a potential preventive measure against monkeypox and related viruses however, further experimental validation is required to confirm these findings.

Antifungal plant flavonoids identified in silico with potential to control rice blast disease caused by Magnaporthe oryzae.

Rice blast disease, caused by the fungus Magnaporthe oryzae, poses a severe threat to rice production, particularly in Asia where rice is a staple food. Concerns over fungicide resistance and environmental impact have sparked interest in exploring natural fungicides as potential alternatives. This study aimed to identify highly potent natural fungicides against M. oryzae to combat rice blast disease, using advanced molecular dynamics techniques. Four key proteins (CATALASE PEROXIDASES 2, HYBRID PKS-NRPS SYNTHETASE TAS1, MANGANESE LIPOXYGENASE, and PRE-MRNA-SPLICING FACTOR CEF1) involved in M. oryzae's infection process were identified. A list of 30 plant metabolites with documented antifungal properties was compiled for evaluation as potential fungicides. Molecular docking studies revealed that 2-Coumaroylquinic acid, Myricetin, Rosmarinic Acid, and Quercetin exhibited superior binding affinities compared to reference fungicides (Azoxystrobin and Tricyclazole). High throughput molecular dynamics simulations were performed, analyzing parameters like RMSD, RMSF, Rg, SASA, hydrogen bonds, contact analysis, Gibbs free energy, and cluster analysis. The results revealed stable interactions between the selected metabolites and the target proteins, involving important hydrogen bonds and contacts. The SwissADME server analysis indicated that the metabolites possess fungicide properties, making them effective and safe fungicides with low toxicity to the environment and living beings. Additionally, bioactivity assays confirmed their biological activity as nuclear receptor ligands and enzyme inhibitors. Overall, this study offers valuable insights into potential natural fungicides for combating rice blast disease, with 2-Coumaroylquinic acid, Myricetin, Rosmarinic Acid, and Quercetin standing out as promising and environmentally friendly alternatives to conventional fungicides. These findings have significant implications for developing crop protection strategies and enhancing global food security, particularly in rice-dependent regions.

Designing a polyvalent vaccine targeting multiple strains of varicella zoster virus using integrated bioinformatics approaches.

The Varicella Zoster Virus (VZV) presents a global health challenge due to its dual manifestations of chickenpox and shingles. Despite vaccination efforts, incomplete coverage, and waning immunity lead to recurrent infections, especially in aging and immunocompromised individuals. Existing vaccines prevent chickenpox but can trigger the reactivation of shingles. To address these limitations, we propose a polyvalent multiepitope subunit vaccine targeting key envelope glycoproteins of VZV. Through bioinformatics approaches, we selected six glycoproteins that are crucial for viral infection. Epitope mapping led to the identification of cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B cell linear (LBL) epitopes. Incorporating strong immunostimulants, we designed two vaccine constructs, demonstrating high antigenicity, solubility, stability, and compatibility with Toll-like receptors (TLRs). Molecular docking and dynamics simulations underscored the stability and affinity of the vaccine constructs with TLRs. These findings lay the foundation for a comprehensive solution to VZV infections, addressing the challenges of incomplete immunity and shingles reactivation. By employing advanced immunoinformatics and dynamics strategies, we have developed a promising polyvalent multiepitope subunit vaccine candidate, poised to enhance protection against VZV and its associated diseases. Further validation through in vivo studies is crucial to confirm the effectiveness and potential of the vaccine to curb the spread of VZV. This innovative approach not only contributes to VZV control but also offers insights into tailored vaccine design strategies against complex viral pathogens.

Tracking SARS-CoV-2 variants through pandemic waves using RT-PCR testing in low-resource settings.

