Rapid Diagnostic Tool Research Articles

Rapid detection of Brucella cells using a gold nanoparticle-based aptasensor via a simple colorimetric method.

Brucellosis is a major worldwide zoonotic disease that is caused by Brucella spp. and threatens the health of communities. Novel methods for rapid detection of Brucella bacteria are beneficial and necessary in preventing infection and subsequent economic losses. Constructing biosensors with nanoparticles is a promising approach for identification of pathogenic bacteria in a short time. This study aimed to introduce a new detection method of Brucella cells using a biosensor, based on gold nanoparticles and a specific aptamer, via a colorimetric reaction. In this work, gold nanoparticles (GNPs) were synthesized and attached to the aptamer through electrostatic bonding. The binding of aptamer to gold nanoparticles was confirmed by Uv/vis spectrophotometry, FT-IR, transmission electron microscope (TEM) and zeta sizer (DLS). In the presence of the bacterial cells, aptamers were bound to their targets, and the surfaces of the nanoparticles were depleted from aptamers resulting in intensified peroxidation activity of GNPs, and with the addition of 3, 3', 5, 5'-tetramethylbenzidine (TMB), the color of the solution was changed from red to purple, which indicated the presence of Brucella. The sensitivity of the aptasensor was investigated using different concentrations of Brucella cells and its specificity was confirmed against several species of bacteria. The results showed that the designed aptasensor was more sensitive compared to PCR assay method with the ability to detect 1.5 × 101 CFU/mL of the bacterial cells. These findings indicate that the designed aptasensor can be used as a simple and rapid diagnostic tool to detect Brucella cells without need to experts and expensive laboratory equipment.

A novel, rapid, ultrasensitive diagnosis platform for detecting Candida albicans using restriction endonuclease‐mediated real-time loop-mediated isothermal amplification

IntroductionCandida albicans (C. albicans, CA) is an essential invasive fungus in clinical diagnosis. Although several detection methods exist, none meet the need for early diagnosis. A rapid, sensitive, and specific diagnostic tool is crucial for effective prevention and control of C. albicans infections.MethodsThis study aimed to develop a new, rapid, and ultrasensitive diagnostic tool for C. albicans detection based on restriction endonuclease-mediated real-time loop-mediated isothermal amplification (ERT-LAMP-CA). The ERT-LAMP-CA technology combines LAMP amplification, restriction endonuclease cleavage, and real-time fluorescence detection in a single reaction tube, which can complete a diagnosis of C. albicans in a short time (approximately 1 h).ResultsHerein, we developed the primer sequences required for ERT-LAMP-CA based on the ITS2 gene of C. albicans and found that ERT-LAMP-CA limit of detection was approximately 500 ag/μL genomic DNA and can present negative results for non-C. albicans templates. We tested sputum samples from 64 patients with suspected C. albicans infections to validate ERT-LAMP-CA applicability in clinical sample testing and found that ERT-LAMP-CA was consistent with multiplex PCR-capillary electrophoresis.DiscussionIn conclusion, ERT-LAMP-CA is a rapid, accurate, and sensitive assay with excellent potential for clinical and basic laboratory diagnosis and an efficient screening strategy.

Assessing sepsis-induced immunosuppression to predict positive blood cultures

IntroductionBacteremia is a life-threatening condition that can progress to sepsis and septic shock, leading to significant mortality in the emergency department (ED). The standard diagnostic method, blood culture, is time-consuming and prone to false positives and false negatives. Although not widely accepted, several clinical and artificial intelligence-based algorithms have been recently developed to predict bacteremia. However, these strategies require further identification of new variables to improve their diagnostic accuracy. This study proposes a novel strategy to predict positive blood cultures by assessing sepsis-induced immunosuppression status through endotoxin tolerance assessment.MethodsOptimal assay conditions have been explored and tested in sepsis-suspected patients meeting the Sepsis-3 criteria. Blood samples were collected at ED admission, and endotoxin (lipopolysaccharide, LPS) challenge was performed to evaluate the innate immune response through cytokine profiling.ResultsClinical variables, immune cell population biomarkers, and cytokine levels (tumor necrosis factor [TNFα], IL-1β, IL-6, IL-8, and IL-10) were measured. Patients with positive blood cultures exhibited significantly lower TNFα production after LPS challenge than did those with negative blood cultures. The study also included a validation cohort to confirm that the response was consistent.DiscussionThe results of this study highlight the innate immune system immunosuppression state as a critical parameter for sepsis diagnosis. Notably, the present study identified a reduction in monocyte populations and specific cytokine profiles as potential predictive markers. This study showed that the LPS challenge can be used to effectively distinguish between patients with bloodstream infection leading to sepsis and those whose blood cultures are negative, providinga rapid and reliable diagnostic tool to predict positive blood cultures. The potential applicability of these findings could enhance clinical practice in terms of the accuracy and promptness of sepsis diagnosis in the ED, improving patient outcomes through timely and appropriate treatment.

