scholarly journals A Nanoparticle-Based Biosensor Combined With Multiple Cross Displacement Amplification for the Rapid and Visual Diagnosis of Neisseria gonorrhoeae in Clinical Application

2021 ◽  
Vol 12 ◽  
Author(s):  
Xu Chen ◽  
Liming Huang ◽  
Qingxue Zhou ◽  
Yan Tan ◽  
Xuhong Tan ◽  
...  
Keyword(s):  
Cost Saving ◽  
Neisseria Gonorrhoeae ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Transmitted Disease ◽  
Health Concern ◽  
Clinical Samples ◽  
Specific Gene ◽  
Sexually Transmitted ◽  
Multiple Cross

Gonorrhea is a sexually transmitted disease caused by the host-adapted human pathogen, Neisseria gonorrhoeae. The morbidity is increasing and poses a major public health concern, especially in resource-scarce regions. Therefore, a rapid, visual, sensitive, specific, cost-saving, and simple assay for N. gonorrhoeae detection is critical for prompt treatment and the prevention of further transmission. Here, for the first time, we report a novel assay called the multiple cross displacement amplification combined with gold nanoparticle-based lateral flow biosensor (MCDA-LFB), which we constructed for the rapid and visual identification of N. gonorrhoeae in clinical samples. We successfully devised a set of MCDA primers based on the N. gonorrhoeae-specific gene, orf1. Optimal assay conditions were determined at 67°C, including genomic DNA preparation (∼15 min), MCDA amplification (30 min), and LFB reading (∼2 min), which can be completed within 50 min. The limit of detection (LoD) of the assay was 20 copies/test (in a 25-μl reaction mixture). Assay specificity was 100%, with no cross-reactions with other pathogens. Thus, our N. gonorrhoeae-MCDA-LFB is a rapid, specific, visual, cost-saving, and easy-to-use assay for N. gonorrhoeae diagnostics, and may have great potential for point-of-care (POC) testing in clinical settings, especially in resource-limited regions.

scholarly journals Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shan Wei ◽  
Esther Kohl ◽  
Alexandre Djandji ◽  
Stephanie Morgan ◽  
Susan Whittier ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Cost Effective ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Percentage Agreement ◽  
Testing Method ◽  
Viral Transport

AbstractThe COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/μl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.

scholarly journals Dynamics of Neisseria gonorrhoeae Attachment: Microcolony Development, Cortical Plaque Formation, and Cytoprotection

2007 ◽  
Vol 75 (10) ◽  
pp. 4743-4753 ◽  
Author(s):  
Dustin L. Higashi ◽  
Shaun W. Lee ◽  
Aurelie Snyder ◽  
Nathan J. Weyand ◽  
Antony Bakke ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Transmitted Disease ◽  
Virus Transmission ◽  
Twitching Motility ◽  
Sexually Transmitted ◽  
Type Iv ◽  
Immunodeficiency Virus ◽  
Live Cell Microscopy ◽  
Induced Apoptosis ◽  
First Time

ABSTRACT Neisseria gonorrhoeae is the bacterium that causes gonorrhea, a major sexually transmitted disease and a significant cofactor for human immunodeficiency virus transmission. The retactile N. gonorrhoeae type IV pilus (Tfp) mediates twitching motility and attachment. Using live-cell microscopy, we reveal for the first time the dynamics of twitching motility by N. gonorrhoeae in its natural environment, human epithelial cells. Bacteria aggregate into microcolonies on the cell surface and induce a massive remodeling of the microvillus architecture. Surprisingly, the microcolonies are motile, and they fuse to form progressively larger structures that undergo rapid reorganization, suggesting that bacteria communicate with each other during infection. As reported, actin plaques form beneath microcolonies. Here, we show that cortical plaques comigrate with motile microcolonies. These activities are dependent on pilT, the Tfp retraction locus. Cultures infected with a pilT mutant have significantly higher numbers of apoptotic cells than cultures infected with the wild-type strain. Inducing pilT expression with isopropyl-β-d-thiogalactopyranoside partially rescues cells from infection-induced apoptosis, demonstrating that Tfp retraction is intrinsically cytoprotective for the host. Tfp-mediated attachment is therefore a continuum of microcolony motility and force stimulation of host cell signaling, leading to a cytoprotective effect.

scholarly journals Epidemiology, molecular characterisation and antimicrobial susceptibility of Neisseria gonorrhoeae isolates in Madrid, Spain, in 2016

