Clinical Swab Samples Research Articles

Facile and Unplugged Surface Plasmon Resonance Biosensor with NIR-Emitting Perovskite Nanocomposites for Fast Detection of SARS-CoV-2.

The emergence of the coronavirus disease 2019 (COVID-19) pandemic prompted researchers to develop portable biosensing platforms, anticipating to detect the analyte in a label-free, direct, and simple manner, for deploying on site to prevent the spread of the infectious disease. Herein, we developed a facile wavelength-based SPR sensor built with the aid of a 3D printing technology and synthesized air-stable NIR-emitting perovskite nanocomposites as the light source. The simple synthesis processes for the perovskite quantum dots enabled low-cost and large-area production and good emission stability. The integration of the two technologies enabled the proposed SPR sensor to exhibit the characteristics of lightweight, compactness, and being without a plug, just fitting the requirements of on-site detection. Experimentally, the detection limit of the proposed NIR SPR biosensor for refractive index change reached the 10-6 RIU level, comparable with that of state-of-the-art portable SPR sensors. In addition, the bio-applicability of the platform was validated by incorporating a homemade high-affinity polyclonal antibody toward the SARS-CoV-2 spike protein. The results demonstrated that the proposed system was capable of discriminating between clinical swab samples collected from COVID-19 patients and healthy subjects because the used polyclonal antibody exhibited high specificity against SARS-CoV-2. Most importantly, the whole measurement process not only took less than 15 min but also needed no complex procedures or multiple reagents. We believe that the findings disclosed in this work can open an avenue in the field of on-site detection for highly pathogenic viruses.

A SERS-signalled, CRISPR/Cas-powered bioassay for amplification-free and anti-interference detection of SARS-CoV-2 in foods and environmental samples using a single tube-in-tube vessel

The pandemic of COVID-19 creates an imperative need for sensitive and portable detection of SARS-CoV-2. We devised a SERS-read, CRISPR/Cas-powered nanobioassay, termed as OVER-SARS-CoV-2 (One-Vessel Enhanced RNA test on SARS-CoV-2), which enabled supersensitive, ultrafast, accurate and portable detection of SARS-CoV-2 in a single vessel in an amplification-free and anti-interference manner. The SERS nanoprobes were constructed by conjugating gold nanoparticles with Raman reporting molecular and single-stranded DNA (ssDNA) probes, whose aggregation-to-dispersion changes can be finely tuned by target-activated Cas12a though trans-cleavage of linker ssDNA. As such, the nucleic acid signals could be dexterously converted and amplified to SERS signals. By customizing an ingenious vessel, the steps of RNA reverse transcription, Cas12a trans-cleavage and SERS nanoprobes crosslinking can be integrated into a single and disposal vessel. It was proved that our proposed nanobioassay was able to detect SARS-CoV-2 as low as 200 copies/mL without any pre-amplification within 45 min. In addition, the proposed nanobioassay was confirmed by clinical swab samples and challenged for SARS-CoV-2 detection in simulated complex environmental and food samples. This work enriches the arsenal of CRISPR-based diagnostics (CRISPR-Dx) and provides a novel and robust platform for SARS-CoV-2 decentralized detection, which can be put into practice in the near future.

Highly sensitive and rapid identification of coxsackievirus A16 based on reverse transcription multiple cross displacement amplification combined with nanoparticle-based lateral flow biosensor assay.

One of the main pathogens responsible for human hand, foot, and mouth disease (HFMD), coxsackievirus A16, has put young children's health at danger, especially in countries in the Asia-Pacific region. Early quick identification is essential for the avoidance and control of the disorder since there are no vaccinations or antiviral medications available to prevent and manage CVA16 infection. Here, we describe the creation of an easy, speedy, and accurate CVA16 infection detection approach using lateral flow biosensors (LFB) and reverse transcriptionmultiple cross displacement amplification (RT-MCDA). A group of 10 primers was developed for the RT-MCDA system in order to amplify the genes in an isothermal amplification device while targeting the highly conserved region of the CVA16 VP1 gene. Then, without requiring any extra tools, RT-MCDA amplification reaction products might well be detected by visual detection reagent (VDR) and LFB. The outcomes showed that 64°C within 40 min was the ideal reaction setting for the CVA16-MCDA test. Target sequences with <40 copies might be found using the CVA16-MCDA. There was no cross-reaction among CVA16 strains and other strains. The findings demonstrated that the CVA16-MCDA test could promptly and successfully identify all of the CVA16-positive (46/220) samples identified by the traditional real-time quantitative polymerase chain reaction (qRT-PCR) assays for 220 clinical anal swab samples. The whole process, such as the processing of the sample (15 min), the MCDA reaction (40 min), and the documenting of the results (2 min), could be finished in 1 h. The CVA16-MCDA-LFB assay, which targeted the VP1 gene, was an efficient, simple, and highly specific examination that might be used extensively in rural regions' basic healthcare institutions and point-of-care settings.