Development of a Multiplex PCR Assay to Detect Neofusicoccum parvum and Botryosphaeria dothidea in Walnut.

In walnut orchards, frequent symptoms of cankers and dieback (fruit blight, twig and branch cankers up to tree death) are caused by different agents, in particular by Botryosphaeriaceae, primarily Neofusicoccum parvum and Botryosphaeria dothidea. This study aimed at developing a sensitive, rapid, specific and internally controlled multiplex PCR assay for the detection of these species. The ability of the multiplex PCR, with an internal inhibition control (i.e. E. coli DNA), to specifically and successfully detect members of the two targeted species was validated using 11 different isolates for each fungal target (inclusivity) and 20 tree-associated fungal species different from B. dothidea or N. parvum (exclusivity). Applicability to plant materials was further investigated and showed an absence of amplification for asymptomatic husk or twig samples while the amplification profiles of symptomatic tissues ranged from no amplification to amplification of both species, in correlation with the observed fungal contamination level. In conclusion, we developed a rapid diagnostic tool for simultaneous detection of two major fungal pathogens of walnut. Although this protocol was tailored for walnut husks and twigs, applicability to any plant sample contaminated by these pathogens can be considered.

On the Use of Reflection Polarized Optical Microscopy for Rapid Comparison of Crystallinity and Phase Segregation of P3HT:PCBM Thin Films.

Rapid, nondestructive characterization techniques for evaluating the degree of crystallinity and phase segregation of organic semiconductor blend thin films are highly desired for in-line, automated optoelectronic device fabrication facilities. Here, it is demonstrated that reflection polarized optical microscopy (POM), a simple technique capable of imaging local anisotropy of materials, is capable of determining the relative degree of crystallinity and phase segregationof thin films of polymer:fullerene blends. While previous works on POM of organic semiconductors have largely employed the transmission geometry, it is demonstrated that reflection POM provides 3× greater contrast. The optimal configuration is described to maximize contrast from POM images of polymer:fullerene films, which requires Köhler illumination and slightly uncrossed polarizers, with an uncrossing angle of ±3°. It is quantitatively demonstrated that contrast in POM images directly correlates with 1) the degree of polymer crystallinity and 2) the degree of phase segregation between polymer and fullerene domains. The origin of the bright and dark domains in POM is identified as arising from symmetry-broken liquid crystalline phases (i.e., dark conglomerates), and it is proven that they have no correlation with surface topography. The use of reflection POM as a rapid diagnostic tool for automated device fabrication facilities is discussed.

Circular DNA enhanced amplification-free CRISPR/Cas12a assays for end-user friendly ultra-sensitive Porphyromonas gingivalis diagnosis

Periodontitis not only leads to tooth loss but is also associated with various systemic symptoms. Porphyromonas gingivalis (Pg) is the microorganism most closely linked to periodontitis. A rapid and accurate Pg detection method holds significant potential health benefits for patients. While several nucleic acid amplification-based diagnostic methods have been developed, there is a current lack of rapid diagnostic tools for Pg, particularly at chair-side level. Here, two detection methods for the Pg 16S gene based on the CRISPR/Cas12a system have been developed. To achieve ultra-high sensitivity in the developed CRISPR/Cas12a assays without pre-amplification, we designed a circular DNA molecule (termed as Cir-amplifier) which amplifies the signal output of the Cas12a reactions in a cascade response manner. The Cir-amplifier enhanced Fluorescence-Cas12a reaction achieved a minimum detectable limit of 0.39 CFU of Pg within 80-minute under room-temperature. In addition, the Cir-amplifier enhanced Lateral Flow Detection (LFD)-Cas12a reaction has demonstrated the ability to detect a minimum of 3.9 CFU of Pg in 90 min, with results observable by the naked eye. The potential of the two methods for Pg detection in clinical samples has been demonstrated, showing 100 % sensitivity compared to the traditional real-time PCR method. In summary, we have established two efficient detection methods for Pg that eliminate the need for pre-amplification, hence no amplicon contaminations, making them promising candidates for chair-side Pg diagnosis. Furthermore, the Cir-amplifier has been demonstrated to be adaptable to CRISPR/Cas systems producing various signal formats. The Cir-amplifier is expected to become a universal CRISPR/Cas enhancer.

