scholarly journals OC 8173 RAPID DETECTION OF MYCOBACTERIUM ULCERANS BY RECOMBINASE POLYMERASE AMPLIFICATION

2019 ◽  
Vol 4 (Suppl 3) ◽  
pp. A2.1-A2
Author(s):  
Michael Frimpong ◽  
Hubert Ahor ◽  
Francisca Sarpong ◽  
Ken Laing ◽  
Mark Wansbrough-Jones ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Point Of Care ◽  
Clinical Signs ◽  
Buruli Ulcer ◽  
Cross Reactivity ◽  
Current Control ◽  
Mycobacterium Ulcerans ◽  
Recombinase Polymerase Amplification ◽  
Clinical Samples ◽  
Clinical Sensitivity

BackgroundThere are no primary measures to prevent people from contracting Buruli ulcer, mainly due to poor understanding of its epidemiology. The current control strategy emphasises early diagnosis and prompt treatment, with the goal of avoiding the complications associated with advanced stages of the disease. There is no diagnostic test for the disease appropriate for use at the primary health care level where most cases are detected and treated. Diagnosis based on clinical signs is unreliable in inexperienced hands and complicated by infections that have similar presentations. This study was to develop and evaluate the use of recombinase polymerase amplification (RPA) assay for the detection of Mycobacterium ulcerans at the point of patient care.MethodsA specific fragment of IS2404 of M. ulcerans was amplified in 15 min at a constant temperature of 42°C, using the RPA assay and analysed on a portable fluorometre. The’method was tested for sensitivity and specificity with molecular standard of IS2404 DNA fragment, various M.’ulcerans strains, other mycobacteria and environmentally associated bacteria. Additionally, the assay performance as a diagnostic tool was tested with archived DNA from symptomatic patients. All results were compared with that of a highly sensitive IS2404 PCR.ResultsThe detection limit was 50 copies of IS2404 in 15 min using plasmid standard and 125 fg with genomic Mu DNA equivalent 25 genomic copies. The assay was highly specific in detecting all strains of M. ulcerans with no observed cross reactivity with other mycobacteria and common skin colonising bacteria. The clinical sensitivity and specificity of the BU-RPA assay using clinical samples was 86% and 100% respectively.ConclusionWe have developed a real-time isothermal RPA assay for the detection of M. ulcerans as a cheaper alternative to PCR. Combining this assay with a simple extraction protocol will maximise its use as point-of-care test for Buruli ulcer.

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 of a reverse transcription recombinase polymerase amplification assay for rapid and direct visual detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245164
Author(s):  
Yee Ling Lau ◽  
Ilyiana binti Ismail ◽  
Nur Izati binti Mustapa ◽  
Meng Yee Lai ◽  
Tuan Suhaila Tuan Soh ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Cross Reactivity ◽  
Recombinase Polymerase Amplification ◽  
Analytical Sensitivity ◽  
Clinical Sensitivity ◽  
Primary Means ◽  
Amplification Assay ◽  
Recombinase Polymerase Amplification Assay

Rapid diagnosis is an important intervention in managing the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak. Real time reverse transcription polymerase chain reaction (RT-qPCR) remains the primary means for diagnosing the new virus strain but it is time consuming and costly. Recombinase polymerase amplification (RPA) is an isothermal amplification assay that does not require a PCR machine. It is an affordable, rapid, and simple assay. In this study, we developed and optimized a sensitive reverse transcription (RT)-RPA assay for the rapid detection of SARS-CoV-2 using SYBR Green I and/or lateral flow (LF) strip. The analytical sensitivity and specificity of the RT-RPA assay were tested by using 10-fold serial diluted synthetic RNA and genomic RNA of similar viruses, respectively. Clinical sensitivity and specificity of the RT-RPA assay were carried out using 78 positive and 35 negative nasopharyngeal samples. The detection limit of both RPA and RT-qPCR assays was 7.659 and 5 copies/μL RNA, respectively with no cross reactivity with other viruses. The clinical sensitivity and specificity of RT-RPA were 98% and 100%, respectively. Our study showed that RT-RPA represents a viable alternative to RT-qPCR for the detection of SARS-CoV-2, especially in areas with limited infrastructure.