COVID-19 resulted in extensive morbidity and mortality worldwide. SARS-CoV-2 evolved rapidly, with increasing transmission due to Variants of Concern (VOC). Identifying VOC became important but genome submissions from low-middle income countries (LMIC) remained low leading to gaps in genomic epidemiology. We demonstrate the use of a specific mutation RT-PCR based approach to identify VOC in SARS-CoV-2 positive samples through the pandemic in Pakistan. We selected 2150 SARS-CoV-2 PCR positive respiratory specimens tested between April 2021 and February 2022, at the Aga Khan University Hospital Clinical Laboratories, Karachi, Pakistan. Commercially available RT-PCR assays were used as required for mutations in Spike protein (N501Y, A570D, E484K, K417N, L452R, P681R and deletion69_70) to identify Alpha, Beta, Gamma, Delta, and Omicron variants respectively. Three pandemic waves associated with Alpha, Delta and Omicron occurred during the study period. Of the samples screened, VOC were identified in 81.7% of cases comprising mainly; Delta (37.2%), Alpha (29.8%) and Omicron (17.1%) variants. During 2021, Alpha variants were predominant in April and May; Beta and Gamma variants emerged in May and peaked in June; the Delta variant peaked in July and remained predominant until November. Omicron (BA.1) emerged in December 2021 and remained predominant until February 2022. The CT values of Alpha, Beta, Gamma and Delta were all significantly higher than that of Omicron variants (p<0.0001). We observed VOC through the pandemic waves using spike mutation specific RT-PCR assays. We show the spike mutation specific RT-PCR assay is a rapid, low-cost and adaptable for the identification of VOC as an adjunct approach to NGS to effectively inform the public health response. Further, by associating the VOC with CT values of its diagnostic PCR we gain information regarding the viral load of samples and therefore the level of transmission and disease severity in the population.

Higher entropy observed in SARS-CoV-2 genomes from the first COVID-19 wave in Pakistan.

BACKGROUND: We investigated the genome diversity of SARS-CoV-2 associated with the early COVID-19 period to investigate evolution of the virus in Pakistan. MATERIALS AND METHODS: We studied ninety SARS-CoV-2 strains isolated between March and October 2020. Whole genome sequences from our laboratory and available genomes were used to investigate phylogeny, genetic variantion and mutation rates of SARS-CoV-2 strains in Pakistan. Site specific entropy analysis compared mutation rates between strains isolated before and after June 2020. RESULTS: In March, strains belonging to L, S, V and GH clades were observed but by October, only L and GH strains were present. The highest diversity of clades was present in Sindh and Islamabad Capital Territory and the least in Punjab province. Initial introductions of SARS-CoV-2 GH (B.1.255, B.1) and S (A) clades were associated with overseas travelers. Additionally, GH (B.1.255, B.1, B.1.160, B.1.36), L (B, B.6, B.4), V (B.4) and S (A) clades were transmitted locally. SARS-CoV-2 genomes clustered with global strains except for ten which matched Pakistani isolates. RNA substitution rates were estimated at 5.86 x10-4. The most frequent mutations were 5' UTR 241C > T, Spike glycoprotein D614G, RNA dependent RNA polymerase (RdRp) P4715L and Orf3a Q57H. Strains up until June 2020 exhibited an overall higher mean and site-specific entropy as compared with sequences after June. Relative entropy was higher across GH as compared with GR and L clades. More sites were under selection pressure in GH strains but this was not significant for any particular site. CONCLUSIONS: The higher entropy and diversity observed in early pandemic as compared with later strains suggests increasing stability of the genomes in subsequent COVID-19 waves. This would likely lead to the selection of site-specific changes that are advantageous to the virus, as has been currently observed through the pandemic.

Synthesis and degradation of acyl peptide using enzyme from Pseudomonas aeruginosa.

The detailed properties of the enzyme from Pseudomonas aeruginosa, which catalyzes the N-acyl linkage between myristic acid and the N-terminal glycine residue of the octapeptide GNAAAARR-NH(2) (PKA) in aqueous solution without ATP and CoA, were studied. The substrate specificity for the acyl peptide in the synthetic reaction was examined, and it was found that at least eight amino acid residues are required for the reaction and that the N-terminal glycine residue is not absolutely essential for the reaction because the activity was detected using the octapeptide that has an N-terminal alanine. The activity was also strongly affected by the amino acid sequence because the activity was very weak in the reaction using GARASVLS-NH(2) (HIV-1p17(gag)). The substrate specificity for fatty acids was also examined. In the reactions using lauric acid and decanoic acid, only slight activities were detected; however, those activities were very small compared with the activity in the reaction using myristic acid. In addition, the degradation of myristoyl PKA by the enzyme was detected, although there are only a few reports on demyristoylation. The optimum pH and temperature of the degradation reaction were consistent with those of the synthetic reaction. The degradation reaction was inhibited by divalent cations.