Feasibility study in quantitative identification of fresh and old osteoporotic vertebral compression fracture by vertebral CT value and CT value difference

BackgroundThis study aims to assess the diagnostic utility of vertebral CT value and CT value difference in distinguishing between fresh and old osteoporotic vertebral compression fractures (OVCF).MethodsA retrospective analysis was conducted on 118 patients with OVCF who underwent both MRI and CT examinations at our hospital. The nature of the fractured vertebra was determined according to MRI. The CT value of the fractured vertebrae and the mean CT value of the adjacent normal vertebrae were measured separately, and the differences between these values were calculated. Independent samples t-tests were used to compare CT value and CT value difference among all groups. The receiver operating characteristic (ROC) curve was employed to determine the optimal cut-off value for both CT value and CT value difference in differentiating fresh and old fractures.ResultsThe study included a total of 163 fractured vertebrae from 118 patients. The CT value of fresh fractured vertebrae was significantly higher than those of adjacent normal vertebrae, which was statistically different (P < 0.001). In contrast, the difference between CT value of old fractured vertebrae and those of adjacent normal vertebrae was not statistically significant (P > 0.05). There were significant differences in CT value and CT value difference between fresh fractured vertebrae and old fractured vertebrae (P < 0.001). The ROC curve analysis showed that the optimal cut-off value of CT value for fresh fractures and old fractures was 103.40 HU. The optimal cut-off value of CT value difference was 39.81 HU.ConclusionVertebral CT value and CT value difference offer a certain reference value for distinguishing between fresh and old OVCF. These parameters can serve as a rapid diagnostic tool when MRI is unavailable or impractical, aiding in the timely assessment of OVCF.

A Glycopolymer Sensor Array That Differentiates Lectins and Bacteria.

Identification of bacterial lectins offers an attractive route to the development of new diagnostics, but the design of specific sensors is complicated by the low selectivity of carbohydrate-lectin interactions. Here we describe a glycopolymer-based sensor array which can identify a selection of lectins with similar carbohydrate recognition preferences through a pattern-based approach. Receptors were generated using a polymer scaffold functionalized with an environmentally sensitive fluorophore, along with simple carbohydrate motifs. Exposure to lectins induced changes in the emission profiles of the receptors, enabling the discrimination of analytes using linear discriminant analysis. The resultant algorithm was used for lectin identification across a range of concentrations and within complex mixtures of proteins. The sensor array was shown to discriminate different strains of pathogenic bacteria, demonstrating its potential application as a rapid diagnostic tool to characterize bacterial infections and identify bacterial virulence factors such as production of adhesins and antibiotic resistance.

Advances in thyroid peroxidase (TPO) and thyroid stimulating hormone receptor (TSHR) biomarkers for autoimmune thyroid diseases

The development of immunological methods in the 1970s and 1980s led to the identification of thyroid peroxidase (TPO) and thyroid stimulating hormone receptor (TSHR) as key players in the onset of autoimmune thyroid diseases (AITD). TSHR is primarily associated with hyperthyroidism, while TPO is linked to hypothyroidism. Advances in understanding TPO and TSHR as biomarkers over recent decades have enhanced their clinical application. This review explores the molecular basis of TPO and TSHR as biomarkers, highlighting their importance in diagnosing AITD and their potential to guide effective therapeutic strategies. Additionally, it discusses the development of lateral flow assay (LFA) kits developed by the authors, which show promise as rapid and reliable diagnostic tools for AITD.

Detection Methods for Pine Wilt Disease: A Comprehensive Review.