scholarly journals Development and Clinical Validation of a Sensitive Lateral Flow Assay for Rapid Urine Fentanyl Screening in the Emergency Department

2020 ◽  
Vol 66 (2) ◽  
pp. 324-332 ◽  
Author(s):  
Zhao Li ◽  
Hui Chen ◽  
Sheng Feng ◽  
Kengku Liu ◽  
Ping Wang
Keyword(s):  
Emergency Department ◽  
Sensitivity And Specificity ◽  
Point Of Care ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Rapid Screening ◽  
Single Digit ◽  
Predictive Values ◽  
Clinical Sensitivity

Abstract Background Rapid identification of fentanyl at the point-of-care is critical. Urine fentanyl concentrations in overdose cases start at single-digit nanograms per milliliter. No fentanyl point-of-care assay with a cutoff at single-digit nanograms per milliliter is available. Methods A competitive lateral flow assay (LFA) was developed using gold nanoparticles and optimized for rapid screening of fentanyl in 5 minutes. Urine samples from 2 cohorts of emergency department (ED) patients were tested using the LFA and LC-MS/MS. The 2 cohorts consisted of 218 consecutive ED patients with urine drug-of-abuse screen orders and 7 ED patients with clinically suspected fentanyl overdose, respectively. Results The LFA detected fentanyl (≥1 ng/mL) and the major metabolite norfentanyl (≥10 ng/mL) with high precision. There was no cross-reactivity with amphetamine, cocaine, morphine, tetrahydrocannabinol, methadone, buprenorphine, naloxone, and acetaminophen at 1000 ng/mL and 0.03%, 0.4%, and 0.05% cross-reactivity with carfentanil, risperidone, and 9-hydroxyrisperidone, respectively. In 218 consecutive ED patients, the prevalence of cases with fentanyl ≥1 ng/mL or norfentanyl ≥10 ng/mL was 5.5%. The clinical sensitivity and specificity of the LFA were 100% (95% CI, 75.8–100%) and 99.5% (95% CI, 97.3–99.9%), respectively. The positive and negative predictive values were 92.3% (95% CI, 66.7–98.6%) and 100% (95% CI, 98.2–100%), respectively. The concordance between the LFA and LC-MS/MS was 100% in the 7 suspected fentanyl overdose cases (5 positive, 2 negative). Conclusions The LFA can detect fentanyl and norfentanyl with high clinical sensitivity and specificity in the ED population with rapid fentanyl screening needs.

scholarly journals Rapid Extraction Method of Mycobacterium ulcerans DNA from Clinical Samples of Suspected Buruli Ulcer Patients

Diagnostics ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 204
Author(s):  
Michael Frimpong ◽  
Hubert Senanu Ahor ◽  
Samuel Asamoah Sakyi ◽  
Bernadette Agbavor ◽  
Emmanuel Akowuah ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Procedure ◽  
Buruli Ulcer ◽  
Molecular Techniques ◽  
Isothermal Amplification ◽  
Mycobacterium Ulcerans ◽  
Recombinase Polymerase Amplification ◽  
Clinical Samples ◽  
Rapid Extraction ◽  
Extraction Protocol

Isothermal amplification techniques such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) for diagnosing Buruli ulcer, a necrotic skin disease caused by Mycobacterium ulcerans, have renewed hope for the molecular diagnosis of clinically suspected Buruli ulcer cases in endemic districts. If these techniques are applied at district-level hospitals or clinics, they will help facilitate early case detection with prompt treatment, thereby reducing disability and associated costs of disease management. The accuracy as well as the application of these molecular techniques at point of need is dependent on simple and fast DNA extraction. We have modified and tested a rapid extraction protocol for use with an already developed recombinase polymerase amplification assay. The entire procedure from “sample in, extraction and DNA amplification” was conducted in a mobile suitcase laboratory within 40 min. The DNA extraction procedure was performed within 15 min, with only two manipulation/pipetting steps needed. The diagnostic sensitivity and specificity of this extraction protocol together with M. ulcerans RPA in comparison with standard DNA extraction with real-time PCR was 87% (n = 26) and 100% (n = 13), respectively. We have established a simple, fast and efficient protocol for the extraction and detection of M. ulcerans DNA in clinical samples that is adaptable to field conditions.