Development and Application of Real-Time PCR Assay for Detection of Salmonella Abortusequi.

Salmonella enterica subsp. enterica serovar Abortusequi is a major pathogen in horse and donkey herds, causing abortion in pregnant equids and resulting in enormous economic losses. A rapid and reliable method is urgently needed to detect S. Abortusequi in herds where the disease is suspected. To achieve this goal, a TaqMan-based real-time PCR assay targeting the gene for the flagellin protein phase 2 antigen FljB was developed. This real-time PCR assay had high specificity, sensitivity, and reproducibility. The detection limit of the assay was 30 copies/μL of standard plasmid and 10 CFU/μL of bacterial DNA. Furthermore, 540 clinical samples, including 162 tissue, 192 plasma, and 186 vaginal swab samples collected between 2018 and 2021 in China, were tested to assess the performance of the developed assay. Compared to the gold standard method of bacterial isolation, the real-time PCR assay exhibited 100% positive agreement for all tissue, plasma and vaginal swab tests. Additionally, this assay detected DNA from S. Abortusequi from 56.7% (34/60) culture-negative tissue and 22.9% (41/179) culture-negative vaginal swab samples from infected equids. Receiver operating characteristic analysis demonstrated that the results of the developed real-time PCR assays were in significant agreement with those of the culture method. The real-time PCR assay can be completed within 45 min of extraction of DNA from samples. Our results show that this assay could serve as a reliable tool for the rapid detection of S. Abortusequi in tissue, plasma, and vaginal swab clinical samples.

A Low-Cost Handheld Reconfigurable Impedimetric Readout System for Diagnostics of Viral Infections

This paper presents a portable impedimetric biosensor for detecting infectious diseases such as SARS-CoV-2 Infections. A bio-ready sensing electrode functionalized with SARS-CoV-2 nucleocapsid antibody was employed to quantitatively convert the concentration of nucleocapsid protein (N-protein) into impedance changes. In this paper, we proposed a readout system with a dynamic input range of 200 Ω to 1 MΩ magnitude and 0 to 180° phase. The resolution of this device is 1% and 6.5° for measuring the magnitude and phase, respectively. Herein we demonstrate and discuss the proposed system’s functionality, sensitivity, and selectivity using the clinical swab samples. As per these results, this readout system is suitable for the detection of N-protein ranging up to 10,000 pg/mL with a resolution of 56 fg/mL. The proposed impedimetric sensing system can be adopted for the detection of infectious diseases in the future. This low-cost (<$80) device using off-the-shelf is a unique candidate for batch production purposes during urgent pandemic situations.

Dual-target nucleic acid sequences responsive electrochemiluminescence biosensor using single type carbon dots as probe for SARS-CoV-2 detection based on series catalytic hairpin assembly amplification

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is rampant all over the world, and rapid and effective virus detection is the best auxiliary to curb the spread of the epidemic. A diagnosis can only be made if two or more different nucleic acid sequences are confirmed at the same time, and in most of traditional detection technologies, these target sequences have been detected separately. In this work, an electrochemiluminescent (ECL) biosensor employing a single ECL probe as signal output and responding to dual-target simultaneously is proposed for the first time. Taking the two sequences located in ORF 1ab region and N region of SARS-CoV-2 gene sequence as the model target and nitrogen doped carbon quantum dots (CDs) as ECL beacon, supplemented with catalytic hairpin assembly (CHA) reaction for signal amplification, the presented strategy has been successfully applied to the rapid detection of SARS-CoV-2. The developed SARS-CoV-2 biosensor based on the series CHA systems can realize the quantitative determination of SARS-CoV-2 in the range of 50 fM to 200 pM within 40 min. Moreover, the clinical validity of this method has been verified by the high consistency between the detection results of using this method and those using RT-qPCR for seven clinical pharyngeal swab samples.