2019 ◽  
Vol 147 ◽  
Author(s):  
M. D. Guerrero-Torres ◽  
M. B. Menéndez ◽  
C. S. Guerras ◽  
E. Tello ◽  
J. Ballesteros ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Antimicrobial Susceptibility ◽  
Strong Association ◽  
Clinical Samples ◽  
Transmitted Infection ◽  
Sexually Transmitted ◽  
High Incidence ◽  
Ciprofloxacin Resistance ◽  
Sequence Types ◽  
Infection Clinic

Abstract With the aim to elucidate gonococcal antimicrobial resistance (AMR)–risk factors, we undertook a retrospective analysis of the molecular epidemiology and AMR of 104 Neisseria gonorrhoeae isolates from clinical samples (urethra, rectum, pharynx and cervix) of 94 individuals attending a sexually transmitted infection clinic in Madrid (Spain) from July to October 2016, and explored potential links with socio-demographic, behavioural and clinical factors of patients. Antimicrobial susceptibility was determined by E-tests, and isolates were characterised by N. gonorrhoeae multi-antigen sequence typing. Penicillin resistance was recorded for 15.4% of isolates, and most were susceptible to tetracycline, cefixime and azithromycin; a high incidence of ciprofloxacin resistance (~40%) was found. Isolates were grouped into 51 different sequence types (STs) and 10 genogroups (G), with G2400, ST5441, ST2318, ST12547 and G2992 being the most prevalent. A significant association (P = 0.015) was evident between HIV-positive MSM individuals and having a ciprofloxacin-resistant strain. Likewise, a strong association (P = 0.047) was found between patient age of MSM and carriage of isolates expressing decreased susceptibility to azithromycin. A decrease in the incidence of AMR gonococcal strains and a change in the strain populations previously reported from other parts of Spain were observed. Of note, the prevalent multi-drug resistant genogroup G1407 was represented by only three strains in our study, while the pan-susceptible clones such as ST5441, and ST2318, associated with extragenital body sites were the most prevalent.

scholarly journals Performance evaluation of the point-of-care SAMBA II SARS-CoV-2 Test for detection of SARS-CoV-2

2020 ◽  
Author(s):  
Sonny M Assennato ◽  
Allyson V Ritchie ◽  
Cesar Nadala ◽  
Neha Goel ◽  
Hongyi Zhang ◽  
...  
Keyword(s):  
Public Health ◽  
Infection Control ◽  
Sensitivity And Specificity ◽  
Patient Management ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Point Of Care Testing ◽  
Clinical Sensitivity

AbstractNucleic acid amplification for the detection of SARS-CoV-2 RNA in respiratory samples is the standard method for diagnosis. These tests are centralised and therefore turnaround times can be 2-5 days. Point-of-care testing with rapid turnaround times would allow more effective triage in settings where patient management and infection control decisions need to be made rapidly.Inclusivity and specificity of the SAMBA II SARS-CoV-2 assay was determined by in silico analyses of the primers and probes. Analytical and clinical sensitivity and specificity of the SAMBA II SARS-CoV-2 Test was evaluated for analytical sensitivity and specificity. Clinical performance was evaluated in residual clinical samples compared to the Public Health England reference tests.The limit of detection of the SAMBA II SARS-CoV-2 Test is 250 cp/mL and is specific for detection of 2 regions of the SARS-CoV-2 genome. The clinical sensitivity was evaluated in 172 clinical samples provided by the Clinical Microbiology and Public Health Laboratory, Addenbrooke’s Hospital, Cambridge (CMPHL), which showed a sensitivity of 98.9% (95% CI 94.03-99.97%), specificity of 100% (95% CI 95.55-100%), PPV of 100% and NPV of 98.78% (92.02-99.82%) compared to testing by CMPHLSAMBA detected 3 positive samples that were initially negative by PHE Test. The data shows that the SAMBA II SARS-CoV-2 Test performs equivalently to the centralised testing methods with a much quicker turnaround time. Point of care testing, such as SAMBA, should enable rapid patient management and effective implementation of infection control measures.

scholarly journals Development and validation of direct RT-LAMP for SARS-CoV-2

2020 ◽  
Author(s):  
Abu Naser Mohon ◽  
Jana Hundt ◽  
Guido van Marle ◽  
Kanti Pabbaraju ◽  
Byron Berenger ◽  
...  
Keyword(s):  
Sindbis Virus ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Extraction Procedure ◽  
Lamp Assay ◽  
Cold Chain ◽  
Clinical Samples ◽  
Lamp Method ◽  
Rt Pcr ◽  
Percent Agreement