scholarly journals Evaluation of mobile real-time polymerase chain reaction tests for the detection of severe acute respiratory syndrome coronavirus 2

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chukwunonso Onyilagha ◽  
Henna Mistry ◽  
Peter Marszal ◽  
Mathieu Pinette ◽  
Darwyn Kobasa ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Point Of Care ◽  
Middle Eastern ◽  
Turnaround Time ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Diarrhea Virus ◽  
Highly Sensitive ◽  
Transmissible Gastroenteritis

AbstractThe coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), calls for prompt and accurate diagnosis and rapid turnaround time for test results to limit transmission. Here, we evaluated two independent molecular assays, the Biomeme SARS-CoV-2 test, and the Precision Biomonitoring TripleLock SARS-CoV-2 test on a field-deployable point-of-care real-time PCR instrument, Franklin three9, in combination with Biomeme M1 Sample Prep Cartridge Kit for RNA 2.0 (M1) manual extraction system for rapid, specific, and sensitive detection of SARS-COV-2 in cell culture, human, and animal clinical samples. The Biomeme SARS-CoV-2 assay, which simultaneously detects two viral targets, the orf1ab and S genes, and the Precision Biomonitoring TripleLock SARS-CoV-2 assay that targets the 5′ untranslated region (5′ UTR) and the envelope (E) gene of SARS-CoV-2 were highly sensitive and detected as low as 15 SARS-CoV-2 genome copies per reaction. In addition, the two assays were specific and showed no cross-reactivity with Middle Eastern respiratory syndrome coronavirus (MERS-CoV), infectious bronchitis virus (IBV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis (TGE) virus, and other common human respiratory viruses and bacterial pathogens. Also, both assays were highly reproducible across different operators and instruments. When used to test animal samples, both assays equally detected SARS-CoV-2 genetic materials in the swabs from SARS-CoV-2-infected hamsters. The M1 lysis buffer completely inactivated SARS-CoV-2 within 10 min at room temperature enabling safe handling of clinical samples. Collectively, these results show that the Biomeme and Precision Biomonitoring TripleLock SARS-CoV-2 mobile testing platforms could reliably and promptly detect SARS-CoV-2 in both human and animal clinical samples in approximately an hour and can be used in remote areas or health care settings not traditionally serviced by a microbiology laboratory.

scholarly journals First isolation of Mycobacterium ulcerans from Swabs and Fine-Needle-Aspiration Specimens in Togo.

2019 ◽  
Author(s):  
Tchalare Kondi Makagni ◽  
Maman Issaka ◽  
Piten Ebekalisai ◽  
Disse Kodjo ◽  
Essossimna A. Kadanga ◽  
...  
Keyword(s):  
Fine Needle Aspiration ◽  
Slow Growth ◽  
Buruli Ulcer ◽  
Needle Aspiration ◽  
Mycobacterium Ulcerans ◽  
Clinical Samples ◽  
Direct Examination ◽  
Fine Needle ◽  
Pcr Targeting