Pine wilt disease (PWD), caused by the nematode Bursaphelenchus xylophilus, is a highly destructive forest disease that necessitates rapid and precise identification for effective management and control. This study evaluates various detection methods for PWD, including morphological diagnosis, molecular techniques, and remote sensing. While traditional methods are economical, they are limited by their inability to detect subtle or early changes and require considerable time and expertise. To overcome these challenges, this study emphasizes advanced molecular approaches such as real-time polymerase chain reaction (RT-PCR), droplet digital PCR (ddPCR), and loop-mediated isothermal amplification (LAMP) coupled with CRISPR/Cas12a, which offer fast and accurate pathogen detection. Additionally, DNA barcoding and microarrays facilitate species identification, and proteomics can provide insights into infection-specific protein signatures. The study also highlights remote sensing technologies, including satellite imagery and unmanned aerial vehicle (UAV)-based hyperspectral analysis, for their capability to monitor PWD by detecting asymptomatic diseases through changes in the spectral signatures of trees. Future research should focus on combining traditional and innovative techniques, refining visual inspection processes, developing rapid and portable diagnostic tools for field application, and exploring the potential of volatile organic compound analysis and machine learning algorithms for early disease detection. Integrating diverse methods and adopting innovative technologies are crucial to effectively control this lethal forest disease.

MALDI-TOF MS profiling to predict resistance or biofilm production in gram-positive ESKAPE pathogens from healthcare-associated infections

Antimicrobial resistance and biofilm production in healthcare-associated infections is a health issue worldwide. This study aimed to identify potential biomarker peaks for resistance or biofilm production in ESKAPE pathogens using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility and biofilm production were assessed on selected isolates. Biomarker peaks were identified using MALDI Biotyper and ClinProTools software. Among resistant strains, 90.0 % were carbapenem-resistant Acinetobacter baumannii (CRAB), 39.0 % were methicillin-resistant Staphylococcus aureus (MRSA), 17.98 % were multidrug-resistant (MDR) Pseudomonas aeruginosa, 21.6 % were vancomycinresistant Enterococcus (VRE), and 2.55 % were carbapenem-resistant Enterobacterales (CRE). Biofilm production was 40.0 % in VRE and 45.8 % in MRSA. Although no potential biomarker peaks for biofilm production were detected, several potential biomarker peaks for drug resistance in VRE (n=5), MRSA (n=4), and MDR P. aeruginosa (n=4) were detected, suggesting avenues for the development of rapid diagnostic tools.

Recent advances in surface plasmon resonance for the detection of ovarian cancer biomarkers: a thorough review.

Early detection of ovarian cancer (OC) is crucial for effective management and treatment, as well as reducing mortality rates. However, the current diagnostic methods for OC are time-consuming and have low accuracy. Surface plasmon resonance (SPR) biosensors offer a promising alternative to conventional techniques, as they enable rapid and less invasive screening of various circulating indicators. These biosensors are widely used for biomolecular interaction analysis and detecting tumor markers, and they are currently being investigated as a rapid diagnostic tool for early-stage cancer detection. Our main focus is on the fundamental concepts and performance characteristics of SPR biosensors. We also discuss the latest advancements in SPR biosensors that enhance their sensitivity and enable high-throughput quantification of OC biomarkers, including CA125, HE4, CEA, and CA19-9. Finally, we address the future challenges that need to be overcome to advance SPR biosensors from research to clinical applications. The ultimate goal is to facilitate the translation of SPR biosensors into routine clinical practice for the early detection and management of OC.

Utility of automated CT perfusion software in acute ischemic stroke with large and medium vessel occlusion.

Early diagnosis of large vessel occlusion (LVO) in acute stroke often requires CT angiography (CTA). Automated CT perfusion (CTP) software, which identifies blood flow abnormalities, enhances LVO diagnosis and patient selection for endovascular thrombectomy (EVT). This study evaluates the sensitivity of automated CTP images in detecting perfusion abnormalities in patients with acute ischemic stroke (AIS) and LVO or medium vessel occlusion (MeVO), compared to CTA. We screened acute ischemic stroke patients presenting within 24 h who underwent CT, CTA, and CTP as per institutional protocol. RAPID AI software processed CTP images, while neuroradiologists reviewed CTA for intracranial arterial occlusions. Sensitivity, specificity, and accuracy of automated CTP maps in detecting occlusions were assessed. Of 790 screened patients, 31 were excluded due to lack of RAPID CTP data or poor-quality scans, leaving 759 for analysis. The median age was 71 years (IQR: 61-81), with 47% female. Among them, 678 had AIS, and 81 had AIS ruled out. CTA identified arterial occlusion in 562 patients (74%), with corresponding CTP abnormalities in 537 patients (Tmax > 6 sec). In the 197 without occlusion, CTP was negative in 161. Automated CTP maps had a sensitivity of 95.55% (CI 95: 93.50-97.10%), specificity of 81.73% (CI 95: 75.61-86.86%), negative predictive value of 98.22% (CI 95: 97.39-98.79%), positive predictive value of 63.54% (CI 95: 56.46-70.09%), and overall accuracy of 85.18% (CI 95: 82.45-87.64%). Automated CTP maps demonstrated high sensitivity and negative predictive value for LVOs and MeVOs, suggesting their usefulness as a rapid diagnostic tool, especially in settings without expert neuroradiologists.

