The Evolution of Nucleic Acid-Based Diagnosis Methods from the (pre-)CRISPR to CRISPR era and the Associated Machine/Deep Learning Approaches in Relevant RNA Design.

Nucleic acid tests (NATs) are considered as gold standard in molecular diagnosis. To meet the demand for onsite, point-of-care, specific and sensitive, trace and genotype detection of pathogens and pathogenic variants, various types of NATs have been developed since the discovery of PCR. As alternatives to traditional NATs (e.g., PCR), isothermal nucleic acid amplification techniques (INAATs) such as LAMP, RPA, SDA, HDR, NASBA, and HCA were invented gradually. PCR and most of these techniques highly depend on efficient and optimal primer and probe design to deliver accurate and specific results. This chapter starts with a discussion of traditional NATs and INAATs in concert with the description of computational tools available to aid the process of primer/probe design for NATs and INAATs. Besides briefly covering nanoparticles-assisted NATs, a more comprehensive presentation is given on the role CRISPR-based technologies have played in molecular diagnosis. Here we provide examples of a few groundbreaking CRISPR assays that have been developed to counter epidemics and pandemics and outline CRISPR biology, highlighting the role of CRISPR guide RNA and its design in any successful CRISPR-based application. In this respect, we tabularize computational tools that are available to aid the design of guide RNAs in CRISPR-based applications. In the second part of our chapter, we discuss machine learning (ML)- and deep learning (DL)-based computational approaches that facilitate the design of efficient primer and probe for NATs/INAATs and guide RNAs for CRISPR-based applications. Given the role of microRNA (miRNAs) as potential future biomarkers of disease diagnosis, we have also discussed ML/DL-based computational approaches for miRNA-target predictions. Our chapter presents the evolution of nucleic acid-based diagnosis techniques from PCR and INAATs to more advanced CRISPR/Cas-based methodologies in concert with the evolution of deep learning (DL)- and machine learning (ml)-based computational tools in the most relevant application domains.

Weight of Evidence: Criteria and Applications.

Toxicologists and authorities evaluate substances that in the traditional way refer to data and knowledge on the toxic mechanism. Non-testing methods (NTMs) proved to be a valuable resource for risk assessment of chemical substances. Indeed, they can be particularly useful when the information provided by different sources is integrated to increase confidence in the result. In this chapter, we will address data obtained from NTM, including in silico models and read-across. Typically, these two approaches are used separately, but in this way, we lose some information. The integration of different results can be sometimes difficult because different methods can lead to conflicting results and because a clear guideline for integrating information from different sources was not available in the recent past. In this chapter, we present and discuss the recently published guideline from EFSA for integrating and weighting evidence for scientific assessment, and how to proceed, manually or through novel integrated tool, SWAN. Moreover, practical examples of the application of these integration principles on evidence from different in silico models are shown. These examples represent a demonstration of the suitability and effectiveness of in silico methods for risk assessment, as well as a practical guide to end users to perform similar analyses on likely hazardous chemicals.

Toxicity Potential of Nutraceuticals.

During the past few decades and especially during and after the COVID-19 pandemic, the use of nutraceuticals has become increasingly popular in both humans and animals due to their easy access, cost-effectiveness, and tolerability with a wide margin of safety. While some nutraceuticals are safe, others have an inherent toxic potential. For a large number of nutraceuticals, no toxicity/safety data are available due to a lack of pharmacological/toxicological studies. The safety of some nutraceuticals can be compromised via contamination with toxic plants, metals, mycotoxins, pesticides, fertilizers, drugs of abuse, etc. Knowledge of pharmacokinetic/toxicokinetic studies and biomarkers of exposure, effect, and susceptibility appears to play a pivotal role in the safety and toxicity assessment of nutraceuticals. Interaction studies are essential to determine efficacy, safety, and toxicity when nutraceuticals and therapeutic drugs are used concomitantly or when polypharmacy is involved. This chapter describes various aspects of nutraceuticals, particularly their toxic potential, and the factors that influence their safety.

