Pneumolysin as a target for new therapies against pneumococcal infections: A systematic review.

BACKGROUND: This systematic review evaluates pneumolysin (PLY) as a target for new treatments against pneumococcal infections. Pneumolysin is one of the main virulence factors produced by all types of pneumococci. This toxin (53 kDa) is a highly conserved protein that binds to cholesterol in eukaryotic cells, forming pores that lead to cell destruction. METHODS: The databases consulted were MEDLINE, Web of Science, and Scopus. Articles were independently screened by title, abstract, and full text by two researchers, and using consensus to resolve any disagreements that occurred. Articles in other languages different from English, patents, cases report, notes, chapter books and reviews were excluded. Searches were restricted to the years 2000 to 2021. Methodological quality was evaluated using OHAT framework. RESULTS: Forty-one articles describing the effects of different molecules that inhibit PLY were reviewed. Briefly, the inhibitory molecules found were classified into three main groups: those exerting a direct effect by binding and/or blocking PLY, those acting indirectly by preventing its effects on host cells, and those whose mechanisms are unknown. Although many molecules are proposed as toxin blockers, only some of them, such as antibiotics, peptides, sterols, and statins, have the probability of being implemented as clinical treatment. In contrast, for other molecules, there are limited studies that demonstrate efficacy in animal models with sufficient reliability. DISCUSSION: Most of the studies reviewed has a good level of confidence. However, one of the limitations of this systematic review is the lack of homogeneity of the studies, what prevented to carry out a statistical comparison of the results or meta-analysis. CONCLUSION: A panel of molecules blocking PLY activity are associated with the improvement of the inflammatory process triggered by the pneumococcal infection. Some molecules have already been used in humans for other purposes, so they could be safe for use in patients with pneumococcal infections. These patients might benefit from a second line treatment during the initial stages of the infection preventing acute respiratory distress syndrome and invasive pneumococcal diseases. Additional research using the presented set of compounds might further improve the clinical management of these patients.

Electrocatalytic Palladium Nanoclusters as Versatile Indicators of Bioassays: Rapid Electroanalytical Detection of SARS-CoV-2 by Reverse Transcription Loop-Mediated Isothermal Amplification.

Quantitative polymerase chain reaction (qPCR) is considered the gold standard for pathogen detection. However, improvement is still required, especially regarding the possibilities of decentralization. Apart from other reasons, infectious diseases demand on-site analysis to avoid pathogen spreading and increase treatment efficacy. In this paper, the detection of SARS-CoV-2 is carried out by reverse transcription loop-mediated isothermal amplification, which has the advantage of requiring simple equipment, easily adaptable to decentralized analysis. It is proposed, for the first time, the use of palladium nanoclusters (PdNCs) as indicators of the amplification reaction at end point. The pH of the medium decreases during the reaction and, in turn, a variation in the catalytic activity of PdNCs on the oxygen reduction reaction (ORR) can be electrochemically observed. For the detection, flexible and small-size screen-printed electrodes can be premodified with PdNCs, which together with the use of a simple and small electrochemical equipment would greatly facilitates their integration in field-deployable devices. This would allow a faster detection of SARS-CoV-2 as well as of other future microbial threats after an easy adaptation.

Electrochemical Detection for Isothermal Loop-Mediated Amplification of Pneumolysin Gene of Streptococcus pneumoniae Based on the Oxidation of Phenol Red Indicator.

A highly sensitive electrochemical methodology for end-point detection of loop-mediated isothermal nucleic acid amplification reactions was developed. It is based on the oxidation process of phenol red (PR), commonly used as a visual indicator. The dependence of its redox process on pH, which changes during amplification, allows performing quantitative measurements. Thus, the change in the oxidation potential of PR during the amplification is used, for the first time, as the analytical signal that correlates with the number of initial DNA copies. As a proof-of-concept, the amplification of the pneumolysin gene from Streptococcus pneumoniae, one of the main pathogens causing community-acquired pneumonia, is performed. Combination of isothermal amplification with electrochemical detection, performed on small-size flexible electrodes, allows easy decentralization. Adaptation to the detection of other pathogens causing infectious diseases would be very useful in the prevention of future epidemics.

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.

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.

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.

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.

Protein Expression Analysis by Western Blot and Protein-Protein Interactions.

Western blot analysis is widely used for detecting protein expression, analysis of protein-protein interactions, and searching for new biomarkers. Also, it is a diagnostic tool used for detection of human diseases and microorganism infections.Some Streptococcus pneumoniae proteins are important virulence factors and a few of them are diagnostic markers. Here, we describe the detection of two pneumococcal proteins, pneumolysin and PpmA, in human urine by using monoclonal and polyclonal antibodies.

