Rapid Diagnostic Method Research Articles

Создание атласа эталонных культур для оценки микробиологического статуса глазной поверхности методом сканирующей электронной микроскопии с лантаноидным контрастированием

Relevance. In modern ophthalmology, for the diagnosis of inflammatory diseases of the ocular surface, a new technique is needed that allows significantly reducing the study time, increasing the informative value of visual data. Scanning electron microscopy can become such a method, provided that a determinant atlas is created with a set of reference cultures for preliminary identification of microorganisms detected on the ocular surface. Purpose. The purpose of our study is to create an atlas of microorganisms, samples of which were prepared by the method of lanthanide contrast for SEM. Material and methods. We used cultures of microorganisms of the following species Achromobacter xylosoxidans, Acinetobacter baumannii, Bacillus cereus, Burkholderia cepacia, Corinebacterium diphthereae, Escherichia coli, Klebsiella pneumoniae, Moraxella catarhalis, Pseudomonas aeruginosa, Rothia mucilaginosa, Salmonella enterica, Serratia marcescens, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus pneumoniae, Candida albicans, Pneumocystis jirovecii, Mycobacterium abscessus. Samples of reference cultures of microorganisms were prepared by the method of lanthanide contrast for SEM. Further, the obtained images of microorganisms were used to compile illustrations of the atlas and analyze the features that are conservative for the defined groups. Results and discussions. 45 images were obtained with a standard resolution of 58.1 nm/px at 1024x768 px and 452 images with a standard resolution of 21 nm/px at 1024x768 px. Preliminary data of typing indicate that with the help of the proposed atlas-determinant, a satisfactory diagnosis up to the genus-level clusters is possible. Conclusion. The atlas with illustrations of reference cultures, the most common microorganisms on the ocular surface, will be the first step for the introduction of a new technique as an express diagnosis of infectious diseases of the eye. Keywords: scanning electron microscopy, lanthanide contrast, rapid diagnostic method, inflammatory diseases of the ocular surface.

Label-free electrochemical immunosensor based on l-cysteine-functionalized AuNP on reduced graphene oxide for the detection of dengue virus E-protein in dengue blood serum

Dengue fever is one of the most deadly viruses, and it has become a recurring public health problem in tropical areas. The increase in dengue cases worldwide has increased the demand for rapid and accurate diagnostic methods. In order to detect the content of dengue virus type E-proteins (DENV-E protein), we fabricated an efficient label-free electrochemical immunosensor platform that was constructed based on in situ reduction and functionalized gold nanoparticles decorated heteroatom-doped reduced graphene oxides nanocomposites (AuNPs/NSG). Here, L-cystein (L-cys) is introduced to green reducing and stabilizing agent of Au(III) and graphene oxide . The sensing platform offers a suitable higher number of antibody immobilization by providing L-cys. The highly crystalline AuNP was unfoirmly grafting on 2D graphene sheet . Under the optimized conditions, the as-designed immunosensor exhibited a wide linear working range from 0.01–100 ng mL −1 with a low detection limit of 1.6 pg mL −1 for DENV-E detection. The proposed immunosensor shows high selective for discriminating DENV-E against antibodies of their antibodies, including the closely related DENV. The reliability of fabricated immunosensor had been used to detect the viral E-protein contents in serum samples collected from patients, and obtained results were compared with the enzyme-linked immunosorbend assay. Furthermore, the developed AuNPs/NSG immunosensor can be used as a potential probe for future prospective clinical diagnostic applications. The electrochemical detection of dengue virus type E-proteins (DENV–E protein), the efficient label-free electrochemical immunosensor platform was constructed based on in situ reduction and functionalized gold nanoparticles decorated heteroatom doped reduced graphene oxides nanocomposites. The fabricated electrode exhibits lower detection limit and higher sensing and selective detection of DENV-E protein in dengue blood serum. • Label-free, highly selective, and an ultrasensitive label-free electrochemical immunosensor platform for DENV–E protein detection. • In situ reduction and functionalized AuNPs/NSG was synthesized via l -cysteine to achieve excellent electrochemical properties. • l -Cysteine on the nanocomposites to bind covalently with anti-DENV–E protein immobilization. • Immunosensor had a low detection limit of 1.6 pg mL −1 for DENV-E detection. • Successful detection of DENV–E protein in human blood serum samples.