Scabies.

Scabies is one of the most common and highest-burden skin diseases globally. Estimates suggest that >200 million people worldwide have scabies at any one time, with an annual prevalence of 455 million people, with children in impoverished and overcrowded settings being the most affected. Scabies infection is highly contagious and leads to considerable morbidity. Secondary bacterial infections are common and can cause severe health complications, including sepsis or necrotizing soft-tissue infection, renal damage and rheumatic heart disease. There is no vaccine or preventive treatment against scabies and, for the past 30 years, only few broad-spectrum antiparasitic drugs (mainly topical permethrin and oral ivermectin) have been widely available. Treatment failure is common because drugs have short half-lives and do not kill all developmental stages of the scabies parasite. At least two consecutive treatments are needed, which is difficult to achieve in resource-poor and itinerant populations. Another key issue is the lack of a practical, rapid, cheap and accurate diagnostic tool for the timely detection of scabies, which could prevent the cycle of exacerbation and disease persistence in communities. Scabies control will require a multifaceted approach, aided by improved diagnostics and surveillance, new treatments, and increased public awareness.

Diagnostic Accuracy of the LabTurbo QuadAIO Common Flu Assay for Detecting Influenza A Virus, Influenza B Virus, RSV, and SARS-CoV-2.

Background: The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for rapid and accurate diagnostic tools for upper respiratory tract infections (URTIs). Nucleic acid amplification tests (NAATs) have transformed URTI diagnostics by enabling the rapid detection of multiple pathogens simultaneously, thereby improving patient management and infection control. This study aimed to evaluate the diagnostic accuracy of the LabTurbo QuadAIO Common Flu Assay compared to that of the Xpert Xpress CoV-2/Flu/RSV Plus Assay for detecting SARS-CoV-2, Influenza A, Influenza B, and respiratory syncytial virus (RSV). Methods: A retrospective diagnostic accuracy study was conducted using nasopharyngeal samples from patients. Samples were tested using the LabTurbo QuadAIO Common Flu Assay and the comparator Xpert Xpress CoV-2/Flu/RSV Plus Assay. Positive and negative percent agreements (PPA and NPA) were calculated. Results: The LabTurbo Assay demonstrated a PPA of 100% and an NPA of 100% for SARS-CoV-2, Influenza A, and Influenza B, whereas it showed a PPA of 100% and an NPA of 98.3% for RSV. Conclusions: The LabTurbo QuadAIO Assay exhibited high diagnostic accuracy for detecting multiple respiratory pathogens, including SARS-CoV-2, Influenza A, Influenza B, and RSV. Despite the slight discrepancy in the NPA for RSV, the overall performance of the LabTurbo Assay supports its integration into routine diagnostic workflows to enhance patient management and infection control.

