Rapid DNA detection of Mycobacterium tuberculosis-towards single cell sensitivity in point-of-care diagnosis
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ABSTRACT:
SUBMITTER: Ng B
PROVIDER: S-EPMC4602229 | biostudies-literature | 2015 Jan
REPOSITORIES: biostudies-literature
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Project description:The implementation of endotype-driven effective intervention strategies is now considered as an essential component for sepsis management. Rapid screening and frequent monitoring of immune responses are critical for evidence-based informed decisions in the early hours of patient arrival. Current technologies focus on pathogen identification that lack rapid testing of the patient immune response, impeding clinicians from providing appropriate sepsis treatment. Herein, we demonstrate a first-of-its-kind novel point-of-care device that uses a unique approach by directly monitoring a panel of five cytokine biomarkers (IL-6, IL-8, IL-10, TRAIL & IP-10), that is attributed as a sign of the body's host immune response to sepsis. The developed point-of-care device encompasses a disposable sensor cartridge attached to an electrochemical reader. High sensitivity is achieved owing to the unique sensor design with an array of nanofilm semiconducting/metal electrode interface, functionalized with specific capture probes to measure target biomarkers simultaneously using non-faradaic electrochemical impedance spectroscopy. The sensor has a detection limit of ~1 pg/mL and provides results in less than five minutes from a single drop of undiluted plasma sample. Furthermore, the sensor demonstrates an excellent correlation (Pearson's r > 0.90) with the reference method for a total n = 40 clinical samples, and the sensor's performance is ~30 times faster compared to the standard reference technique. We have demonstrated the sensor's effectiveness to enhance diagnosis with a mechanistic biomarker-guided approach that can help disease endotypying for effective clinical management of sepsis at the patient bedside.
| S-EPMC7569407 | biostudies-literature
Project description:Early diagnosis of tuberculosis can dramatically reduce both its transmission and the associated death rate. The extremely slow growth rate of the causative pathogen, Mycobacterium tuberculosis (Mtb), however, makes this challenging at the point of care, particularly in resource-limited settings. Here we report the use of BlaC (an enzyme naturally expressed/secreted by tubercle bacilli) as a marker and the design of BlaC-specific fluorogenic substrates as probes for Mtb detection. These probes showed an enhancement by 100-200 times in fluorescence emission on BlaC activation and a greater than 1,000-fold selectivity for BlaC over TEM-1 ?-lactamase, an important factor in reducing false-positive diagnoses. Insight into the BlaC specificity was revealed by successful co-crystallization of the probe/enzyme mutant complex. A refined green fluorescent probe (CDG-OMe) enabled the successful detection of live pathogen in less than ten minutes, even in unprocessed human sputum. This system offers the opportunity for the rapid, accurate detection of very low numbers of Mtb for the clinical diagnosis of tuberculosis in sputum and other specimens.
| S-EPMC4136554 | biostudies-literature
Project description:BackgroundDespite major public health initiatives and the existence of efficacious treatment regimes, tuberculosis (TB) remains a threat, particularly in resource-limited settings. A significant part of the problem is the difficulty of rapidly identifying infected individuals, and as a result, there has been renewed interest in developing better diagnostics for infection or disease caused by Mycobacterium tuberculosis. Many of the existing tools, however, have limitations such as poor sensitivity or specificity, or the need for well-equipped laboratories to function effectively. Serodiagnostic approaches in particular have long drawn attention, due to their potential utility in large field studies, particularly in resource-poor settings. Unfortunately none of the serodiagnostic approaches have so far proven useful under field conditions.ResultsWe screened a large panel of antigens with serodiagnostic potential by ELISA and selected a subpanel that was strongly and broadly recognised by TB patients, but not by controls. These antigens were then formulated into a simple immuno-chromatographic lateral flow assay format, suitable for field use, and tested against panels of plasma and blood samples from individuals with different clinical status (confirmed TB patients, household contacts, and apparently healthy community controls), recruited from Ethiopia (a highly TB-endemic country) and Turkey (a TB meso-endemic country). While specificity was good (97-100% in non TB-endemic controls), the sensitivity was not as high as expected (46-54% in pulmonary TB, 25-29% in extra-pulmonary TB).ConclusionsThough below the level of sensitivity the consortium had set for commercial development, the assay specifically identified M. tuberculosis-infected individuals, and provides a valuable proof of concept.