Electrochemical Biosensor for Rapid Detection of Listeria monocytogenes

Listeria monocytogenes (L. monocytogenes) is a common foodborne pathogen that has been responsible for many foodborne illness outbreaks in recent years. Currently, Listeria contamination in food products is identified only through molecular tests conducted in diagnostic laboratories. No established phage based diagnostic methods for L. monocytogenes during food production or processing are used. Here we report a potentially disruptive rapid diagnostic method based on electrochemical biosensing principles that use bacteriophages as bioreceptors for selective identification and quantification of L. monocytogenes. Electrochemical biosensors are good alternatives to molecular detection methods due to their ease of use, high specificity, sensitivity, and low cost. Bacteriophages can serve as excellent biorecognition elements in biosensors due to their robust stability in a range of environmental conditions and their ability to distinguish between live and dead bacterial cells. The impedimetric biosensing platform for L. monocytogenes detection was developed by immobilizing P100 bacteriophage onto quarternized polyethylenimine modified carbon nanotubes using an in-house developed molecular tethering method. The resulting sensor showed high selectivity and sensitivity toward L. monocytogenes with a limit of detection of 8.4 CFU/ml. Initial results demonstrate that the biosensing platform is highly reliable in its selectivity towards its target analyte, L. monocytogenes.

Pneumonia Caused by Coinfection with Cytomegalovirus and Pneumocystis jirovecii in an HIV-Negative Infant Diagnosed by Metagenomic Next-Generation Sequencing

BackgroundPneumonia produced by coinfection with Pneumocystis jirovecii (PJ) and cytomegalovirus (CMV) in infants and young children without timely diagnosis and treatment is often fatal due to the limitations of traditional tests. More accurate and rapid diagnostic methods for multiple infections are urgently needed.Case PresentationHere, we report a case of a 2-month-old boy with pneumonia caused by Pneumocystis jirovecii (PJ) and cytomegalovirus (CMV) without HIV infection. Chest computed tomography (CT) showed massive exudative consolidation in both lungs. Microscopic examination of stained sputum and smear specimens and bacterial and fungal culture tests were all negative, and CMV nucleic acid and antibody tests were positive. After a period of antiviral and anti-infective therapy, pulmonary inflammation was not relieved. Subsequently, sputum and venous blood samples were analysed by metagenomic next-generation sequencing (mNGS), and the sequences of PJ and CMV were acquired. The patient was finally diagnosed with pneumonia caused by PJ and CMV coinfection. Anti-fungal combined with anti-viral therapy was given immediately. mNGS re-examination of bronchoalveolar lavage fluid (BALF) also revealed the same primary pathogen. Therapy was stopped due to the request of the patient’s guardian. Hence, the child was discharged from the hospital and eventually died.ConclusionThis case emphasizes the combined use of mNGS and traditional tests in the clinical diagnosis of mixed lung infections in infants without HIV infection. mNGS is a new adjunctive diagnostic method that can rapidly discriminate multiple causes of pneumonia.

High Asymptomatic Cases of Babesiosis in Dogs and Comparison of Diagnostic Performance of Conventional PCR vs Blood Smears.

Babesiosis is one of the most important globally extended and quickly spreading tick-borne infections of dogs. Diagnosis of babesiosis in Sri Lanka is based on clinical signs followed by thin blood smears which could be error-prone due to undetected early infections, absence of clinical signs or low parasitemia. The present study investigated the prevalence of babesiosis in dogs presented to the Veterinary Teaching Hospital (VTH) at the Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, for treatments, vaccinations, and regular check-ups, and compared the diagnosis methods of microscopy and molecular analysis. Blood samples from dogs were collected from January to June 2019. First, Giemsa stained blood smears were prepared, and then the blood samples were subjected to PCR using genus-specific primers to amplify a 411-450bp region in the 18S rRNA gene. Twenty samples from PCR amplified products were sequenced for species identification and phylogenetic analysis. Clinical signs of the dogs were noted down, and ticks were also collected from dogs if any. Results show a very high prevalence of canine babesiosis (78.6%) among the dogs brought to the VTH. The parasite was identified microscopically and genetically as Babesia gibsoni. A large percentage (66.7%) of infections was asymptomatic. Out of 42 blood samples, 19 (45.2%) were microscopically positive for babesiosis while 33 (78.6%) were PCR positive, showing a significant difference in the two methods of diagnosis (chi-square test, χ2 = 9.462, p = 0.002). Three tick species: Rhipicephalus haemaphysaloides, Haemaphysalis bispinosa, and Rhipicephalus sanguineus were found attached to the dogs. This study shows a very high prevalence of canine babesiosis among dogs in the Kandy area. Most of these infections might go undetected if only microscopy was used to diagnose. An improved, rapid diagnostic method such as the novel, PCR-based point-of-care diagnostic method that detects very low parasitemia within 30min is needed. Moreover, as most infected dogs did not show clinical signs, they may act as reservoirs of infection. The ability of asymptomatic dogs to spread babesiosis should be investigated.