AbstractWe have developed a reverse-transcriptase loop mediated amplification (RT-LAMP) method targeting genes encoding the Spike (S) protein and RNA-dependent RNA polymerase (RdRP) of SARS-CoV-2. The LAMP assay achieves comparable limit of detection as commonly used RT-PCR protocols based on artificial targets, recombinant Sindbis virus, and clinical samples. Clinical validation of single-target (S gene) LAMP (N=120) showed a positive percent agreement (PPA) of 41/42 (97.62%) and negative percent agreement (NPA) of 77/78 (98.72%) compared to reference RT-PCR. Dual-target RT-LAMP (S and RdRP gene) achieved a PPA of 44/48 (91.97%) and NPA 72/72 (100%) when including discrepant samples. The assay can be performed without a formal extraction procedure, with lyophilized reagents which do need cold chain, and is amenable to point-of-care application with visual detection.

scholarly journals Neisseria gonorrhoeae subverts formin-dependent actin polymerization to colonize human macrophages

2021 ◽  
Vol 17 (12) ◽  
pp. e1010184
Author(s):  
Stanimir S. Ivanov ◽  
Reneau Castore ◽  
Maria Dolores Juarez Rodriguez ◽  
Magdalena Circu ◽  
Ana-Maria Dragoi
Keyword(s):  
Plasma Membrane ◽  
Neisseria Gonorrhoeae ◽  
Actin Polymerization ◽  
Bacterial Pathogens ◽  
Transmitted Disease ◽  
Bacterial Pathogen ◽  
Host Cells ◽  
Human Macrophages ◽  
Sexually Transmitted ◽  
Bacterial Replication

Dynamic reorganization of the actin cytoskeleton dictates plasma membrane morphogenesis and is frequently subverted by bacterial pathogens for entry and colonization of host cells. The human-adapted bacterial pathogen Neisseria gonorrhoeae can colonize and replicate when cultured with human macrophages, however the basic understanding of how this process occurs is incomplete. N. gonorrhoeae is the etiological agent of the sexually transmitted disease gonorrhea and tissue resident macrophages are present in the urogenital mucosa, which is colonized by the bacteria. We uncovered that when gonococci colonize macrophages, they can establish an intracellular or a cell surface-associated niche that support bacterial replication independently. Unlike other intracellular bacterial pathogens, which enter host cells as single bacterium, establish an intracellular niche and then replicate, gonococci invade human macrophages as a colony. Individual diplococci are rapidly phagocytosed by macrophages and transported to lysosomes for degradation. However, we found that surface-associated gonococcal colonies of various sizes can invade macrophages by triggering actin skeleton rearrangement resulting in plasma membrane invaginations that slowly engulf the colony. The resulting intracellular membrane-bound organelle supports robust bacterial replication. The gonococci-occupied vacuoles evaded fusion with the endosomal compartment and were enveloped by a network of actin filaments. We demonstrate that gonococcal colonies invade macrophages via a process mechanistically distinct from phagocytosis that is regulated by the actin nucleating factor FMNL3 and is independent of the Arp2/3 complex. Our work provides insights into the gonococci life-cycle in association with human macrophages and defines key host determinants for macrophage colonization.

scholarly journals Identification of Acinetobacter baumannii and its carbapenem-resistant gene blaOXA-23-like by multiple cross displacement amplification combined with lateral flow biosensor

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Shoukui Hu ◽  
Lina Niu ◽  
Fan Zhao ◽  
Linlin Yan ◽  
Jinqing Nong ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Limit Of Detection ◽  
Lateral Flow ◽  
Clinical Samples ◽  
Conventional Culture ◽  
Carbapenem Resistant ◽  
Multiple Cross ◽  
Pure Cultures ◽  
Amplification Technique ◽  
Sensitivity Specificity

AbstractAcinetobacter baumannii is a frequent cause of the nosocomial infections. Herein, a novel isothermal amplification technique, multiple cross displacement amplification (MCDA) is employed for detecting all A. baumannii strains and identifying the strains harboring blaOXA-23-like gene. The duplex MCDA assay, which targets the pgaD and blaOXA-23-like genes, could identify the A. baumannii isolates and differentiate these isolates harboring blaOXA-23-like gene. The disposable lateral flow biosensors (LFB) were used for analyzing the MCDA products. A total of sixty-eight isolates, include fifty-three A. baumannii strains and fifteen non-A. baumannii strains, were employed to optimize MCDA methods and determine the sensitivity, specificity and feasibility. The optimal reaction condition is found to be 63 °C within 1 h, with limit of detection at 100 fg templates per tube for pgaD and blaOXA-23-like genes in pure cultures. The specificity of this assay is 100%. Moreover, the practical application of the duplex MCDA-LFB assay was evaluated using clinical samples, and the results obtained from duplex MCDA-LFB method were consistent with conventional culture-based technique. In sum, the duplex MCDA-LFB assay appears to be a reliable, rapid and specific technique to detect all A. baumannii strains and identify these strains harboring blaOXA-23-like gene for appropriate antibiotic therapy.

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