Performance of a pneumolysin enzyme-linked immunosorbent assay for diagnosis of pneumococcal infections.

A pneumolysin-specific enzyme-linked immunosorbent assay (PLY-ELISA) for the detection of pneumolysin in urine was developed and evaluated in comparison with the commercially available Binax Now Streptococcus pneumoniae test (Binax, Portland, ME) for the diagnosis of pneumococcal infections. Assay sensitivity was evaluated using urine from 108 patients with culture-confirmed pneumococcal infections. In adults, the sensitivity and specificity of the PLY-ELISA were 56.6% and 92.2%, respectively. In children with nasopharyngeal pneumococcal carriage, PLY-ELISA and Binax Now S. pneumoniae test sensitivities were 62.5% and 87.5%, respectively, while specificities were 94.4% and 27.8%, respectively. In children with nonnasopharyngeal pneumococcal carriage, PLY-ELISA and Binax Now S. pneumoniae test sensitivities were 68.7% and 93.7%, respectively, and test specificities were 94.1% and 41.2%, respectively. The persistence of pneumolysin in urine of pneumococcal pneumonia patients decreased significantly after 4 to 6 days of treatment. Our data suggest that combining the high specificity of the PLY-ELISA with the high sensitivity of the Binax Now S. pneumoniae test would enable pneumococcal infections to be accurately diagnosed in children.

Protection against pneumococcal pneumonia in mice by monoclonal antibodies to pneumolysin.

Pneumolysin (PLY) is an important virulence factor of Streptococcus pneumoniae. We examined the ability of three murine monoclonal antibodies (MAbs) to PLY (PLY-4, PLY-5, and PLY-7) to affect the course of pneumococcal pneumonia in mice. The intravenous administration of antibodies PLY-4 and PLY-7 protected the mice from the lethal effect of the purified toxin. Mice treated with PLY-4 before intranasal inoculation of S. pneumoniae type 2 survived longer (median survival time, 100 h) than did untreated animals (median survival time, 60 h) (P < 0.0001). The median survival time for mice treated with a combination of PLY-4 and PLY-7 was 130 h, significantly longer than that for mice given isotype-matched indifferent MAbs (P = 0.0288) or nontreated mice (P = 0.0002). The median survival time for mice treated with a combination of three MAbs was significantly longer (>480 h) than that for mice treated with PLY-5 (48 h; P < 0.0001), PLY-7 (78 h; P = 0.0007), or PLY-4 (100 h; P = 0.0443) alone. Similarly, the survival rate for mice treated with three MAbs (10 of 20 mice) was significantly higher than the survival rate obtained with PLY-5 (1 of 20; P = 0.0033), PLY-4 (2 of 20; P = 0.0138), or PLY-7 (3 of 20; P = 0.0407) alone. These results suggest that anti-PLY MAbs act with a synergistic effect. Furthermore, MAb administration was associated with a significant decrease in bacterial lung colonization and lower frequencies of bacteremia and tissue injury with respect to the results for the control groups.

Immunodetection of pneumolysin in human urine by ELISA.

An ELISA test has been employed for the detection of pneumolysin (PLY) in urine from 14 pneumococcal pneumonia patients and from 11 healthy adult volunteers. The urines of all the 11 healthy adult volunteers developed signals around the mean of the blanks, whereas all the pneumococcal pneumonia patient urines rendered signals at least five times this mean. Chemiluminescent Western blot analyses of these urines, carried out with the PLY-specific rabbit polyclonal IgG preparation used in ELISA, were negative. The 30-kDa filtrates of three high-signal urines were ELISA negative, suggesting that this ELISA test mainly detected high molecular weight forms in urine rather than free PLY-derived antigenic fragments. The urine sample, which rendered the highest ELISA signal, was then concentrated by filtration through a 10-kDa filter. When this concentrate was subjected to Western blot with the ELISA-capture monoclonal antibody, a major band was developed. Its relative molecular mass was similar to that of recombinant PLY and its peptide mass fingerprinting showed peptides corresponding to amino acid stretches from the four domains of the PLY molecule. When the pool of PLY-negative urines was sham-contaminated with purified recombinant pneumolysin, a conspicuous matrix effect was observed; nevertheless, this ELISA test was still reproducible and highly sensitive, detecting pneumolysin in the order of picograms per milliliter. A comparison was also made between this PLY-ELISA and the Binax NOW Streptococcus pneumoniae Urinary Antigen Test in analysing bacterial isolates. On the basis of the minimum number of pneumococci examined, both tests were shown to have similar potency, but strain-dependent discrepancies were observed. This ELISA could provide an alternative to the Binax NOW Streptococcus pneumoniae Urinary Antigen Test in the diagnosis of pneumococcal pneumonia.