Comparable outcomes from long and short read random sequencing of total RNA for detection of pathogens in chicken respiratory samples.

Co-infections of avian species with different RNA viruses and pathogenic bacteria are often misdiagnosed or incompletely characterized using targeted diagnostic methods, which could affect the accurate management of clinical disease. A non-targeted sequencing approach with rapid and precise characterization of pathogens should help respiratory disease management by providing a comprehensive view of the causes of disease. Long-read portable sequencers have significant potential advantages over established short-read sequencers due to portability, speed, and lower cost. The applicability of short reads random sequencing for direct detection of pathogens in clinical poultry samples has been previously demonstrated. Here we demonstrate the feasibility of long read random sequencing approaches to identify disease agents in clinical samples. Experimental oropharyngeal swab samples (n = 12) from chickens infected with infectious bronchitis virus (IBV), avian influenza virus (AIV) and Mycoplasma synoviae (MS) and field-collected clinical oropharyngeal swab samples (n = 11) from Kenyan live bird markets previously testing positive for Newcastle disease virus (NDV) were randomly sequenced on the MinION platform and results validated by comparing to real time PCR and short read random sequencing in the Illumina MiSeq platform. In the swabs from experimental infections, each of three agents in every RT-qPCR-positive sample (Ct range 19-34) was detectable within 1 h on the MinION platform, except for AIV one agent in one sample (Ct = 36.21). Nine of 12 IBV-positive samples were assigned genotypes within 1 h, as were five of 11 AIV-positive samples. MinION relative abundances of the test agent (AIV, IBV and MS) were highly correlated with RT-qPCR Ct values (R range-0.82 to-0.98). In field-collected clinical swab samples, NDV (Ct range 12-37) was detected in all eleven samples within 1 h of MinION sequencing, with 10 of 11 samples accurately genotyped within 1 h. All NDV-positive field samples were found to be co-infected with one or more additional respiratory agents. These results demonstrate that MinION sequencing can provide rapid, and sensitive non-targeted detection and genetic characterization of co-existing respiratory pathogens in clinical samples with similar performance to the Illumina MiSeq.

Gold Nanourchins Improve Virus Targeting and Plasmonic Coupling for Virus Diagnosis on a Smartphone Platform.

Point-of-care detection of pathogens is critical to monitor and combat viral infections. The plasmonic coupling assay (PCA) is a homogeneous assay and allows rapid, one-step, and colorimetric detection of intact viruses. However, PCA lacks sufficient sensitivity, necessitating further mechanistic studies to improve the detection performance of PCA. Here, we demonstrate that gold nanourchins (AuNUs) provide significantly improved colorimetric detection of viruses in PCA. Using respiratory syncytial virus (RSV) as a target, we demonstrate that the AuNU-based PCA achieves a detection limit of 1400 PFU/mL, or 17 genome equivalent copies/μL. Mechanistic studies suggest that the improved detection sensitivity arises from the higher virus-binding capability and stronger plasmonic coupling at long distances (∼10 nm) by AuNU probes. Furthermore, we demonstrate the virus detection with a portable smartphone-based spectrometer using RSV-spiked nasal swab clinical samples. Our study uncovers important mechanisms for the sensitive detection of intact viruses in PCA and provides a potential toolkit at the point of care.

Counter-propagating Gaussian beam enhanced Raman spectroscopy for rapid reagentless detection of respiratory pathogens in nasal swab samples

Co-circulation of respiratory viruses compounded by similarities in clinical presentation and mode of transmission underscores the need for broad range pathogen detection. Accurate identification and diagnosis at the point-of-need is critical to limiting disease spread. A novel point-of-need Raman spectroscopy-based platform is described for rapid detection of multiple respiratory pathogens in nasal swab samples with high sensitivity and specificity. The system takes advantage of a counter-propagating Gaussian beam focused within the sample chamber that augments the Raman signal of pathogens. Combined with multiclass machine learning spectral analysis via Gradient Boosting Machine, accurate identification of SARS-CoV-2, human coronaviruses OC43, NL63, 229E, Influenza A (H1N1), respiratory syncytial virus, and Streptococcus pyogenes in spiked clinical nasal swab samples was demonstrated at 99% sensitivity and 93% specificity. The limit of detection was assessed using binary class Support Vector Machine with SARS-CoV-2 in nasal swab samples against negative control at 2.2 × 104 virions/swab. The spectrometer can be operated by minimally trained personnel with software-generated diagnostic yes/no results in 2 min or less, making it well suited for point-of-need applications. Furthermore, adaptive algorithms can detect and differentiate new and emerging variants using a Raman spectral database.