Fast, Simple, and Highly Specific Molecular Detection of Porphyromonas gingivalis Using Isothermal Amplification and Lateral Flow Strip Methods.

Porphyromonas gingivalis is an important oral pathogen that causes periodontal disease and is difficult to culture under conventional conditions. Therefore, a reliable technique for detecting this pathogenic bacterium is required. Here, isothermal recombinase polymerase amplification (RPA), a new nucleic acid amplification method, was combined with a visualization method based on nanoparticle-based lateral flow strips (LFS) for the rapid detection of P. gingivalis. The species-specific 16S rRNA sequence of P. gingivalis was used as the target for RPA, and a set of specific primer–probe combinations were designed and screened to amplify the target sequences. As a thermostatic amplification method, the RPA reaction, under optimized conditions, takes only 30 min to complete at a constant temperature (37°C). The amplification reaction products can be detected visually by LFS without any need for special equipment. The RPA-LFS method established for the detection of P. gingivalis was shown to be highly specific in distinguishing P. gingivalis from other pathogenic organisms by using 20 clinical isolates of P. gingivalis and 23 common pathogenic microorganisms. Susceptibility measurements and probit regression analysis were performed with gradient dilutions of P. gingivalis genomic DNA. The method was obtained to be highly sensitive, with a detection limit of 9.27 CFU per reaction at 95% probability. By analyzing the gingival sulcus fluid specimens from 130 patients with chronic periodontitis, the results showed that the RPA-LFS method detected 118 positive cases and 12 negative cases of P. gingivalis, and the results obtained were consistent with those of a conventional PCR assay. The RPA–LFS method is an efficient, rapid, and convenient diagnostic method that simplifies the tedious process of detecting P. gingivalis.

Exhaled Breath Volatile Organic Compound Analysis for the Detection of Lung Cancer- A Systematic Review

A rapid and effective diagnostic method is essential for lung cancer since it shows symptoms only at its advanced stage. Research is being carried out in the area of exhaled breath analysis for the diagnosis of various pulmonary diseases including lung cancer. In this method exhaled breath volatile organic compounds (VOC) are analyzed with various techniques such as gas chromatography-mass spectrometry, ion mobility spectrometry, and electronic noses. The VOC analysis is suitable for lung cancer detection since it is non-invasive, fast, and also a low-cost method. In addition, this technique can detect primary stage nodules. This paper presents a systematic review of the various method employed by researchers in the breath analysis field. The articles were selected through various search engines like EMBASE, Google Scholar, Pubmed, and Google. In the initial screening process, 214 research papers were selected using various inclusion and exclusion criteria and finally, 55 articles were selected for the review. The results of the reviewed studies show that detection of lung cancer can be effectively done using the VOC analysis of exhaled breath. The results also show that this method can be used for detecting the different stages and histology of lung cancer. The exhaled breath VOC analysis technique will be popular in the future, bypassing the existing imaging techniques. This systematic review conveys the recent research opportunities, obstacles, difficulties, motivations, and suggestions associated with the breath analysis method for lung cancer detection.

Development of a Real-Time Recombinase Polymerase Amplification Assay for the Rapid Detection of African Swine Fever Virus Genotype I and II.