| S-EPMC3680158 | biostudies-literature
Project description:Outside of the ongoing COVID-19 pandemic, tuberculosis is the leading cause of infectious disease mortality globally. Currently, there is no commercially available point-of-care diagnostic that is rapid, inexpensive, and highly sensitive for the diagnosis of active tuberculosis disease. Here we describe the development and optimization of a novel, highly sensitive prototype bioelectronic tuberculosis antigen (BETA) assay to detect tuberculosis-specific antigen, CFP10, in small-volume serum and urine samples. In this proof-of-concept study we evaluated the performance of the BETA assay using clinical specimens collected from presumptive tuberculosis patients from three independent cohorts. Circulating CFP10 antigen was detected in ALL serum (n = 19) and urine (n = 3) samples from bacteriologically confirmed tuberculosis patients who were untreated or had less than one week of treatment at time of serum collection, successfully identifying all culture positive tuberculosis patients. No CFP10 antigen was detected in serum (n = 7) or urine (n = 6) samples from individuals who were determined to be negative for tuberculosis disease. Additionally, antigen quantification using the BETA assay of paired serum samples collected from tuberculosis patients (n = 8) both before and after treatment initiation, indicate consistently declining within-person levels of CFP10 antigen during treatment. This novel, low-cost assay demonstrates potential as a rapid, non-sputum-based, point-of-care tool for the diagnosis of tuberculosis disease.
| S-EPMC8478938 | biostudies-literature
Project description:BackgroundTuberculosis (TB) is a serious infectious disease caused by Mycobacterium tuberculosis (MTB). An estimated 1.7 billion people worldwide are infected with Mycobacterium tuberculosis (LTBI) during the incubation period without any obvious symptoms. Because of MTB's high infection and mortality rates, there is an urgent need to develop a fast, portable, and sensitive diagnostic technology for its detection.MethodsWe included research from PubMed, Cochrane Library, Web of Science, and Embase and extracted the data. MetaDisc and STATA were used to build forest plots, Deek's funnel plot, Fagan plot, and bivariate boxplot for analysis.ResultsForty-six articles were analyzed, the results of which are as follows: sensitivity and specificity were 0.92 (0.91-0.93) and 0.95 (0.94-0.95) respectively. The NLR and PLR were 0.04 (95% CI 0.03-0.07) and 25.32 (95% CI 12.38-51.78) respectively. DOR was 639.60 (243.04-1683.18). The area under the SROC curve (AUC) was 0.99.ConclusionsMPT64 exhibits good diagnostic efficiency for MTB. There is no obvious heterogeneity between the three commercial kits.
| S-EPMC8035777 | biostudies-literature
Project description:Improved access to effective tests for diagnosing tuberculosis (TB) has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA) is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC) DNA in <20 minutes at 39 °C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110) and 20 fg (IS1081)were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9) and 86.1% (95%CI: 78.1, 94.1) respectively (n = 71). Specificities were 100% and 88.6% (95% CI: 80.8, 96.1) respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2) and 70.8% (95%CI: 62.9, 78.7) were obtained (n = 90). Specificities were 95.4 (95% CI: 92.3,98.1) and 88% (95% CI: 83.6, 92.4) respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB assays could be of use for integration into a point-of-care test for use in resource constrained settings.
| S-EPMC4138011 | biostudies-literature
Project description:Background: Non-malaria febrile illnesses such as bacterial bloodstream infections (BSI) are a leading cause of disease and mortality in the tropics. However, there are no reliable, simple diagnostic tests for identifying BSI or other severe non-malaria febrile illnesses. We hypothesized that different infectious agents responsible for severe febrile illness would impact on the host metabololome in different ways, and investigated the potential of plasma metabolites for diagnosis of non-malaria febrile illness. </p> Methodology: We conducted a comprehensive mass-spectrometry based metabolomics analysis of the plasma of 61 children with severe febrile illness from a malaria-endemic rural African setting. Metabolite features characteristic for non-malaria febrile illness, BSI, severe anemia and poor clinical outcome were identified by receiver operating curve analysis. </p> Principal Findings: The plasma metabolome profile of malaria and non-malaria patients revealed fundamental differences in host response, including a differential activation of the hypothalamic-pituitary-adrenal axis. A simple corticosteroid signature was a good classifier of severe malaria and non-malaria febrile patients (AUC 0.82, 95% CI: 0.70-0.93). Patients with BSI were characterized by upregulated plasma bile metabolites; a signature of two bile metabolites was estimated to have a sensitivity of 98.1% (95% CI: 80.2-100) and a specificity of 82.9% (95% CI: 54.7-99.9) to detect BSI in children younger than 5 years. This BSI signature demonstrates that host metabolites can have a superior diagnostic sensitivity compared to pathogen-detecting tests to identify infections characterized by low pathogen load such as BSI. </p> Conclusions: This study demonstrates the potential use of plasma metabolites to identify causality in children with severe febrile illness in malaria-endemic settings.