Rapid Adaptation of Established High-Throughput Molecular Testing Infrastructure for Monkeypox Virus Detection.

Beginning in May 2022, a rising number of monkeypox cases were reported in non–monkeypox-endemic countries in the Northern Hemisphere. We adapted 2 published quantitative PCRs for use as a dual-target monkeypox virus test on widely used automated high-throughput PCR systems. We determined analytic performance by serial dilutions of monkeypox virus reference material, which we quantified by digital PCR. We found the lower limit of detection for the combined assays was 4.795 (95% CI 3.6–8.6) copies/mL. We compared clinical performance against a commercial manual orthopoxvirus research use only PCR kit by using clinical remnant swab samples. Our assay showed 100% positive (n = 11) and 100% negative (n = 56) agreement. Timely and scalable PCR tests are crucial for limiting further spread of monkeypox. The assay we provide streamlines high-throughput molecular testing for monkeypox virus on existing broadly established platforms used for SARS-CoV-2 diagnostic testing.

Multiplexed lateral flow assay integrated with orthogonal CRISPR-Cas system for SARS-CoV-2 detection

The development of field-deployable detection platform amenable for multiplexed genes testing will significantly improve the efficiency and reliability during point-of-care testing (POCT) applications. In this regard, an orthogonal CRISPR-Cas-mediated multiplexed lateral flow assay (designated as OC-MLFA) is proposed for SARS-CoV-2 genome detection. Taking the advantage of activation and cleavage preferences between Cas12a and Cas13a, orthogonal (two-independent-channel signal readout) CRISPR-Cas system is investigated. Lateral flow strips with two target lines are designed to accommodate the orthogonal CRISPR system. The interference between Cas12a and Cas13a channels can be effectively eliminated via the elaborate nucleic acids and lateral flow strips design. The high preamplification efficiency from reverse transcription recombinase polymerase amplification (RT-RPA) and Cas enzyme mediated trans-cleavage process bring the sensitivity of our OC-MLFA method to 10 copies per test (30 μL). Nasopharyngeal swab clinical samples with different cycle threshold (Ct) values according to the RT-PCR method were analyzed with the proposed OC-MLFA, during which 76 out of 76 detection accuracy was obtained. Featured with the multiplexed genes detection simultaneously in one reaction and colorimetric readout through single strip, the OC-MLFA we proposed herein ensures great accuracy and efficiency, which endows promising field-deployable POCT application feasibility.

The Role of Emergency Departments in Cross-Contamination: The Case of VRE

Colonized surfaces, equipment, individuals, and infected patients can be sources for the hospital spread of vancomycin-resistant enterococci (VRE), which is one of the important nosocomial pathogens. The basic epidemiological tools for the prevention and control of hospital-acquired infections are the typing methods. Pulsed-field gel electrophoresis (PFGE) is a highly discriminating method used frequently to detect clonal associations in epidemics and hospital-acquired VRE infections. This study aimed to investigate the presence of cross-contamination, which service or services come to the forefront in case of possible cross-contamination and clonal relationship between VRE strains isolated from rectal swab, clinical and environmental swab samples taken in two different periods in various clinics of Istanbul University Istanbul Medical Faculty Hospital by PFGE method. A total of 125 VREs isolated in two different periods were included in the study. Rectal and environmental swab samples were inoculated on Enterococcosel agar and sodium azide broth, urine samples were inoculated on chromogenic agar, and other clinical samples were inoculated on 5% sheep blood agar and incubated for 18-24 hours. VITEK 2 Compact automation system GP panel (bioMerieux, MarcyL'Etoile, France) was used for the species identification of catalase-negative, gram-positive colonies and disc diffusion and minimum inhibitory concentration (MIC) gradient tests were used to determine vancomycin susceptibility. Multiplex polymerase chain reaction was used to search for vanA and vanB resistance genes in isolates identified as VRE, and PFGE was used to determine clonal association. All isolates were identified as Enterococcus faecium with the vanA resistance gene. It was shown that PFGE clones were divided into six types with 65% similarity (A-F), and in this polyclonal spread, the major type was type A, which contained 108 isolates in 37 subtypes existed in the hospital for years. In patients from whom similar isolates were obtained, the high rate of hospitalizations in the same emergency room or in different emergency services in the same building drew attention. Our results showed that the presence of VRE was established in our hospital, new isolates were added from time to time, and there was a cross-contamination. It was observed that emergency services, where infection control measures were neglected due to working conditions, were among the high-risk areas for VRE contamination. In order to better understand the importance of emergency services in cross-contamination, it would be useful to conduct surveillance studies among patients hospitalized in emergency services and monitor the rate of VRE in the services where positive patients were transferred.