African swine fever (ASF) is a contagious viral disease in pigs and wild boars which poses a major threat to the pig industry. Rapid and accurate diagnosis is necessary to control ASF. Hence, we developed a rapid diagnostic method using a recombinase polymerase amplification (RPA) assay targeting the conserved sequences of CP204L (p30) thatcan rapidly detect ASF virus (ASFV) genotype strains I and II. The lower detection limit of the real-time RPA assay was 5 × 101 copies per reaction. The real-time RPA assay effectively detected ASFV isolates and clinical specimens belonging to ASFV genotypes I and II. The sensitivity and specificity of the assay were 96.8% (95% confidence interval (CI): 83.3–99.9) and 100% (95% CI: 88.4–100.0), respectively. The agreement between the real-time RPA assay and a reference commercial real-time quantitative polymerase chain reaction (qPCR) was 100%. The real-time RPA assay had a detection time of 6.0 min (95% CI: 5.7–6.2), which was significantly shorter than that of qPCR (49 min; 95% CI: 47.4–50.6; p < 0.001). Thus, the developed real-time RPA assay is a rapid and accurate diagnostic tool for detecting ASFV genotypes I and II.

Rapid and Visual Detection of Barley Yellow Dwarf Virus by Reverse Transcription Recombinase Polymerase Amplification with Lateral Flow Strips.

Barley yellow dwarf virus (BYDV) has been a major viral pathogen causing significant losses of cereal crops including oats worldwide. It spreads naturally through aphids, and a rapid, specific, and reliable diagnostic method is imperative for disease monitoring and management. Here, we established a rapid and reliable method for isothermal reverse transcription recombinase polymerase amplification (RT-RPA) combined with a lateral flow strips (LFS) assay for the detection of BYDV-infected oat samples based on the conserved sequences of the BYDV coat protein gene. Specific primers and a probe for RT-RPA reacted and optimally incubated at 42°C for 10 min, and the end-labeled amplification products were visualized on LFS within 10 min. The RT-RPA-LFS assay showed no cross-reactivity with other major cereal viruses, including barley mild mosaic virus, barley yellow mosaic virus, and rice black streaked dwarf virus, indicating high specificity of the assay. The sensitivity of the RT-RPA-LFS assay was similar to that of reverse transcription polymerase chain reaction, and it was successfully validated to detect BYDV in oat samples from six different regions and in individual aphids. These results confirm the outstanding potential of the RT-RPA-LFS assay for rapid detection of BYDV.

The smartphone biosensors for point-of-care detection of human infectious diseases: Overview and perspectives—A systematic review

The accurate and fast diagnosis of infectious diseases plays a critical role in the timely and tailored treatment of the patients and preventing the infections from the further spread. This is especially necessary in regions with low- and middle-income of the world. According to the smartphone-based sensory applications such as the inimitable ability to obtain portable, user-friendly, accurate, and effective functions, that in turn, converts lab-on-hardware an interesting area of new investigations. In the present review, original research articles published in English were collected from various databases during January 2011–July 2021. Also, the reference lists of the articles have been searched. In between 121 electronically searched citations, 35 articles had the inclusion criteria. The maximum potential and extended utilization of smartphone-based biosensors in the diagnosis of human infectious agents belonged to colorimetric smartphone biosensors. Also, because of their unique futures in easy visualization, comfortable analyzing and processing by smartphone, they are easier to use for point-of-care products. Based on the results, smartphone-based biosensors are rapid, precise, and low-cost diagnostic methods and increasing their quality in limited-resource settings is of major importance. In addition, their abilities to connect to the “cloud” data storage systems and the Internet of Things (IoT) networks for collecting and sharing medical analytical data provide a trending novel research field in medical information technology. • Smartphone biosensory applications for medical monitoring, data storage and transferring them. • Medical data sharing through internet-of-medical things (IoMT) in Smartphone biosensors. • Integration of optomechanical units with smartphone biosensors converts them into field-portable and cost-effective colorimetric or fluorometric readers. • Recording high-definition videos for human infectious disease diagnostics using the modern “Smartphone videoscopy”. Schematic representation of smartphone biosensors for infectious diseases.

IL-6 and IL-10 Are Associated With Gram-Negative and Gram-Positive Bacteria Infection in Lymphoma.