The role of respiratory function tests in infants with stridor: diagnosis at glance and follow-up.

BACKGROUND: Recently, the development of advanced, noninvasive methods has allowed the study of respiratory function even in uncooperative infants. To date, there is still little data on the application of this technique in infants with suspected airway obstruction. THE AIMS OF OUR STUDY WERE: - To evaluate the role of respiratory function testing (PFR) in the diagnosis and follow-up of infants with stridor - To evaluate the differences between patients with inspiratory stridor and expiratory stridor. - To evaluate the concordance between PFR and endoscopy. METHODS: We enrolled infants aged < 1 year with a diagnosis of inspiratory and/or expiratory chronic stridor and a group of healthy controls. For each patient we performed PFR at diagnosis (T0) and for cases at follow-up, at 3 months (T1), 6 months (T2), 12 months (T3). At T0, all patients were classified according to a clinical score, and at follow-up, stature-ponderal growth was assessed. When clinically indicated, patients underwent bronchoscopy. RESULTS: We enrolled 48 cases (42 diagnosed with inspiratory stridor and 6 expiratory stridor) and 26 healthy controls. At T0, patients with stridor had increased inspiratory time (p < 0.0001) and expiratory time (p < 0.001) than healthy controls and abnormal curve morphology depending on the type of stridor. At T0, patients with expiratory stridor had a reduced Peak expiratory flow (p < 0.023) and a longer expiratory time (p < 0.004) than patients with inspiratory stridor. We showed an excellent concordance between PFR and endoscopic examination (k = 0.885, p < 0.0001). At follow-up, we showed a progressive increase of the respiratory parameters in line with the growth. CONCLUSIONS: PFR could help improve the management of these patients through rapid and noninvasive diagnosis, careful monitoring, and early detection of those most at risk.

Diagnostic value for methylation in cervical cancer based on a small-molecule fluorescent probe targeting DNMT1.

BACKGROUND: Value analysis of a small-molecule fluorescent probe for methylation detection in different cervical lesions. MATERIALS AND METHODS: (1) The grayscale values of distinct lesion tissues were remarkably distinct among the four groups (p < 0.05). The comparison of the grayscale value between the two groups showed that the CA group noticeably exceeded the LSIL and cervicitis groups, and the HSIL group was apparently higher than the LSIL and cervicitis groups (p < 0.05); (2) The mean grayscale values of the enrolled subjects were calculated with 55.21 as the midline, with >55.21 as positive and ≤55.21 as negative. RESULTS: The results showed that the positive rate of the cervicitis group was 0.00%, the LSIL group 67.74%, the HSIL group 83.33%, and the CA group 100.00%. The results among the four groups were notably distinct (p < 0.05); (3) The comparison among DAPI, probe, bright, and merged images of cervicitis, LSIL, HSIL, and CA indicated that different cervical lesions were with quite various stains. CONCLUSION: The grayscale value, positive rate, and stained picture of distinct cervical lesions were remarkably different. The small-molecule fluorescent probe has a good value in differentiating cervical lesions and can be considered for popularization and application.

Parent and patient knowledge and attitudes about cancer predisposition syndrome genetic testing in pediatric oncology: Understanding sociodemographic and parent-child differences.