A quantitative RT-qLAMP for the detection of SARS-CoV-2 and human gene in clinical application.

Reverse transcription (RT) – loop‐mediated isothermal amplification (LAMP) assay is a rapid and one‐step method to detect SARS‐CoV‐2 in the pandemic. Quantitative estimation of the viral load of SARS‐CoV‐2 in patient samples could help physicians make decisions on clinical treatment and patient management. Here, we propose to use a quantitative LAMP (qLAMP) method to evaluate the viral load of SARS‐CoV‐2 in samples. We used threshold time (TT) values of qLAMP, the isothermal incubation time required for the fluorescent or colorimetric signal to reach the threshold, to indicate the viral load of clinical samples. Similar to the cycle threshold (C t) values in conventional qPCR, TT values of qLAMP show a linear relationship to the copy numbers of SARS‐CoV‐2. The higher the viral loadings, the lower qLAMP TT values are. The RT‐qLAMP assay was demonstrated to quantify the viral loads of synthesized full‐length RNA, inactivated viral particles (BBIBP‐CorV), and clinical samples within 15 min by fluorescent reading and 25 min by colorimetric reading. The RT‐qLAMP has been applied to detect Alpha, Beta, Kappa, Delta, and Omicron variants of SARS‐CoV‐2, as well as the human beta‐actin gene, and their TT values showed the linear patterns. The RT‐qLAMP assays were evaluated by 64 clinical samples (25 positives and 39 negatives) for the assessment of viral loads, and it was also used to quantify the human beta‐actin gene, which was used as a control and an indicator of sampling quality in clinical swab samples. The result of RT‐qLAMP was in good agreement with the result of RT‐qPCR. The RT‐qLAMP assay detected all clinical samples, including those with C t = 35, within 10 min using fluorescent reading.

NEXT CRISPR: An enhanced CRISPR-based nucleic acid biosensing platform using extended crRNA

CRISPR-based molecular detection has shown great potential for early cancer diagnostics and infectious disease testing at the point of care. Here, we investigated the effect of extending the 5′ end of CRISPR RNA (crRNA) on CRISPR detection and developed a 5′ eNd EXTension CRISPR (termed “NEXT CRISPR”) biosensing platform for rapid, highly sensitive, and specific nucleic acid detection. By taking advantage of a 5′ end-extended crRNA and optimized CRISPR reaction system, we demonstrated an enhanced NEXT CRISPR detection. The NEXT CRISPR is compatible with recombinase polymerase amplification (RPA), and can be easily adapted for both fluorescence detection and lateral flow strip readout. To enable point-of-care testing, we coupled RPA/NEXT CRISPR with a lateral flow assay to develop a NEXT CRISPR biosensing platform for human papillomavirus (HPV) 16 DNA detection with attomolar sensitivity within 30 min. Further, we clinically validated our NEXT CRISPR biosensing platform by detecting clinical swab samples, achieving a comparable performance with the conventional PCR method. This rapid, sensitive, and affordable NEXT CRISPR biosensing platform provides a promising tool for point-of-care diagnostics of cancers and infectious diseases.

