A Pentaplex PCR Assay for the Detection and Differentiation of Shigella Species

The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, andrfpB for S. dysenteriae, aswell as one internal control (ompA) gene, was developed in a single reaction to detect and differentiate Shigella spp. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 104 CFU/ml, or approximately 120 CFU per reaction mixture of bacteria. The sensitivity of the pentaplex PCR assay was further improved following preincubation of the stool samples in Gram-negative broth. A preliminary study with 30 diarrhoeal specimens resulted in no cross-reaction with other non-Shigella strains tested. We conclude that the developed pentaplex PCR assay is robust and can provide information about the four target genes that are essential for the identification of the Shigella genus and the three Shigella species responsible for the majority of shigellosis cases

Clinical relevance of molecular testing methods in the diagnosis and guidance of therapy in patients with staphylococcal empyema: a systematic review and meta-analysis

BackgroundEfficient detection tools for determining staphylococcal pleural infection are critical for its eradication. The objective of this meta-analysis was to assess the diagnostic utility of nucleic acid amplification tests (NAAT) in suspected empyema cases to identify staphylococcal strains and avoid unnecessary empiric methicillin-resistant Staphylococcus aureus (MRSA) therapy.MethodsFrom inception to July 24, 2021, relevant records were retrieved from PubMed, Embase, Scopus, Web of Science, and the Cochrane Library. The quality of studies was determined using the QUADAS-2 tool. The pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and hierarchical summary receiver operating characteristic (HSROC) curve for NAAT’s diagnostic performance were evaluated using an HSROC model.ResultsEight studies comprising 424 samples evaluated NAAT accuracy for Staphylococcus aureus (SA) identification, while four studies comprising 317 samples evaluated methicillin-resistant Staphylococcus aureus (MRSA) identification. The pooled NAAT summary estimates for detection of both SA (sensitivity: 0.35 (95% CI 0.19–0.55), specificity: 0.95 (95% CI 0.92–0.97), PLR: 7.92 (95% CI 4.98–12.59), NLR: 0.44 (95% CI 0.14–1.46), and DOR: 24.0 (95% CI 6.59–87.61) ) and MRSA (sensitivity: 0.45 (95% CI 0.15–0.78), specificity: 0.93 (95% CI 0.89–0.95), PLR: 10.06 (95% CI 1.49–67.69), NLR: 0.69 (95% CI 0.41–1.15), and DOR: 27.18 (95% CI 2.97–248.6) ) were comparable. The I2 statistical scores for MRSA and SA identification sensitivity were 13.7% and 74.9%, respectively, indicating mild to substantial heterogeneity. PCR was frequently used among NAA tests, and its diagnostic accuracy coincided well with the overall summary estimates. A meta-regression and subgroup analysis of country, setting, study design, patient selection, and sample condition could not explain the heterogeneity (meta-regression P = 0.66, P = 0.46, P = 0.98, P = 0.68, and P = 0.79, respectively) in diagnostic effectiveness.ConclusionsOur study suggested that the diagnostic accuracy of NAA tests is currently inadequate to substitute culture as a principal screening test. NAAT could be used in conjunction with microbiological culture due to the advantage of faster results and in situations where culture tests are not doable

Incidence of Colorectal Carcinoma in the Remote Area of Sindh, Pakistan

Background: Colorectal carcinoma (CRC) is the third most common cancer in the world that show malignant growth in the colon, rectum, and or appendix. CRC is the second most common malignancy in females and third in males. This study aimed to ascertain the incidence of colorectal carcinoma (CRC) in a population in a remote area of Sindh, Pakistan, and also compared and correlated the sociodemographic characteristics and different parameters such as diagnosis, grade, and histopathology of the CRC cases.Methods: The study was conducted from February 2012 to 2019 at Pakistan Atomic Energy Cancer Hospital (NORIN) Nawabshah Sindh, Pakistan. All cases were evaluated through a detailed history, clinical examination, radiological examination, and histopathology-proven cases. The sociodemographic parameters, diagnosis grades, and histopathology of the CRC were statistically compared and correlated by SPSS version 21.Results: Out of a total of 10,848 reported patients, 424 (3.9%) were of CRC. The CRC was further comprised of 63.9% males and 36.1% females. We classified our patients into two age groups, ≤ 40 years (Group-A), and > 40 years (Group B). Group-A comprised 41% of patients, and their mean age was 28±6.98 years, while Group B comprised 59% of patients with a mean age of 43±8.3 years. Histological specimens divulged that most of the common specimens were of adenocarcinoma. Early diagnosis was very strenuous due to no signs and symptoms. Thus, the majority of the patients, approximately 39%, were found to be of grade 2 carcinoma.Conclusion: Our study unveils an increased number of patients with CDC at an age of <40 belonging to rural areas. Further studies are needed to elucidate the cause of this high incidence with a particular focus on genetic and molecular risk factors.Keywords: Active lifestyle, Colorectal Carcinoma; Age group; Colorectal Adenocarcinoma