Abstract Background Buruli ulcer is a skin disease caused by a mycobacterium called Mycobacterium ulcerans . It is prevalent in more than 33 countries on several continents but West Africa is the most affected. The isolation in culture of the bacteria is difficult because of its slow growth and the facilities required. In Togo, studies have been done on the risk factors for Mycobacterium ulcerans infection and the detection of cases by the Ziehl-Neelsen and PCR technique on clinical and environmental samples, but to date no data of isolates from clinical samples are available. The purpose of this study was to perform an in vitro culture of M. ulcerans from swab and fine needle aspiration samples through the confirmation stages of direct examination and IS2404 -PCR. Method A total of 70 clinical samples from Togo and 10 clinical isolates from Benin are analyzed by the three techniques indicated in the diagnosis, in particular the direct examination of acid-fast bacilli (AFB) using the Ziehl-Neelsen staining, the PCR targeting the IS2404 sequence, and the culture after transport of the samples in a transport medium made of Middlebrook 7H9 medium supplemented with a mixture of PANTA and OADC and decontamination by the modified Petroff method. Results The application of the three techniques of diagnosis for clinical samples yielded 44.28% of positivity rates on direct examination of AFB, 35.71% on culture and 77.14% on qPCR IS2404 with a significantly higher rate for qPCR (0.0001). All samples positive for Ziehl-Neelsen staining and culture were also positive for qPCR. Conclusion : Our results show that the culture, despite it difficulty and the slow growth of the bacteria, can be carried out with recommended tools of the mycobacteria culture and a good method of decontamination of the samples can improve the positivity rates. Its realization will allow the assessment of the in vitro sensitivity to the antibiotics used in the treatment and the discovery of new strains of Mycobacterium ulcerans .

scholarly journals Multiplexed Detection of Sepsis Markers in Whole Blood using Nanocomposite Coated Electrochemical Sensors

2020 ◽  
Author(s):  
Uroš Zupančič ◽  
Pawan Jolly ◽  
Pedro Estrela ◽  
Despina Moschou ◽  
Donald E. Ingber
Keyword(s):  
Electrochemical Detection ◽  
Whole Blood ◽  
Electrochemical Sensors ◽  
Point Of Care ◽  
Sample Volume ◽  
Nanocomposite Coating ◽  
Cross Reactivity ◽  
Detection Methods ◽  
Clinical Samples ◽  
Undiluted Serum

ABSTRACTSepsis is a leading cause of mortality worldwide that is difficult to diagnose and manage because this requires simultaneous analysis of multiple biomarkers. Electrochemical detection methods could potentially provide a way to accurately quantify multiple sepsis biomarkers in a multiplexed manner as they have very low limits of detection and require minimal sensor instrumentation; however, affinity-based electrochemical sensors are usually hampered by biological fouling. Here we describe development of an electrochemical detection platform that enables detection of multiple sepsis biomarkers simultaneously by incorporating a recently developed nanocomposite coating composed of crosslinked bovine serum albumin containing a network of reduced graphene oxide nanoparticles that prevents biofouling. Using nanocomposite coated planar gold electrodes, we constructed a procalcitonin sensor and demonstrated sensitive PCT detection in undiluted serum and clinical samples, as well as excellent correlation with a conventional ELISA (adjusted r2 = 0.95). Sensors for two additional sepsis biomarkers — C-reactive protein and pathogen-associated molecular patterns — were developed on the same multiplexed platform and tested in whole blood. Due to the excellent antifouling properties of the nanocomposite coating, all three sensors exhibited specific responses within the clinically significant range without any cross-reactivity in the same channel with low sample volume. This platform enables sensitive simultaneous electrochemical detection of multiple analytes in human whole blood, which can be expanded further to any target analyte with an appropriate antibody pair or capturing probe, and thus, may offer a potentially valuable tool for development of clinical point-of-care diagnostics.GRAPHICAL ABSTRACT

scholarly journals Performance of the Molecular Alere i Influenza A&B Test Compared to That of the Xpert Flu A/B Assay

2014 ◽  
Vol 53 (2) ◽  
pp. 706-709 ◽  
Author(s):  
Kimberle C. Chapin ◽  
Estefany J. Flores-Cortez
Keyword(s):  
Influenza Virus ◽  
Sensitivity And Specificity ◽  
Influenza A ◽  
Point Of Care ◽  
Type A ◽  
Influenza B ◽  
Clinical Sensitivity ◽  
Transport Medium ◽  
The Poor ◽  
B Test

Data on the performance of rapid molecular point-of-care use platforms for diagnosis of influenza are lacking. We validated nasopharyngeal (NP) flocked specimens in universal transport medium (UTM) and evaluated the clinical sensitivity and specificity of the Alere i influenza A&B test compared to those of the Xpert flu A/B assay. The Alere i influenza A&B test had an overall sensitivity and specificity of 93.8% and 62.5% for influenza A, respectively, and of 91.8% and 53.6% for influenza B, respectively. The poor specificity was due to influenza virus samples determined positive for both type A and B.