A highly sensitive and versatile fluorescent biosensor for pathogen nucleic acid detection based on toehold-mediated strand displacement initiated primer exchange reaction

Existing detection methods for pathogen nucleic acid detection, such as polymerase chain reaction (PCR), are complicated and expensive to perform. Here, we report a simple and versatile strategy for highly sensitive detection of pathogen nucleic acid based on toehold-mediated strand displacement initiated primer exchange amplification (t-PER). In the presence of the target, the blocked hairpin substrate is released by toehold-mediated strand displacement, which triggers the primer exchange reaction amplification. Then, multiple long tandem-repeat single-strands generated by PER open the molecular beacon to recover the fluorescence signal. The t-PER protocol also successfully directly detected human papilloma virus from clinical cervical swab samples, with consistent results compared to real time-polymerase chain reaction (RT-PCR). Moreover, the versatility and clinical feasibility of this method was further confirmed by measuring Epstein-Barr virus, hepatitis B virus, and Ureaplasma urealyticum from different clinical samples (serum samples and urine samples). This simple platform enabled specific and sensitive detection of pathogen nucleic acid in a format that might hold great potential for point-of-care infection diagnosis.

Fluorescence spectrophotometry for COVID-19 determination in clinical swab samples.

Considering the limitations of the assays currently available for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its emerging variants, a simple and rapid method using fluorescence spectrophotometry was developed to detect coronavirus disease 2019 (COVID-19). Forty clinical swab samples were collected from the nasopharyngeal and oropharyngeal cavities of COVID-19-positive and -negative. Each sample was divided into two parts. The first part of the samples was analyzed using reverse transcription-polymerase chain reaction (RT-qPCR) as the control method to identify COVID-19-positive and -negative samples. The second part of the samples was analyzed using fluorescence spectrophotometry. Fluorescence measurements were performed at excitation and emission wavelengths ranging from 200 to 800 nm. Twenty COVID-19-positive samples and twenty COVID-19-negative samples were detected based on RT-qPCR results. The fluorescence spectrum data indicated that the COVID-19-positive and -negative samples had significantly different characteristics. All positive samples could be distinguished from negative samples by fluorescence spectrophotometry. Principal component analysis showed that COVID-19-positive samples were clustered separately from COVID-19-negative samples. The specificity and accuracy of this experiment reached 100%. Limit of detection (LOD) obtained 42.20 copies/ml (Ct value of 33.65 cycles) for E gene and 63.60 copies/ml (Ct value of 31.36 cycles) for ORF1ab gene. This identification process only required 4 min. Thus, this technique offers an efficient and accurate method to identify an individual with active SARS-CoV-2 infection and can be easily adapted for the early investigation of COVID-19, in general.

Isothermal amplification using sequence-specific fluorescence detection of SARS coronavirus 2 and variants in nasal swabs.

Coronavirus disease 2019 is a public health challenge requiring rapid testing for the detection of infections and transmission. Nucleic acid amplification tests targeting SARS coronavirus 2 (CoV2) are used to detect CoV2 in clinical samples. Real-time reverse transcription quantitative PCR is the standard nucleic acid amplification test for CoV2, although reverse transcription loop-mediated isothermal amplification is used in diagnostics. The authors demonstrate a sequence-specific reverse transcription loop-mediated isothermal amplification-based nucleic acid amplification assay that is finished within 30 min using minimally processed clinical nasal swab samples and describe a fluorescence-quenched reverse transcription loop-mediated isothermal amplification assay using labeled primers and a quencher oligonucleotide. This assay can achieve rapid (30 min) and sensitive (1000 plaque-forming units/ml) fluorescence detection of CoV2 (WA1/2020), B.1.1.7 (Alpha) and variants of concern Delta (B.1.617.2) and Omicron (B.1.1.529) in nasal samples.

Sensitively detecting antigen of SARS-CoV-2 by NIR-II fluorescent nanoparticles.