Proteome level analysis of drug-resistant Prevotella melaninogenica for the identification of novel therapeutic candidates

The management of infectious diseases has become more critical due to the development of novel pathogenic strains with enhanced resistance. Prevotella melaninogenica, a gram-negative bacterium, was found to be involved in various infections of the respiratory tract, aerodigestive tract, and gastrointestinal tract. The need to explore novel drug and vaccine targets against this pathogen was triggered by the emergence of antimicrobial resistance against reported antibiotics to combat P. melaninogenica infections. The study involves core genes acquired from 14 complete P. melaninogenica strain genome sequences, where promiscuous drug and vaccine candidates were explored by state-of-the-art subtractive proteomics and reverse vaccinology approaches. A stringent bioinformatics analysis enlisted 18 targets as novel, essential, and non-homologous to humans and having druggability potential. Moreover, the extracellular and outer membrane proteins were subjected to antigenicity, allergenicity, and physicochemical analysis for the identification of the candidate proteins to design multi-epitope vaccines. Two candidate proteins (ADK95685.1 and ADK97014.1) were selected as the best target for the designing of a vaccine construct. Lead B- and T-cell overlapped epitopes were joined to generate potential chimeric vaccine constructs in combination with adjuvants and linkers. Finally, a prioritized vaccine construct was found to have stable interactions with the human immune cell receptors as confirmed by molecular docking and MD simulation studies. The vaccine construct was found to have cloning and expression ability in the bacterial cloning system. Immune simulation ensured the elicitation of significant immune responses against the designed vaccine. In conclusion, our study reported novel drug and vaccine targets and designed a multi-epitope vaccine against the P. melaninogenica infection. Further experimental validation will help open new avenues in the treatment of this multi-drug-resistant pathogen

Colorimetric sensing of uric acid based on sawdust-deposited silver nanoparticles via an eco-friendly and cost-effective approach

Uric acid is directly linked to gout, arthritis, neurological, cardiovascular, and kidney-related disorders. It is a byproduct obtained from the breakdown of purines and a significant indicator of hyperuricemia observed in both urine and blood. In the absence of any enzyme, it's quite difficult to develop a novel, cost-effective, and clinical method for uric acid detection. Herein, we report a very simple, low-cost, and non-enzymatic method for the selective identification and quantification of uric acid using green synthesized silver nanoparticles (Ag NPs). The desired Ag NPs were synthesized by the hydrothermal method using Erythrina suberosa sawdust as a deagglomeration agent and Psidium guajava extract as a reductant. The synthesis of the sensing platform, i.e., sawdust-deposited Ag NPs, was confirmed through different techniques such as UV-Vis spectrophotometer, FTIR, XRD, EDX, and scanning electron microscopy (SEM). Sawdust can offer a good, environmentally friendly, and cost-effective strategy to overcome the problem of agglomeration in nanoparticles. The enzyme mimic, with the help of H2O2, oxidizes the colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to oxidized TMB with a blue-green color. The addition of uric acid reduces the oxidized TMB to a colorless product, resulting in a colorimetric change. For quality improvement, different reaction parameters, including pH, time, TMB, and NPs concentration, were optimized. Our proposed sensor responds in linear ranges of 0.04–0.360 μM, with a limit of quantification of 0.01 μM and a limit of detection of 0.004 μM. The suggested enzyme mimic detected uric acid in blood samples, with particular specificity in the presence of competitive analytes

mRNA Vaccine Designing Using Chikungunya Virus E Glycoprotein through Immunoinformatics-Guided Approaches

Chikungunya virus is an alphavirus transmitted by mosquitos that develops into chikungunya fever and joint pain in humans. This virus’ name originated from a Makonde term used to describe an illness that changes the joints and refers to the posture of afflicted patients who are affected by excruciating joint pain. There is currently no commercially available drug or vaccine for chikungunya virus infection and the treatment is performed by symptom reduction. Herein, we have developed a computationally constructed mRNA vaccine construct featuring envelope glycoprotein as the target molecule to aid in the treatment process. We have utilized the reverse vaccinology approach to determine epitopes that would generate adaptive immune reactions. The resulting T and B lymphocytes epitopes were screened by various immunoinformatic tools and a peptide vaccine construct was designed. It was validated by proceeding to docking and MD simulation studies. The following design was then back-translated in nucleotide sequence and codons were optimized according to the expression host system (H. sapiens). Various sequences, including 3′ and 5′ UTR regions, Kozak sequence, poly (A) tail, etc., were introduced into the sequence for the construction of the final mRNA vaccine construct. The secondary structure was generated for validation of the mRNA vaccine construct sequence. Additionally, in silico cloning was also performed to design a vector for proceeding towards in vitro experimentation. The proposed designed vaccine construct may proceed with experimental testing for further efficacy verification and the final development of a vaccine against chikungunya virus infection