Recent Developments in Phenotypic and Molecular Diagnostic Methods for Antimicrobial Resistance Detection in Staphylococcus aureus: A Narrative Review

Staphylococcus aureus is an opportunistic pathogen responsible for a wide range of infections in humans, such as skin and soft tissue infections, pneumonia, food poisoning or sepsis. Historically, S. aureus was able to rapidly adapt to anti-staphylococcal antibiotics and become resistant to several classes of antibiotics. Today, methicillin-resistant S. aureus (MRSA) is a multidrug-resistant pathogen and is one of the most common bacteria responsible for hospital-acquired infections and outbreaks, in community settings as well. The rapid and accurate diagnosis of antimicrobial resistance in S. aureus is crucial to the early initiation of directed antibiotic therapy and to improve clinical outcomes for patients. In this narrative review, I provide an overview of recent phenotypic and molecular diagnostic methods for antimicrobial resistance detection in S. aureus, with a particular focus on MRSA detection. I consider methods for resistance detection in both clinical samples and isolated S. aureus cultures, along with a brief discussion of the advantages and the challenges of implementing such methods in routine diagnostics.

Steroid hormone profiles and molecular diagnostic tools in pediatric patients with non-CAH primary adrenal insufficiency

Background There is a significant challenge of attributing specific diagnoses to patients with primary adrenal insufficiency of unknown etiology other than congenital adrenal hyperplasia (non-CAH PAI). Specific diagnoses per se may guide personalized treatment or may illuminate pathophysiology. Objective Investigation of the efficacy of steroid hormone profiles and high-throughput sequencing methods in establishing the etiology in non-CAH PAI of unknown origin. Design Paediatric patients with non-CAH PAI whose etiology could not be established by clinical and biochemical characteristics were enrolled. Genetic analysis was performed using targetedgene panel sequencing (TPS) and whole-exome sequencing (WES). Plasma adrenal steroids were quantified by liquid chromatography-mass spectrometry and compared to that of controls. Setting Eighteen pediatric endocrinology clinics. Patients Forty-one patients (17 females, median age: 3 months, range: 0-8 years) with non-CAH PAI of unknown etiology. Results A genetic diagnosis was obtained in 29 (70.7%) patients by TPS. Further molecular diagnosis could not be achieved by WES. Compared to healthy control group, patients showed lower steroid concentrations, most significantly in cortisone, cortisol, and corticosterone (p<0.0001, area under the ROC curve: 0.96, 0.88, 0.87, respectively). Plasma cortisol<4 ng/mL, cortisone<11 ng/mL, and corticosterone<0.11 ng/mL had >95% specificity to ensure the diagnosis of non-CAH PAI of unknown etiology. Conclusion Steroid hormone profiles are highly sensitive for the diagnosis of non-CAH PAI of unknown etiology, while they are unlikely to point out a specific molecular diagnosis. TPS is an optimal approach in the molecular diagnosis of these patients with high efficacy, while little additional benefit is expected from WES.

A novel molecular diagnostic method for the gut content analysis of Philaenus DNA

Philaenus spumarius is a vector of Xylella fastidiosa, one of the most dangerous plants pathogenic bacteria worldwide. There is currently no control measure against this pathogen. Thus, the development of vector control strategies, like generalist predators, such as spiders, could be essential to limit the spread of this vector-borne pathogen. In this study, a polymerase chain reaction (PCR)-based approach was developed to principally detect DNA of P. spumarius in the spider’s gut. Accordingly, 20 primer pairs, targeting the mitochondrial cytochrome oxidase I (COI) and cytochrome b (cytB) genes, were tested for specificity, sensitivity, and efficiency in detecting P. spumarius DNA. Overall, two primer sets, targeting COI gene (COI_Ph71F/COI_Ph941R) and the cytB gene (cytB_Ph85F/cytB_Ph635R), showed the highest specificity and sensitivity, being able to amplify 870 pb and 550 bp fragments, respectively, with P. spumarius DNA concentrations 100-fold lower than that of the DNA of non-target species. Among these two primer sets, the cytB_Ph85F/cytB_Ph635R was able to detect P. spumarius in the spider Xysticus acerbus, reaching 50% detection success 82 h after feeding. The feasibility of this primer set to detect predation of P. spumarius by spiders was confirmed in the field, where 20% of the collected spiders presented positive amplifications.

Clinical Performance of the cobas Liat SARS-CoV-2 & Influenza AB for the Detection of SARS-CoV-2 in Nasal Samples

Background and Objective: Point-of-care type molecular diagnostic tests have been used for detecting SARS-CoV-2, although their clinical utility with nasal samples has yet to be established. This study evaluated the clinical performance of the cobas Liat SARS-CoV-2 & Influenza AB (Liat) in nasal samples. Methods: Nasal and nasopharyngeal samples were collected and were tested using the Liat, the cobas 6800 system and the cobas SARS-CoV-2 & Influenza AB (cobas), and a method developed by National Institute of Infectious Diseases, Japan (NIID). Results: A total of 814 nasal samples were collected. The Liat assay was positive for SARS-CoV-2 in 113 (13.9%). The total, positive, and negative concordance rate between the Liat and cobas/NIID assays were 99.3%/98.4%, 99.1%/100%, and 99.3%/98.2%, respectively. Five samples were positive only using the Liat assay. Their Ct values ranged from 31.9 to 37.2. The Ct values of the Liat assay were significantly lower (p < 0.001) but were correlated (p < 0.001) with those of other molecular assays. In the participants who tested positive for SARS-CoV-2 on the Liat assay using nasopharyngeal samples, 88.2% of their nasal samples also tested positive using the Liat assay. Conclusion: The Liat assay showed high concordance with other molecular assays in nasal samples. Some discordance occurred in samples with Ct values > 30 on the Liat assay.

