Magnetic Nanoclusters Increase the Sensitivity of Lateral Flow Immunoassays for Protein Detection: Application to Pneumolysin as a Biomarker for Streptococcus pneumoniae

Lateral flow immunoassays for detecting biomarkers in body fluids are simple, quick, inexpensive point-of-care tests widely used in disease surveillance, such as during the coronavirus disease 2019 (COVID-19) pandemic. Improvements in sensitivity would increase their utility in healthcare, food safety, and environmental control. Recently, biofunctional magnetic nanoclusters have been used to selectively label target proteins, which allows their detection and quantification with a magneto-inductive sensor. This type of detector is easily integrated with the lateral flow immunoassay format. Pneumolysin is a cholesterol-dependent cytolysin and one of the most important protein virulence factors of pneumonia produced by Streptococcus pneumoniae. It is recognized as an important biomarker for diagnosis in urine samples. Pneumonia is the infectious disease that causes the most deaths globally, especially among children under five years and adults over 65 years, most of them in low- and middle-income countries. There especially, a rapid diagnostic urine test for pneumococcal pneumonia with high sensitivity and specificity would be helpful in primary care. In this work, a lateral flow immunoassay with magnetic nanoclusters conjugated to anti-pneumolysin antibodies was combined with two strategies to increase the technique's performance. First, magnetic concentration of the protein before the immunoassay was followed by quantification by means of a mobile telephone camera, and the inductive sensor resulted in detection limits as low as 0.57 ng (telephone camera) and 0.24 ng (inductive sensor) of pneumolysin per milliliter. Second, magnetic relocation of the particles within the test strip after the immunoassay was completed increased the detected signal by 20%. Such results obtained with portable devices are promising when compared to non-portable conventional pneumolysin detection techniques such as enzyme-linked immunosorbent assays. The combination and optimization of these approaches would have excellent application in point-of-care biodetection to reduce antibiotic misuse, hospitalizations, and deaths from community-acquired pneumonia

Detection of Streptococcus pneumoniae in Urine by Loop-Mediated Isothermal Amplification

Objective To assess the loop-mediated isothermal amplification (LAMP) to detect cell-free DNA from Streptococcus pneumoniae in urine samples from children with pneumococcal pneumonia. Methods LAMP reactions using four primers (backward inner primer, forward inner primer, B3, and F3) targeting conserved regions of the S. pneumoniae ply gene and DNA from the recombinant plasmid pTrc99A-ply were optimized for temperature (65 degrees C) and MgSO4 concentration (8mM) conditions. Urine samples from 71 patients with symptoms of pneumonia and from 17 healthy children were tested side by side using the isothermal methodology LAMP and the commercial urinary antigen test, BinaxNOW S. pneumoniae assay. Percentages of sensitivity, specificity, positive predictive value (PPV), negative predictive value, and positive (LR) were calculated to compare both tests. Results The specificity of the LAMP reaction was confirmed against several species of bacteria and yeast that can cause pneumonia or urine infections. The suitability of the LAMP assay was evaluated in urine samples from 71 patients and 17 healthy children. All patients (100%) with confirmed pneumococcal pneumonia were positive for the LAMP assay. Among patients with possible/probable pneumonia, 74.1% were identified as positive using the LAMP test. Notably, a higher specificity (95.4%), PPV ( 94.1%) and positive LR (21.7) were found compared with the urinary antigen test. Conclusion The presence of S. pneumoniae cell-free DNA in urine samples of pediatric patients can be used as a specific diagnostic biomarker for community-acquired pneumonia by using the LAMP methodology

Developing Diagnostic and Therapeutic Approaches to Bacterial Infections for a New Era: Implications of Globalization

In just a few months, the current coronavirus pandemic has exposed the need for a more global approach to human health. Indeed, the quick spread of infectious diseases and their unpredictable consequences, in terms of human lives and economic losses, will require a change in our strategy, both at the clinical and the research level. Ultimately, we should be ready to fight against infectious diseases affecting a huge number of people in different parts of the world. This new scenario will require rapid, inexpensive diagnostic systems, applicable anywhere in the world and, preferably, without the need for specialized personnel. Also, treatments for these diseases must be versatile, easily scalable, cheap, and easy to apply. All this will only be possible with joint support of the governments, which will have to make the requirements for the approval of new therapies more flexible. Meanwhile, the pharmaceutical sector must commit to prioritizing products of global interest over the most profitable ones. Extreme circumstances demand a vehement response, and any profit losses may well pay dividends going forward. Here, we summarize the developing technologies destined to face the current and future health challenges derived from infectious diseases and discuss which ones have more possibilities of being implemented

Gram-positive pneumonia: Possibilities offered by phage therapy

Pneumonia is an acute pulmonary infection whose high hospitalization and mortality rates can, on occasion, bring healthcare systems to the brink of collapse. Both viral and bacterial pneumonia are uncovering many gaps in our understanding of host–pathogen interactions, and are testing the effectiveness of the currently available antimicrobial strategies. In the case of bacterial pneumonia, the main challenge is antibiotic resistance, which is only expected to increase during the current pandemic due to the widespread use of antibiotics to prevent secondary infections in COVID-19 patients. As a result, alternative therapeutics will be necessary to keep this disease under control. This review evaluates the advantages of phage therapy to treat lung bacterial infections, in particular those caused by the Gram-positive bacteria Streptococcus pneumoniae and Staphylococcus aureus, while also highlighting the regulatory impediments that hamper its clinical use and the difficulties associated with phage research

Identification of Pneumococcal Serotypes by PCR–Restriction Fragment Length Polymorphism

Streptococcus pneumoniae shows more than 90 capsular serotypes that can be distinguished by their reactivity against antisera. The main objective of this work was the development of a molecular method for serotyping without the use of antisera. A computer program containing an algorithm was used to search in a database for potentially useful enzymes for Restriction Fragment Length Polymorphism-RFLP typing, in order to maximize the discrimination between different serotypes. DNA sequences of 90 serotypes for the region between dexB and aliA genes were compiled, and a computer screening of restriction enzymes was performed. The wzg–wzh–wzd–wze region and Sse9I restriction predicted unique PCR-RFLP patterns for 39 serotypes and eight serogroups. A second restriction enzyme resolved fragment specific patterns for 25 serotypes. The method was tested with 98 serotype-unknown clinical isolates. PCR-RFLP analysis deduced correct serotypes that were confirmed by Quellung reaction for 78.5% of the isolates

Toxicity prediction based on artificial intelligence: A multidisciplinary overview

The use and production of chemical compounds are subjected to strong legislative pressure. Chemical toxicity and adverse effects derived from exposure to chemicals are key regulatory aspects for a multitude of industries, such as chemical, pharmaceutical, or food, due to direct harm to humans, animals, plants, or the environment. Simultaneously, there are growing demands on the authorities to replace traditional in vivo toxicity tests carried out on laboratory animals (e.g., European Union REACH/3R principles, Tox21 and ToxCast by the U.S. government, etc.) with in silica computational models. This is not only for ethical aspects, but also because of its greater economic and time efficiency, as well as more recently because of their superior reliability and robustness than in vivo tests, mainly since the entry into the scene of artificial intelligence (AI)-based models, promoting and setting the necessary requirements that these new in silico methodologies must meet. This review offers a multidisciplinary overview of the state of the art in the application of AI-based methodologies for the fulfillment of regulatory-related toxicological issues. This article is categorized under: Data Science > Chemoinformatics Data Science > Artificial Intelligence/Machine Learning