BACKGROUND: Cancer predisposition syndromes (CPS) impact about 10% of patients with pediatric cancer. Genetic testing (CPS-GT) has multiple benefits, but few studies have described parent and child knowledge and attitudes regarding CPS-GT decision-making. This study examined parent and patient CPS-GT decision-making knowledge and attitudes. PROCEDURE: English- or Spanish-speaking parents of children with pediatric cancer and patients with pediatric cancer ages 15-18 within 12 months of diagnosis or relapse were eligible to participate. Seventy-five parents and 19 parent-patient dyads (N = 94 parents, 77.7% female, 43.6% Latino/a/Hispanic; 19 patients, 31.6% female) completed surveys measuring CPS-GT-related beliefs. Independent samples t-tests compared parent responses across sociodemographic characteristics and parent-patient responses within dyads. RESULTS: Spanish-speaking parents were significantly more likely than English-speaking parents to believe that CPS-GT not being helpful (p < .001) and possibly causing personal distress (p = .002) were important considerations for deciding whether to obtain CPS-GT. Parents with less than four-year university education, income less than $75,000, or Medicaid (vs. private insurance) were significantly more likely to endorse that CPS-GT not being helpful was an important consideration for deciding whether to obtain CPS-GT (p < .001). Parents felt more strongly than patients that they understood what CPS-GT was (p = .01) and that parents should decide whether patients under 18 should receive CPS-GT (p = .002). CONCLUSIONS: Spanish-speaking parents and parents with lower socioeconomic statuses were more strongly influenced by the potential disadvantages of CPS-GT in CPS-GT decision-making. Parents felt more strongly than patients that parents should make CPS-GT decisions. Future studies should investigate mechanisms behind these differences and how to best support CPS-GT knowledge and decision-making.

Stem cell-based approaches for developmental neurotoxicity testing.

Neurotoxicants are substances that can lead to adverse structural or functional effects on the nervous system. These can be chemical, biological, or physical agents that can cross the blood brain barrier to damage neurons or interfere with complex interactions between the nervous system and other organs. With concerns regarding social policy, public health, and medicine, there is a need to ensure rigorous testing for neurotoxicity. While the most common neurotoxicity tests involve using animal models, a shift towards stem cell-based platforms can potentially provide a more biologically accurate alternative in both clinical and pharmaceutical research. With this in mind, the objective of this article is to review both current technologies and recent advancements in evaluating neurotoxicants using stem cell-based approaches, with an emphasis on developmental neurotoxicants (DNTs) as these have the most potential to lead to irreversible critical damage on brain function. In the next section, attempts to develop novel predictive model approaches for the study of both neural cell fate and developmental neurotoxicity are discussed. Finally, this article concludes with a discussion of the future use of in silico methods within developmental neurotoxicity testing, and the role of regulatory bodies in promoting advancements within the space.

Physical and psychological distress amongst patients undergoing neoadjuvant chemotherapy for advanced ovarian cancer.

OBJECTIVE: This study compares baseline clinical characteristics, physical function testing, and patient-reported outcomes for patients undergoing primary cytoreductive surgery versus neoadjuvant chemotherapy, with the goal of better understanding unique patient needs at diagnosis. METHODS: Patients with suspected advanced stage (IIIC/IV) epithelial ovarian cancer undergoing either primary cytoreductive surgery or neoadjuvant chemotherapy were enrolled in a single-institution, non-randomized prospective behavioral intervention trial of prehabilitation. Baseline clinical characteristics were abstracted. Physical function was evaluated using the Short Physical Performance Battery, Fried Frailty Index, gait speed, and grip strength. Patient-reported outcomes were evaluated using Patient-Reported Outcomes Measurement Information System metrics and the Perceived Stress Scale. RESULTS: There were no significant differences in demographics or clinical characteristics between cohorts at enrollment, with the exception of performance status, clinical stage, and albumin. While gait speed and grip strength were lower amongst neoadjuvant chemotherapy patients, there were no significant differences in physical function using the Short Physical Performance Battery and Fried Frailty Index. Patients in the neoadjuvant chemotherapy cohort reported decreased perception of physical function and increased fatigue on Patient-Reported Outcomes Measurement Information System metrics. A larger proportion of patients in the neoadjuvant cohort reported severe levels of emotional distress and anxiety, as well as greater perceived stress at diagnosis. CONCLUSIONS: Our findings suggest that patients undergoing neoadjuvant chemotherapy for advanced ovarian cancer present with increased psychosocial distress and decreased perception of physical function at diagnosis and may benefit most from early introduction of supportive care.