scholarly journals Comparison of commercial RT-PCR diagnostic kits for COVID-19

2020 ◽  
Author(s):  
Puck B. van Kasteren ◽  
Bas van der Veer ◽  
Sharon van den Brink ◽  
Lisa Wijsman ◽  
Jørgen de Jonge ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Cross Reactivity ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Specific Method ◽  
Preliminary Evaluation ◽  
Rt Pcr ◽  
Accurate Identification ◽  
Clinical Sensitivity ◽  
Pcr Efficiency

ABSTRACTThe final months of 2019 witnessed the emergence of a novel coronavirus in the human population. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has since spread across the globe and is posing a major burden on society. Measures taken to reduce its spread critically depend on timely and accurate identification of virus-infected individuals by the most sensitive and specific method available, i.e. real-time reverse transcriptase PCR (RT-PCR). Many commercial kits have recently become available, but their performance has not yet been independently assessed.The aim of this study was to compare basic analytical and clinical performance of selected RT-PCR kits from seven different manufacturers (Altona Diagnostics, BGI, CerTest Biotec, KH Medical, PrimerDesign, R-Biopharm AG, and Seegene).We used serial dilutions of viral RNA to establish PCR efficiency and estimate the 95% limit of detection (LOD95%). Furthermore, we ran a panel of SARS-CoV-2-positive clinical samples (n=16) for a preliminary evaluation of clinical sensitivity. Finally, we used clinical samples positive for non-coronavirus respiratory viral infections (n=6) and a panel of RNA from related human coronaviruses to evaluate assay specificity.PCR efficiency was ≥96% for all assays and the estimated LOD95% varied within a 6-fold range. Using clinical samples, we observed some variations in detection rate between kits. Importantly, none of the assays showed cross-reactivity with other respiratory (corona)viruses, except as expected for the SARS-CoV-1 E-gene.We conclude that all RT-PCR kits assessed in this study may be used for routine diagnostics of COVID-19 in patients by experienced molecular diagnostic laboratories.

scholarly journals Evaluation of different DNA extraction methods and loop-mediated isothermal amplification primers for the detection of Mycobacterium ulcerans in clinical specimens

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Anthony Ablordey ◽  
Evans Ahotor ◽  
Charles A. Narh ◽  
Sandra A. King ◽  
Isra Cruz ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Point Of Care ◽  
Buruli Ulcer ◽  
Extraction Methods ◽  
Isothermal Amplification ◽  
Mycobacterium Ulcerans ◽  
Loop Mediated Isothermal Amplification ◽  
Dna Extraction Method ◽  
Dna Extraction Methods

Abstract Background Early diagnosis and treatment of Buruli ulcer is critical in order to avoid the debilitating effects of the disease. In this regard, the development of new diagnostic and point of care tools is encouraged. The loop-mediated isothermal amplification for the detection of Mycobacterium ulcerans represents one of the new tools with a good potential of being developed into a point of care test. There is however the need to standardize the assays, reduce sample preparation times, improve the detection/visualization system and optimize them for high-throughput screening, adaptable to low resourced laboratories. Methods In this study, we assessed two DNA extraction protocols (modified Boom and EasyNAT methods), three previously published LAMP primer sets (BURULI, MU 2404 and BU-LAMP), and compared the sensitivity and specificity of LAMP assays on three DNA amplification platforms. Results Our results show that Buruli ulcer diagnosis using primers targeting IS2404 for the LAMP method is sensitive (73.75–91.49%), depending on the DNA extraction method used. Even though the modified Boom DNA extraction method provided the best results, its instrumentation requirement prevent it from being field applicable. The EasyNAT method on the other hand is simpler and may represent the best method for DNA extraction in less resourced settings. Conclusions For further work on the development and use of LAMP tests for Buruli diagnosis, it is recommended that the BURULI sets of primers be used, as these yielded the best results in terms of sensitivity (87.50–91.49%) and specificity (89.23–100%), depending on the DNA extraction methods used.

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