Early detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is an efficient way to prevent the spread of coronavirus disease 2019 (COVID-19). Detecting SARS-CoV-2 antigen can be rapid and convenient, but it is still challenging to develop highly sensitive methods for effective diagnosis. Herein, a lateral flow assay (LFA) based on fluorescent nanoparticles emitting in the second near-infrared (NIR-II) window is developed for sensitive detection of SARS-CoV-2 antigen. Benefiting from the NIR-II fluorescence with high penetration and low autofluorescence, such NIR-II based LFA allows enhanced signal-to-background ratio, and the limit of detection is down to 0.01 ng·mL−1 of SARS-CoV-2 antigen. In the clinical swab sample tests, the NIR-II LFA outperforms the colloidal gold LFA with higher overall percent agreement with the polymerase chain reaction test. The clinical samples with low antigen concentrations (∼ 0.015−∼ 0.068 ng·mL−1) can be successfully detected by the NIR-II LFA, but fail for the colloidal gold LFA. The NIR-II LFA can provide a promising platform for highly sensitive, rapid, and cost-effective method for early diagnosis and mass screening of SARS-CoV-2 infection.Electronic Supplementary MaterialSupplementary material (the operation procedure and cost of the materials needed of NIR-II lateral flow assays, the dynamic light scattering spectrum of the NIR-II nanoparticles, the components and testing principle, optimization of main parameters pertaining to the LFA performance, the colloidal gold LFA strip, the fluorescence intensity distribution curves and the T/C values of the strips for clinical samples by NIR-II LFA, and results of clinical swab samples detected by colloidal gold LFA) is available in the online version of this article at 10.1007/s12274-022-4351-1.

Proof‑of‑concept study to quantify changes in intestinal loads of KPC-producing Klebsiella pneumoniae in colonised patients following selective digestive decontamination with oral gentamicin

To monitor quantitatively the extent of intestinal colonisation by KPC-producing Klebsiella pneumoniae (KPC-Kp) in colonised patients who receive selective digestive decontamination (SDD) with oral gentamicin. We developed a real-time quantitative PCR(qPCR) method for determination of the relative load of blaKPC (RLKPC) within the gut microbiota. Clinical validation was performed using a culture method as the gold standard and receiver operating curve (ROC) analysis. Fifteen patients were observationally and prospectively followed for one year. Clinical, microbiological variables and rectal swab samples were collected at 0 (baseline), 14 and 30 days and monthly thereafter. Clinical validation performed on 111 rectal swab samples demonstrated that the PCR method detected 17% more positives than theculture method. ROC curve analysis documented excellent agreement between both methods (area under the curve, 0.96; 95% confidence interval 0.93-0.99). The RLKPC decreased in 6/15 (40%) and 7/12 (58.3%) patients on days 14 and 30, respectively. Persistent eradication was observed in 2/12 (16.7%), 3/9 (33.3%), 4/8 (50%) and 7/8 (87.5%) patients at 1, 3, 6 and 12 months, respectively, with a median time of 150 days (range 30-270) to persistent eradication. Gentamicin-resistant KPC-Kp isolates were identified in 4/15 (26.7%) patients. The rates of infections (57.1% vs. 12.5%, P=0.119) and deaths (71.4% vs. 0%, P=0.007) were higher among patients with high baseline RLKPC. Following SDD, a rapid reduction on intestinal load is observed when the colonising KPC-Kp isolate is susceptible to gentamicin; however, persistent eradication at the end of SDD is low. Intestinal carriage of KPC-Kp persists after three months in about one third of patients.

Purely electrical SARS-CoV-2 sensing based on single-molecule counting.

The majority of RNA based COVID-19 diagnostics employ enzymatic amplification to achieve high sensitivity, but this relies on arbitrary thresholding, which complicates the comparison of test results and may lead to false outcomes. Here we introduce solid-state nanopore sensing for label-free quantification of SARS-CoV-2 RNA in clinical nasal swab samples. This PCR-free method involves reverse transcribing a target gene on the viral RNA before enzymatically digesting all but the resulting dsDNA. Ratiometric quantification of RNA abundance is achieved by single-molecule counting and length-based nanopore identification of dsDNA from a SARS-CoV-2 gene and a human reference gene. We graded nasal swab samples from >15 subjects and find that the SARS-CoV-2 ratiometric nanopore index correlates well with the reported RT-qPCR threshold cycle for positive classified samples. Remarkably, nanopore analysis also reports quantitative positive outcomes for clinical samples classified as negative by RT-qPCR, suggesting that the method may be used to diagnose COVID-19 in samples that may evade detection. We show that the sample preparation workflow can be implemented using a compact microfluidic device with integrated thermal control for semi-automated processing of extremely small sample volumes, offering a viable route towards automated, fast and affordable RNA quantification in a small and portable device.