Clinical Impact of Constitutional Genomic Testing on Current Breast Cancer Care.

The most commonly diagnosed cancer in women worldwide is cancer of the breast. Up to 20% of familial cases are attributable to pathogenic mutations in high-penetrance (BReast CAncer gene 1 [BRCA1], BRCA2, tumor protein p53 [TP53], partner and localizer of breast cancer 2 [PALB2]) or moderate-penetrance (checkpoint kinase 2 [CHEK2], Ataxia-telangiectasia mutated [ATM], RAD51C, RAD51D) breast-cancer-predisposing genes. Most of the breast-cancer-predisposing genes are involved in DNA damage repair via homologous recombination pathways. Understanding these pathways can facilitate the development of risk-reducing and therapeutic strategies. The number of breast cancer patients undergoing testing for pathogenic mutations in these genes is rapidly increasing due to various factors. Advances in multigene panel testing have led to increased detection of pathogenic mutation carriers at high risk for developing breast cancer and contralateral breast cancer. However, the lack of long-term clinical outcome data and incomplete understanding of variants, particularly for moderate-risk genes limits clinical application. In this review, we have summarized the key functions, risks, and prognosis of breast-cancer-predisposing genes listed in the National Health Service (NHS) England National Genomic Test Directory for inherited breast cancer and provide an update on current management implications including surgery, radiotherapy, systemic treatments, and post-treatment surveillance.

Enhancing CRC Screening in a Predominantly Hispanic Community: Effectiveness of 1-Day vs. 3-Day Stool-Based Testing Kits.

Colorectal cancer (CRC) is the leading cause of cancer-related mortality among U.S. Hispanics, with screening proven to decrease both incidence and mortality. Despite rising CRC screening rates in the U.S., Hispanic participation remains disproportionately low. Stool-based tests, particularly popular for reaching underserved populations, may enhance screening adherence. This study evaluates the performance of a 1-day versus a 3-day stool-based testing kit in improving screening completion rates and reducing the need for reminder calls in a Hispanic community along the U.S.-Mexico border. In our quasi-experimental observational study, participants aged 45-75 years who were uninsured or underinsured and overdue for CRC screening were recruited. They received colorectal cancer education and no-cost stool-based screening facilitated by promotoras. Participants were randomly assigned to receive a 1-day or 3-day Fecal Immunochemical Test (FIT) kit. The promotoras swapped FIT kit distribution roles midway through the study period to mitigate performance bias. Our analysis covered 6,660 FITs-3,067 using the 3-day kit and 3,593 with the 1-day kit. Results indicated a higher return rate for the 1-day FIT kit (61.3% vs. 58.7%, adjusted odds ratio [aOR] = 1.22, p < 0.001), fewer reminders needed (69.7% vs. 78.1%, aOR = 0.65, p < 0.001), and lower abnormal FIT results (5.3% vs. 8.1%, aOR = 0.61, p < 0.001). Conclusively, the 1-day FIT kit required fewer reminders and significantly improved return rates, suggesting it may be a more effective option for increasing CRC screening completion among hard-to-reach Hispanic populations.

Non-coding variants are a rare cause of recessive developmental disorders in trans with coding variants.

PURPOSE: Identifying pathogenic non-coding variants is challenging. A single protein-altering variant is often identified in a recessive gene in individuals with developmental disorders (DD), but the prevalence of pathogenic non-coding 'second hits' in trans with these is unknown. METHODS: In 4,073 genetically undiagnosed rare disease trio probands from the 100,000 Genomes project, we identified rare heterozygous protein-altering variants in recessive DD-associated genes. We identified rare non-coding variants on the other haplotype in introns, untranslated regions (UTRs), promoters, and candidate enhancer regions. We clinically evaluated the top candidates for phenotypic fit, and performed functional testing where possible. RESULTS: We identified 3,761 rare heterozygous loss-of-function or ClinVar pathogenic variants in recessive DD-associated genes in 2,430 probands. For 1,366 (36.3%) of these, we identified at least one rare non-coding variant in trans. Bioinformatic filtering and clinical review, revealed seven to be a good clinical fit. After detailed characterisation, we identified likely diagnoses for three probands (in GAA, NPHP3, and PKHD1) and candidate diagnoses in a further three (PAH, LAMA2, IGHMBP2). CONCLUSION: We developed a systematic approach to uncover new diagnoses involving compound heterozygous coding/non-coding variants and conclude that this mechanism is likely to be a rare cause of DDs.