A-102 Clinical Evaluation of Novel Markers in Brain Injury

Abstract Background Traumatic brain injury (TBI) is a significant health concern affecting individuals of all ages, demanding precise diagnostic methodologies to guide effective clinical management. According to the 2010 definition, a TBI results from an external force causing an alteration in brain function or other evidence of brain pathology. Current diagnostic tools, i.e. CT scans and the Glasgow Coma Scale (GCS), are inefficient because they are time and labor intensive. The need for rapid diagnostic tools is underscored by the drawbacks of existing methods, such as CT scans, which are expensive, expose patients to large amounts of radiation, and lead to delayed treatment. Recent advances in TBI diagnosis involve novel biomarkers, such as Glial Fibrillary Acidic Protein (GFAP) and Ubiquitin C-Terminal Hydrolase L1 (UCH-L1), which have shown promise in revolutionizing the assessment of head traumas. GFAP, a glial-specific protein, is elevated in circulation after a TBI, distinguishing it from multi-trauma patients without brain injuries. UCH-L1, found in neurons, is significantly elevated in TBI patients compared to uninjured controls. A 2021 study further supports the clinical effectiveness of these biomarkers, demonstrating their capacity to exclude CT-positive injury in acute TBI cases. The primary aim of this study is to explore the clinical utility of the Abbott Alinity TBI assay, specifically biomarkers GFAP and UCH-L1, in emergency department patients with a history of head trauma. Methods A retrospective study involving 238 emergency department patients with a history of head trauma and noncontrast CT scans was conducted using the Abbott Alinity TBI assay. Serum samples were collected, and biomarker values were established and compared to CT scan results. The study was comprised of 94 females and 144 males spanning various age groups. The GFAP and UCH-L1 cutoffs used were FDA approved, at 35.0 ng/L and 400.0 ng/L, respectively. If either marker was above the cutoff the patient ruled at risk for TBI. Results Results demonstrated the assay correctly identified all CT-positive cases (100% sensitivity), placing them in the rule-in group, while identifying 44 patients with a 0 chance of TBI. The negative predictive value for the TBI overall was 100% (95% CI: 91.96%-100%). The GFAP assay alone identified 30/31 positive CT scans, reporting a 98.77% (95% CI: 92.03%-99.82%) negative predictive value. The UCH-L1 assay reported 26/31 positive CT scans with a negative predictive value of 94.68% (95% CI: 88.71-97.58%). Analyzing the data revealed the TBI assay would have reduced CT scans by 18.5%. Conclusions The results of this study did show a potential reduction in the number of patients sent to CT making it a valuable tool for triaging patients presenting with head trauma; however, the complexity of neurological problems may lead to increased false positives, reducing assay specificity. Despite this limitation, this study concludes that the GFAP and UCH-L1 markers could be used to triage patients into CT rule-out and rule-in groups, thereby streamlining clinical management. In conclusion, this assay, incorporating GFAP and UCH-L1 biomarkers, presents as a promising diagnostic tool for emergency departments.

Gastroenteritis: A Comprehensive Review

Gastroenteritis, commonly referred to as stomach flu, is an acute inflammation of the gastrointestinal tract, marked by symptoms including diarrhea, vomiting, abdominal cramps, and fever. This review article provides a comprehensive overview of gastroenteritis, addressing its etiology, epidemiology, pathophysiology, diagnosis, management, and prevention strategies. The condition is caused by a variety of infectious agents such as viruses (noroviruses, rotaviruses), bacteria (Campylobacter, Salmonella), and parasites (Giardia lamblia), with transmission typically occurring through contaminated food, water, or person-to-person contact. Globally, gastroenteritis remains a significant public health issue, with high morbidity and mortality rates, particularly in children under five in developing countries. Diagnosis often relies on clinical evaluation and laboratory tests, while management focuses on rehydration therapy and symptomatic relief. Preventive measures include personal hygiene, food safety practices, environmental sanitation, and vaccination, with rotavirus vaccines significantly reducing severe cases in children. Emerging trends in gastroenteritis research aim at developing rapid diagnostic tools, novel therapeutic approaches, and new vaccines, highlighting the importance of a multidisciplinary approach to mitigate the global impact of this disease.

Development and validation of a new and rapid molecular diagnostic tool based on RT-LAMP for Hepatitis C virus detection at point-of-care

PurposeGlobally, it is estimated that 1.0 million individuals are newly infected by Hepatitis C virus (HCV) every year, and nearly 50 million people live with a chronic infection, according to World Health Organization. To overcome underdiagnosis of HCV infection among hard-to-reach populations, it is essential to develop new rapid and easy-to-use molecular diagnostic systems. In this work, we have developed a pangenotypic diagnostic tool based on Loop-Mediated Isothermal Amplification (LAMP), coupled to a direct sample lysis procedure for molecular detection of HCV at point-of-care (POC). MethodsProcedure validation was performed using 129 different samples from HCV infected patients (116 serum samples, and 13 fresh blood samples), 27 individuals who tested negative for HCV but positive for HIV, and 11 healthy donors. Serum was collected, lysed for 10 min at room temperature, and assayed by RT-LAMP. To achieve this, a set of 9 LAMP-primers was used for the first time. Parallel RT-qPCR assays were conducted for HCV to both validate the procedure and quantify viral loads. ResultsHCV was detected by RT-LAMP in 109/116 HCV positive serum samples, and in 11/13 positive blood samples in less than 40 min. Compared to RT-qPCR results, our RT-LAMP procedure showed a sensitivity of 94 %, 100 % specificity, and a limit of detection of 3.26 log10 IU/mL (10–20 copies per reaction). ConclusionsWe have developed an accurate system, more affordable than the current available rapid tests for HCV. Since no prior RNA purification step from capillary blood is required, we strongly recommend our RT-LAMP system as a valuable and rapid tool for the molecular detection of HCV at POC.