To investigate the Th1/Th2 cytokine profile in patients with lymphoma during the myelosuppression stage of infection. 52 patients with gram-negative bacterial infection (G- group), 49 patients with gram-positive bacterial infection (G+ group), 51 uninfected patients with lymphoma (uninfected group) and 20 healthy controls (healthy group) were enrolled in this study. We evaluated the quantification of Th1/Th2 cytokines with flow cytometry bead assay (CBA) in the sera to explore a rapid diagnostic method to determine the type of infection and anti-infective effect. The levels of procalcitonin (PCT) were also detected simultaneously. The four groups did not differ with regard to IL-2 and IL-4 (P>0.05). The IFN-γ and TNF-α levels of patients with lymphoma were higher than those of healthy controls (P<0.05). There was significantly upregulated IL-6 and IL-10 expression in the G- group (P<0.001). A similar trend was reflected in the IL-6 of the G+ group, which was significantly increased (P<0.001). However, no significant upregulation was observed for IL-10 in the G+ group. According to the different degrees of increased IL-6 and IL-10 levels, We proposed to use the G- Bacterial Infection Cytokine Profile (G- BICP) and the G+ Bacterial Infection Cytokine Profile (G+ BICP) for the first time to differentiate between Gram-negative and Gram-positive (G-/G+) bacterial infection in adults with lymphoma in the myelosuppression stage after chemotherapy. The IL-6, IL-10 and PCT in the G- group and the IL-6, PCT in the G+ group were significantly decreased at day 4 and day 8 compared with those at day 1. IL-6 and IL-10 are closely associated with the severity and treatment efficacy in adults with lymphomas who develop infections after chemotherapy and can help distinguish between G- and G+ bacterial infections at an early stage.

Urinary Antigen Testing for Respiratory Infections: Current Perspectives on Utility and Limitations.

Pneumonia is a leading cause of hospitalization and death due to infection worldwide. Streptococcus pneumoniae and Legionella pneumophila remain among the most commonly identified bacterial pathogens. Unfortunately, more than half of all pneumonia cases today lack an etiologic diagnosis due to limitations in traditional microbiological methods like blood and sputum cultures, which are affected by poor sample collection, prior antibiotic administration, and delayed processing. Urinary antigen tests (UATs) for S. pneumoniae and L. pneumophila have emerged as powerful tools for improving the diagnosis of bacterial respiratory infections, enabling physicians to administer early directed therapy and improve antimicrobial stewardship. UATs are simple, rapid, and non-invasive diagnostic tests with high specificity (>90%) and moderate sensitivity (<80%). The potential impact of urinary antigen testing is especially significant for respiratory infections caused by Legionella. While all recommended community-acquired pneumonia (CAP) therapies are adequate for treating pneumococcal pneumonia, only certain antibiotics are effective against Legionella. Delayed therapy for Legionella is associated with worse clinical outcomes, which underscores the importance of rapid diagnostic methods like UATs. Despite their potential impact, current American Thoracic Society and Infectious Diseases Society of America (ATS/IDSA) guidelines argue against the routine use of urinary antigen testing for S. pneumoniae and L. pneumophila, except in patients with severe CAP and those with epidemiological risk factors for Legionella. Further research is necessary to evaluate the impact of early targeted treatment due to positive UAT results, as well as optimal strategies for UAT utilization. The purpose of this review is to summarize the UATs available for bacterial respiratory infections, describe current guidelines on their usage, and assess their impact on clinical outcomes and targeted therapy.

Generalised diagnostic framework for rapid battery degradation quantification with deep learning

Diagnosing lithium-ion battery degradation is challenging due to the complex, nonlinear, and path-dependent nature of the problem. Here, we develop a generalised and rapid degradation diagnostic method with a deep learning-convolutional neural network that quantifies degradation modes of batteries aged under various conditions in 0.012 s without feature engineering. Rather than performing extensive aging experiments, synthetic aging datasets for network training are generated. This dramatically lowers training cost/time, with these datasets covering almost all the aging paths, enabling a generalised degradation diagnostic framework. We show that the five thermodynamic degradation modes are correlated, and systematically elucidate their correlations. We thus propose a non-invasive comprehensive evaluation method and find the degradation diagnostic errors to be less than 1.22% for three leading commercial battery chemistries. The comparison with the traditional diagnostic methods confirms the high accuracy and fast nature of the proposed approach. Quantification of degradation modes with the partial discharge/charge data using the proposed diagnostic framework validates the real-world feasibility of this approach. This work, therefore, enables the promise of online identification of battery degradation and efficient analysis of large-data sets, unlocking potential for long lifetime energy storage systems.

Mass spectrometry in research laboratories and clinical diagnostic: a new era in medical mycology.