2016-03-17 | MTBLS315 | MetaboLights
Project description:Undifferentiated abdominal pain is a common presentation often requiring immediate medical or surgical intervention. Providing an accurate diagnosis involves a detailed patient history and thorough physical exam. Point of care ultrasound is gaining acceptance as a rapid diagnostic tool that can be used to accurately detect life-threatening conditions while potentially avoiding unnecessary radiation exposure and facilitating rapid treatment. Detection of pneumoperitoneum with point-of-care ultrasound is a simple procedure that relies heavily on the experience of the investigating practitioner. Standard technique involves placing a high-frequency linear-array transducer in the right upper quadrant, where abdominal free air is most likely to accumulate. Detection of the 'gut point', which is the transition of abdominal wall sliding to lack thereof in a single image, is the pathognomonic finding of pneumoperitoneum. If visualization is difficult, moving the patient to the left lateral decubitus position or using the scissors technique can provide additional image views. This representative case report and review highlights the use of abdominal POCUS for the diagnosis of pneumoperitoneum. Ultrasound should continue to be explored by clinicians to narrow the differential diagnosis of acute abdominal pain.
| S-EPMC7721848 | biostudies-literature
Project description:BackgroundTruenat is a novel molecular assay that rapidly detects tuberculosis (TB) and rifampicin-resistance. Due to the portability of its battery-powered testing platform, it may be valuable in peripheral healthcare settings in India.MethodsUsing a microsimulation model, we compared four TB diagnostic strategies for HIV-negative adults with presumptive TB: (1) sputum smear microscopy in designated microscopy centers (DMCs) (SSM); (2) Xpert MTB/RIF in DMCs (Xpert); (3) Truenat in DMCs (Truenat DMC); and (4) Truenat for point-of-care testing in primary healthcare facilities (Truenat POC). We projected life expectancy, costs, incremental cost-effectiveness ratios (ICERs), and 5-year budget impact of deploying Truenat POC in India's public sector. We defined a strategy "cost-effective" if its ICER was <US$990/year-of-life saved (YLS). Model inputs included: TB prevalence, 15% (among those not previously treated for TB) and 27% (among those previously treated for TB); sensitivity for TB detection, 89% (Xpert) and 86% (Truenat); per test cost, $12.63 (Xpert) and $13.20 (Truenat); and linkage-to-care after diagnosis, 84% (DMC) and 95% (POC). We varied these parameters in sensitivity analyses.ResultsCompared to SSM, Truenat POC increased life expectancy by 0.39 years and was cost-effective (ICER $210/YLS). Compared to Xpert, Truenat POC increased life expectancy by 0.08 years due to improved linkage-to-care and was cost-effective (ICER $120/YLS). In sensitivity analysis, the cost-effectiveness of Truenat POC, relative to Xpert, depended on the diagnostic sensitivity of Truenat and linkage-to-care with Truenat. Deploying Truenat POC instead of Xpert increased 5-year expenditures by $270 million, due mostly to treatment costs. Limitations of our study include uncertainty in Truenat's sensitivity for TB and not accounting for the "start-up" costs of implementing Truenat in the field.ConclusionsUsed at the point-of-care in India, Truenat for TB diagnosis should improve linkage-to-care, increase life expectancy, and be cost-effective compared with smear microscopy or Xpert.
| S-EPMC6605662 | biostudies-literature
Project description:There is an urgent need for point-of-care tuberculosis (TB) diagnostic methods that are fast, inexpensive, and operationally simple. Here, we report on a bright solvatochromic dye trehalose conjugate that specifically detects Mycobacterium tuberculosis (Mtb) in minutes. 3-Hydroxychromone (3HC) dyes, known for having high fluorescence quantum yields, exhibit shifts in fluorescence intensity in response to changes in environmental polarity. We synthesized two analogs of 3HC-trehalose conjugates (3HC-2-Tre and 3HC-3-Tre) and determined that 3HC-3-Tre has exceptionally favorable properties for Mtb detection. 3HC-3-Tre-labeled mycobacterial cells displayed a 10-fold increase in fluorescence intensity compared to our previous reports on the dye 4,4-N,N-dimethylaminonapthalimide (DMN-Tre). Excitingly, we detected fluorescent Mtb cells within 10 min of probe treatment. Thus, 3HC-3-Tre permits rapid visualization of mycobacteria that ultimately could empower improved Mtb detection at the point-of-care in low-resource settings.
| S-EPMC8479770 | biostudies-literature