Development of an ultrasensitive electrochemical aptasensor based on aptamer/rGO/SPGE for the detection of Botulinum neurotoxin A (BoTN/A) in food samples

Consumption of Botulinum neurotoxin A (BoTN/A) contaminated foods has increased the public health issues which necessitates the development of rapid and simple diagnostic tools for their sensitive detection. Electrochemical aptasensors can replace existing biosensors and traditional methods of BoTN/A detection in foods. These aptasensors are simple, quick response, economic, precise, sensitive, accurate and reproducible. In the present study, an electrochemical aptasensor was constructed by immobilizing BoTN/A specific aptamer and reduced graphene oxide (rGO) onto screen printed gold electrode (SPGE) for the detection of BoTN/A in spiked food samples. The specificity of this aptasensor was achieved by conjugating BoTN/A specific aptamer on rGO/SPGE. Test sample containing BoTN/A form aptamer-BoTN/A complex onto SPGE which was directly proportional to BoTN/A concentration. Formation of aptamer-BoTN/A complex reduced the surface of working SPGE for redox reactions. Thus decreased the current peaks that was taken as measuring signal of BoTN/A in test food sample. BoTN/A aptasensor showed optimum response at pH 6.5 and 30 °C temperature. The responsetime of BoTN/A/aptamer/rGO/SPGE was 6 s. In optimum conditions aptamer/rGO/SPGE BoTN/A aptasensor exhibited wide linearity ranges from 1-100 pM and 1 pM limit of detection (LOD). The analytical recoveries of aptamer/rGO/SPGE BoTN/A aptasensor at 5 pM and 10 pM BoTN/A were 98.5 % and 99.8 % respectively. The proposed aptasensor showed unique sensitivity, reproducibility and accuracy. Moreover, aptamer/rGO/SPGE aptasensor was used for the detection of BoTN/A in five different types of food samples.

Trichomonas vaginalis: comparison of primers for implementation as an in-house PCR in rural Vellore, South India

BackgroundTrichomonas vaginalis (TV) accounts for the highest burden of curable, non-viral sexually transmitted infections worldwide. Prevalence in India ranges from 0.4 to 27.4% in women and 0.0-5.6% in men. In 2015, the prevalence of TV among pregnant women of rural Vellore was 3.11% using Sekisui OSOM® Trichomonas test and culture methods. Molecular methods are the most sensitive, rapid diagnostic tool for Sexually Transmitted Infection’s (STI) albeit cost hinders implementation of commercial platforms. To determine a sensitive, sustainable molecular method, we compared three targets (Adhesin AP65, cytoskeleton Beta-tubulin BTUB 9/2 and TVK 3/7) with the highest published diagnostic accuracy against microscopy, culture and Real Time PCR (RT- PCR).Materials & methodsSix-hundred adult, sexually active women attending the Obstetrics-Gynaecology rural out-patient clinic the Rural Unit for Health and Social Affairs (RUHSA) from July 2020 - February 2021 were enrolled. A vaginal lateral and posterior fornix specimen was inoculated, onsite, into Biomed InPouch® TV culture and smeared onto a slide for fluorescence microscopy using Acridine orange. A flocked nylon swab specimen for PCR was used to determine the sensitivities of the Adhesin AP65, cytoskeleton Beta-tubulin BTUB 9/2 and TVK 3/7 gene targets. Seegene Allplex™ STI Essential Assay, S.Korea was used to confirm TV positives.ResultsNine specimens (9/600, 1.5%) were positive for TV. There was a 100% correlation between Biomed InPouch TV® culture, PCR with TVK 3/7 and RT-PCR while a correlation of 66.6% with BTUB 9/2 and AP65 gene targets. Clinically, 77.7% (n = 7) presented with white-greenish discharge per vagina, 11% (n = 1) with infertility, 22.2% (n = 2) were asymptomatic. Eight of nine patients (88.9%) had co-infections with other bacterial STIs. Prevalence of TV coinfection with Neisseria gonorrhoea was 1.1%.ConclusionCurrent hospital-based prevalence of TV in rural Vellore was 1.5%. Repetitive DNA target TVK 3/7 was more sensitive than AP65 and BTUB 9/2 primers.