Characterization of the biological effect of BiodentineTM on primary dental pulp stem cells.

BACKGROUND: Biodentine™ is relatively a new tricalcium silicate cement that has gained great attention of the researchers due to its biological potential in comparison with other materials. The aim of this study was to investigate the optimum concentrations of Biodentine in relation to its stimulatory or inhibitory effect on proliferation, migration and adhesion of stem cells of human exfoliated deciduous teeth (SHED). The cell cultures of SHED were treated with Biodentine™ extract at four different concentrations; 20mg/ml, 2mg/ml, 0.2mg/ml and 0.02mg/ml. Cells cultured without Biodentine™ were kept as a blank control. The proliferation potential of SHED cells was evaluated by MTT viability analysis for 6 days. Migration potential was investigated by wound healing and transwell migration assays. The growth, survival and communication potential of these cells was determined by Adhesion assay. RESULTS: A significant increase was observed in the proliferation and migration of SHED at (2mg/ml, 0.2mg/ml and 0.02mg/ml) while higher concentration of Biodentine™ (20mg/ml) exhibited cytotoxic effect on the cells. However, three tested Biodentine™ concentrations were similar in effect (non-significant) to adhesion ability of cells when compared with blank control. CONCLUSION: Our findings suggest that lower concentrations of Biodentine™ can be considered as the optimum concentrations to enhance the stimulatory effect of Biodentine on SHED.

Vibrio cholerae Transmits Through Water Among the Household Contacts of Cholera Patients in Cholera Endemic Coastal Villages of Bangladesh, 2015-2016 (CHoBI7 Trial).

Recurrent cholera causes significant morbidity and mortality in cholera endemic estuarine areas of Bangladesh. There have been limited studies to investigate the transmission patterns of V. cholerae associated with cholera in Bangladesh. In this study, we characterized V. cholerae serogroup O1 isolated from 30 cholera patients, 76 household contacts, 119 stored drinking water samples, and 119 water source samples in Bakerganj and Mathbaria, two cholera endemic coastal regions in Bangladesh. Results of phenotypic and molecular characterization of V. cholerae isolates (n = 56) confirmed them to be toxigenic belonging to serogroup O1 biotype El Tor (ET), and possessing cholera toxin of the classical biotype (altered ET). Molecular fingerprinting of the V. cholerae O1 of clinical and water origins determined by PFGE of Not-I- digested genomic DNA showed them to be closely related, as the PFGE banding patterns were highly homogenous. Phylogenetic analysis using dendrogram of cholera patients, household contacts, and household groundwater sources showed isolates within households to be clonally linked, suggesting water as an important vehicle of transmission of cholera in the coastal villages of Bangladesh. Transmission of toxigenic V. cholerae O1 through drinking water in cholera endemic rural settings underscores the urgent need for evidence based water, sanitation, and hygiene interventions promoting safe drinking water to prevent morbidity and mortality related to cholera and other enteric infections in Bangladesh.

Purification and characterization of enzyme responsible for N-myristoylation of octapeptide in aqueous solution without ATP and coenzyme a from Pseudomonas aeruginosa.

The enzyme that catalyzes N-acyl linkage between myristic acid and the NH(2)-terminal glycine residue of the octapeptide Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg-NH(2) in aqueous solution without ATP and coenzyme A was found in Pseudomonas aeruginosa. The enzyme was purified from cell-free crude extract using DEAE-Cellulose, Sephadex G-200, CM-Sephadex C-50, and hydroxyapatite column chromatographies, and then purified approximately 1900-fold with about 1.5% recovery of enzyme activity from the crude extract. Finally, the purified enzyme showed a main band on SDS polyacrylamide gel electrophoresis after staining with Coomassie Brilliant Blue. The band corresponded to a molecular mass of approximately 60 kDa. The K(m)s of the purified enzyme for the substrate myristic acid and the octapeptide were 0.36 and 2.6 mM, respectively. When myristoyl-CoA instead of myristic acid was used as the substrate for the enzyme reaction, myristoyl octapeptide could be synthesized as observed in the case of myristic acid. The K(m) of myristoyl-CoA was 0.17 mM.