Diagnosis by clinical mycology laboratory plays a critical role in patient care by providing definitive knowledge of the cause of infection and antimicrobial susceptibility data to physicians. Rapid diagnostic methods are likely to improve patient. Aggressive resuscitation bundles, adequate source control, and appropriate antibiotic therapy are cornerstones for success in the treatment of patients. Routine methods for identifying clinical specimen fungal pathogen are based on the cultivation on different media with the subsequent examination of its phenotypic characteristics comprising a combination of microscopic and colony morphologies. As some fungi cannot be readily identified using these methods, molecular diagnostic methods may be required. These methods are fast, but it can cost a lot. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is suitable for high-throughput and rapid diagnostics at low costs. It can be considered an alternative for conventional biochemical and molecular identification systems in a microbiological laboratory. The reliability and accuracy of this method have been scrutinized in many surveys and have been compared with several methods including sequencing and molecular methods. According to these findings, the reliability and accuracy of this method are very high and can be trusted. With all the benefits of this technique, the libraries of MALDI-TOF MS need to be strengthened to enhance its performance. This review provides an overview of the most recent research literature that has investigated the applications and usage of MT-MS to the identification of microorganisms, mycotoxins, antifungal susceptibility examination, and mycobiome research.

Drug resistant TB – latest developments in epidemiology, diagnostics and management

AimThe aim of this review is to inform the reader on the latest developments in epidemiology, diagnostics and management. EpidemiologyDrug-resistant Tuberculosis (DR-TB) continues to be a current global health threat, and is defined by higher morbidity and mortality, sequelae, higher cost and complexity. The WHO classifies drug-resistant TB into 5 categories: isoniazid-resistant TB, rifampicin resistant (RR)-TB and MDR-TB, (TB resistant to isoniazid and rifampicin), pre-extensively drug-resistant TB (pre-XDR-TB) which is MDR-TB with resistance to a fluoroquinolone and finally XDR-TB that is TB resistant to rifampicin, plus any fluoroquinolone, plus at least one further priority A drug (bedaquiline or linezolid). Of 500,000 estimated new cases of RR-TB in 2020, only 157 903 cases are notified. Only about a third of cases are detected and treated annually. DiagnosticsRecently newer rapid diagnostic methods like the GeneXpert, whole genome sequencing and Myc-TB offer solutions for rapid detection of resistance. TreatmentThe availability of new TB drugs and shorter treatment regimens have been recommended for the management of DR-TB. ConclusionDespite advances in diagnostics and treatments we still have to find and treat two thirds of the drug resistant cases that go undetected and therefore go untreated each year. Control of TB and elimination will only occur if cases are detected, diagnosed and treated promptly.

Development of a Rapid and Efficient RPA-CRISPR/Cas12a Assay for Mycoplasma pneumoniae Detection.

Mycoplasma pneumoniae (MP) is a one of most common pathogen in causing respiratory infection in children and adolescents. Rapid and efficient diagnostic methods are crucial for control and treatment of MP infections. Herein, we present an operationally simple, rapid and efficient molecular method for MP identification, which eliminates expensive instruments and specialized personnel. The method combines recombinase polymerase amplification (RPA) with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) 12a-based detection, with an optimal procedure less than 1 h from sample to result including DNA extraction (25 min), RPA reaction (39°C for 15-20 min), CRISPR/Cas12a detection (37°C for 10 min) and visual detection by naked eyes (2 min). This diagnostic method shows high sensitivity (two copies per reaction) and no cross-reactivity against other common pathogenic bacteria. Preliminary evaluation using 201 clinical samples shows sensitivity of 99.1% (107/108), specificity of 100% (93/93) and consistency of 99.5% (200/201), compared with real-time PCR method. The above data demonstrate that our developed method is reliable for rapid diagnosis of MP. In conclusion, the RPA-CRISPR/Cas12a has a great potential to be as a useful tool for reliable and quick diagnosis of MP infection, especially in primary hospitals with limited conditions.

Surface plasmon resonance biosensor with laser heterodyne feedback for highly-sensitive and rapid detection of COVID-19 spike antigen

The ongoing outbreak of the COVID-19 has highlighted the importance of the pandemic prevention and control. A rapid and sensitive antigen assay is crucial in diagnosing and curbing pandemic. Here, we report a novel surface plasmon resonance biosensor based on laser heterodyne feedback interferometry for the detection of SARS-CoV-2 spike antigen, which is achieved by detecting the tiny difference in refractive index between different antigen concentrations. The biosensor converts the refractive index changes at the sensing unit into the intensity changes of light through surface plasmon resonance, achieving label-free and real-time detection of biological samples. Moreover, the gain amplification effect of the laser heterodyne feedback interferometry further improved the sensitivity of this biosensor. The biosensor can rapidly respond to continuous and periodic changes in the refractive index with a high resolution of 3.75 × 10−8 RIU, demonstrating the repeatability of the biosensor. Afterwards, the biosensor is immobilized by the anti-SARS-CoV-2 spike monoclonal antibodies, thus realizing the specific recognition of the antigen. The biosensor exhibited a high sensitivity towards the concentration of the antigen with a linear dynamic range of five orders of magnitude and a resolution of 0.08 pg/mL. These results indicate that this principle can be used as a rapid diagnostic method for COVID-19 antigens without sample labelling.