Fraud in Genetic Testing: Swindling the System.

PURPOSE: Healthcare fraud comprises a sizable portion of United States healthcare expenditure and inflicts undue burden on payors, patients, and the healthcare system overall. The genetic testing industry is rapidly growing which propagates opportunities for healthcare fraud. Although federal organizations have highlighted it as an issue, there is limited research exploring genetic testing fraud. METHODS: A retrospective review of federal websites, news articles, and a legal database resulted in 42 cases of fraud involving outpatient genetic testing published between February 2019 and December 2023. These cases were analyzed for themes via inductive conventional content analysis. RESULTS: Themes of fraudulent activity included submission of fraudulent claims, kickback or bribe payments, minimal or no contact with patients for which testing was ordered, inappropriate billing and documentation practices, and further actions to conceal fraud. Repercussions imposed on defendants included monetary penalty, imprisonment, business restrictions, and seizure of property. CONCLUSION: High rates of medically inappropriate testing in fraud cases highlight the value of genetics experts in ordering or reviewing claims for genetic testing. Examining fraudulent activity in genetic testing can help providers identify and report fraud, and provide awareness of optimal healthcare allocation in the genetic testing industry.

Ablation for Atrial Fibrillation in Patients With Rare Pathogenic Variants in Cardiomyopathy and Arrhythmia Genes.

BACKGROUND: Patients with rare, pathogenic cardiomyopathy (CM) and arrhythmia variants can present with atrial fibrillation (AF). The efficacy of AF ablation in these patients is unknown. OBJECTIVE: This study tested the hypotheses that: 1) patients with a pathogenic variant in any CM or arrhythmia gene have increased recurrence following AF ablation; and 2) patients with a pathogenic variant associated with a specific gene group (arrhythmogenic left ventricular CM [ALVC], arrhythmogenic right ventricular CM, dilated CM, hypertrophic CM, or a channelopathy) have increased recurrence. METHODS: We performed a prospective, observational, cohort study of patients who underwent AF catheter ablation and whole exome sequencing. The primary outcome measure was ≥30 seconds of any atrial tachyarrhythmia that occurred after a 90-day blanking period. RESULTS: Among 1,366 participants, 109 (8.0%) had a pathogenic or likely pathogenic (P/LP) variant in a CM or arrhythmia gene. In multivariable analysis, the presence of a P/LP variant in any gene was not significantly associated with recurrence (HR 1.15; 95% CI 0.84-1.60; P = 0.53). P/LP variants in the ALVC gene group, predominantly LMNA, were associated with increased recurrence (n = 10; HR 3.75; 95% CI 1.84-7.63; P < 0.001), compared with those in the arrhythmogenic right ventricular CM, dilated CM, hypertrophic CM, and channelopathy gene groups. Participants with P/LP TTN variants (n = 46) had no difference in recurrence compared with genotype-negative-controls (HR 0.93; 95% CI 0.54-1.59; P = 0.78). CONCLUSIONS: Our results support the use of AF ablation for most patients with rare pathogenic CM or arrhythmia variants, including TTN. However, patients with ALVC variants, such as LMNA, may be at a significantly higher risk for arrhythmia recurrence.

Can an online battery match in-person cognitive testing in providing information about age-related cortical morphology?