An integrated lab-on-a-chip device for RNA extraction, amplification and CRISPR-Cas12a-assisted detection for COVID-19 screening in resource-limited settings

In response to the ongoing COVID-19 pandemic and disparities of vaccination coverage in low-and middle-income countries, it is vital to adopt a widespread testing and screening programme, combined with contact tracing, to monitor and effectively control the infection dispersion in areas where medical resources are limited. This work presents a lab-on-a-chip platform, namely IFAST-CRISPR, as an affordable, rapid and high-precision molecular diagnostic means for SARS-CoV-2 detection. The herein proposed sample-to-answer platform integrates RNA extraction, amplification and CRISPR-Cas-based detection with lateral flow readout in one device. The microscale dimensions of the device containing immiscible liquids, coupled with the use of silica paramagnetic beads and GuHCl, streamline sample preparation, including RNA concentration, extraction and purification, in 15 min with minimal hands-on steps. By combining RT-LAMP with CRISPR-Cas12 assays targeting the nucleoprotein (N) gene, visual identification of ≥ 470 copies mL-1 genomic SARS-CoV-2 samples was achieved in 45 min, with no cross-reactivity towards HCoV-OC43 nor H1N1. On-chip assays showed the ability to isolate and detect SARS-CoV-2 from 1,000 genome copies mL-1 of replication-deficient viral particles in 1 h. This simple, affordable and integrated platform demonstrated a visual, faster, and yet specificity and sensitivity-comparable alternative to the costly gold-standard RT-PCR assay, requiring only a simple heating source. Further investigations on multiplexing and direct interfacing of the accessible Swan-brand cigarette filter for saliva sample collection could provide a complete work flow for COVID-19 diagnostics from saliva samples suitable for low-resource settings.

Clinical Utility of Oncuria™, a Multiplexed Liquid Biopsy for the Non-Invasive Detection of Bladder Cancer—A Pilot Study

Oncuria™ is a validated quantitative multiplex immunoassay capable of detecting bladder cancer from a voided urine sample. Herein, we sought to determine whether Oncuria™ affects physicians’ use of non-invasive and invasive diagnostic tests for microhematuria, gross hematuria, and bladder cancer surveillance. We conducted a survey-based study to assess physician management of nine clinical scenarios involving real-world data from patients with gross hematuria, microhematuria, and bladder cancer on surveillance. We randomly sampled 15 practicing urologists and generated data including 135 patient-by-urologist interactions and 2160 decision points. Urologists recommended a selection of diagnostic tests and procedures before and after Oncuria™ results were provided. We assessed changes in provider use of non-invasive and invasive diagnostic tests after Oncuria™ results were provided. Over 90% of all urologists changed their diagnostic behavior in at least one patient case with the addition of Oncuria™ results. The total number of diagnostic procedures was reduced by 31% following the disclosure of a negative Oncuria™ test and 27% following the disclosure of a positive Oncuria™ test. This is pilot study has the potential to shed light on the analysis of our four large multicenter international studies deploying OncuriaTM. The Oncuria™ urine-based test, a molecular diagnostic capable of ruling out the presence of bladder cancer, reduces both unnecessary invasive and non-invasive diagnostics and has the potential to reduce costs and improve patient outcomes.

Identification of bacteria strains using the Recombinase Polymerase Amplification assay on a miniaturized solid-state pH sensor

Rapid identification of bacteria based on nucleic acid amplification allows dealing with the detection of pathogens in clinical, food, and environmental samples. Amplification products must be detected and analyzed by external devices or integrated complicated optical systems. Here, we developed a solid-state pH electrode based on iridium oxide (IrO2) films to measure released hydrogen ions (H+) from isothermal nucleic acid (NA) amplification of bacterial samples. By recombinase polymerase amplification (RPA), we achieved rapid (< 15 min) and sensitive (<30 copies) detection with an accuracy of about 0.03 pH. The RPA-based hydrogen ion sensing assay shows higher specificity, sensitivity, and efficiency as the same polymerase chain reaction (PCR) methods. We initially used the RPA-based sensor to detect E. coli species in laboratory samples. Among, 27 random laboratory samples of E. coli samples, 6 were found to be DH5alpha, 9 BL21, 3 HB101, 6 TOP10, and 3 JM109. The electrical detection of amplification provides generally applicable techniques for the detection of nucleic acid amplification, enabling molecular diagnostic tests in the field and integrating data transmission to the mobile device. These results can be future developed into an efficient tool for rapid on-site detection of bacterial pathogens in clinical samples.

Laboratory Methods for the Diagnosis of SARS-Cov-2

The coronavirus disease 2019 (COVID-19) which has become the pandemic par excellence of our time places pressure on various aspects of human endeavor and as such requires detailed study to better combat it. However, diagnostic tests were used to provide data on the incidence of COVID-19 and to assess the immune status of infected individuals. The objective of this chapter is to describe the diagnostic methods currently used to identify SARS-CoV-2 infection. Obtaining the first SARS-CoV-2 genome sequence was decisive for the development of molecular diagnostic assays that currently make it possible to diagnose and screen for the Sars-CoV-2 infection. Their uses depend on the target to be detected. Antigenic tests detect the presence of a virus antigen, which usually makes a proteinaceous part of the virus surface. The serology tests detect the presence of antibodies generated against SARS-CoV-2 and are also a relevant tool for epidemiological studies.