The Role of Colistin in the Era of New β-Lactam/β-Lactamase Inhibitor Combinations.

With the current crisis related to the emergence of carbapenem-resistant Gram-negative bacteria (CR-GNB), classical treatment approaches with so-called “old-fashion antibiotics” are generally unsatisfactory. Newly approved β-lactam/β-lactamase inhibitors (BLBLIs) should be considered as the first-line treatment options for carbapenem-resistant Enterobacterales (CRE) and carbapenem-resistant Pseudomonas aeruginosa (CRPA) infections. However, colistin can be prescribed for uncomplicated lower urinary tract infections caused by CR-GNB by relying on its pharmacokinetic and pharmacodynamic properties. Similarly, colistin can still be regarded as an alternative therapy for infections caused by carbapenem-resistant Acinetobacter baumannii (CRAB) until new and effective agents are approved. Using colistin in combination regimens (i.e., including at least two in vitro active agents) can be considered in CRAB infections, and CRE infections with high risk of mortality. In conclusion, new BLBLIs have largely replaced colistin for the treatment of CR-GNB infections. Nevertheless, colistin may be needed for the treatment of CRAB infections and in the setting where the new BLBLIs are currently unavailable. In addition, with the advent of rapid diagnostic methods and novel antimicrobials, the application of personalized medicine has gained significant importance in the treatment of CRE infections.

Review of Liquid Chromatography-Mass Spectrometry-Based Proteomic Analyses of Body Fluids to Diagnose Infectious Diseases.

Rapid and precise diagnostic methods are required to control emerging infectious diseases effectively. Human body fluids are attractive clinical samples for discovering diagnostic targets because they reflect the clinical statuses of patients and most of them can be obtained with minimally invasive sampling processes. Body fluids are good reservoirs for infectious parasites, bacteria, and viruses. Therefore, recent clinical proteomics methods have focused on body fluids when aiming to discover human- or pathogen-originated diagnostic markers. Cutting-edge liquid chromatography–mass spectrometry (LC-MS)-based proteomics has been applied in this regard; it is considered one of the most sensitive and specific proteomics approaches. Here, the clinical characteristics of each body fluid, recent tandem mass spectroscopy (MS/MS) data-acquisition methods, and applications of body fluids for proteomics regarding infectious diseases (including the coronavirus disease of 2019 [COVID-19]), are summarized and discussed.

PAM-free loop-mediated isothermal amplification coupled with CRISPR/Cas12a cleavage (Cas-PfLAMP) for rapid detection of rice pathogens

Pathogenic disease is an important factor affecting rice growth, yield and quality, and the development and application of rapid diagnostic methods will contribute to the prevention and control of rice disease. Herein, we developed a novel protospacer adjacent motif (PAM)-free loop-mediated isothermal amplification (LAMP) assisted CRISPR/Cas12a cleavage (Cas-PfLAMP) assay for detection of three rice pathogens; Xanthomonas oryzae pv. Oryzae (XOO), rice stripe virus (RSV), and rice black-streaked dwarf virus (RBSDV). The Cas-PfLAMP assay showed high specificity due to doubly specific recognition of LAMP primer sets and FnCas12a/sgRNA, and high sensitivity down to 9 or 3 copies due to LAMP amplification and CRISPR/Cas12a trans cleavage activity. Furthermore, a visual on-spot Cas-PfLAMP platform was established for detection of rice pathogens by combining solid-phase nucleic acid extraction and a lateral flow strip (LFS) test. Analysis of rice leaf field samples confirmed the impressive performance of the Cas-PfLAMP platform, demonstrating its suitability for rapid (∼50 min) on-spot detection of rice diseases. The assay could also be extended to detection of other plant diseases, and other nucleic acid field tests.