Clinical identification of early neurodegenerative changes requires an accurate and accessible characterization of brain and cognition in healthy aging. We assessed whether a brief online cognitive assessment can provide insights into brain morphology comparable to a comprehensive neuropsychological battery. In 141 healthy mid-life and older adults, we compared Creyos, a relatively brief online cognitive battery, to a comprehensive in person cognitive assessment. We used a multivariate technique to study the ability of each test to inform brain morphology as indexed by cortical sulcal width extracted from structural magnetic resonance imaging (sMRI).We found that the online test demonstrated comparable strength of association with cortical sulcal width compared to the comprehensive in-person assessment.These findings suggest that in our at-risk sample online assessments are comparable to the in-person assay in their association with brain morphology. With their cost effectiveness, online cognitive testing could lead to more equitable early detection and intervention for neurodegenerative diseases.

Implementing non-invasive prenatal testing in a national screening program: Lessons learned from the TRIDENT studies.

The Dutch NIPT Consortium, a multidisciplinary collaboration of stakeholders in prenatal care initiated and launched the TRIDENT studies. The goal of the TRIDENT studies was to implement non-invasive prenatal testing (NIPT), first as a contingent (second-tier) and later as a first-tier test, and to evaluate this implementation. This paper describes how NIPT can be successfully implemented in a country or state. Important factors include the significance of forming a consortium and encouraging cooperation among relevant stakeholders, appropriate training for obstetric care professionals, and taking into account the perspectives of pregnant women when implementing prenatal tests. We describe the advantages of high sensitivity and specificity when comparing contingent NIPT with first-tier NIPT. This paper emphasizes the value of pre- and post-test counselling and the requirement for a standardized method of information delivery and value clarification, to assist couples in decision making for prenatal screening.

Usability testing of a novel interlocking three-dimensional miniplate for mandibular angle fractures.

BACKGROUND: We developed a novel interlocking three-dimensional (3D) miniplate design with an adjustable configuration. As this device is new, surgeons must become familiar with its application. This study evaluated the usability and learning curves associated with the novel interlocking 3D miniplate for mandibular fracture fixation. METHODS: The study participants, nine plastic surgeons, were asked to apply an interlocking 3D miniplate and a standard miniplate to polyurethane mandible models. The participants had completed the Basic Craniomaxillofacial Osteosynthesis course during residency and had operated on craniomaxillofacial fractures within the past 5 years. They were instructed to place the interlocking 3D miniplate three times and the standard miniplate once. We assessed the time required for implant placement, the comfort level of the surgeons, and the biomechanical stability of the plates. Biomechanical testing was conducted by subjecting the mandible to forces ranging from 10 to 90 N and the displacement was measured. RESULTS: The results indicate increasing comfort with each attempt at placing the interlocking 3D miniplate, with a significant difference between the first and third attempts. Additionally, a reduction in application time was noted with repeated attempts, suggesting improved efficiency. Biomechanical tests showed comparable stability between the tested plates. CONCLUSION: Multiple attempts at applying the interlocking 3D miniplate resulted in increased comfort and reduced application time. These findings indicate that, despite its novelty, the interlocking 3D miniplate is relatively straightforward to apply and has a short learning curve. However, surgeons must have specific qualifications to ensure proper training and minimize errors during placement.

Diagnostic Strategies in the Era of Monkeypox Resurgence: A Comprehensive Analysis.

The resurgence of monkeypox (Mpox), an orthopoxvirus infection closely related to smallpox, presents a significant global health challenge. This study presents a comprehensive overview of Mpox, focusing on its clinical manifestations, diagnostic strategies, and testing methodologies. A thorough review of the literature and available data on Mpox, emphasizing diagnostic assays, clinical indicators, and laboratory testing, constitutes the core of this analysis. The study involves insights from Mpox patients and healthcare professionals engaged in its diagnosis and management. Contextualizing the research within the global spread of Mpox addresses the complexities associated with the diagnosis of the disease. The findings illuminate diverse Mpox diagnostic techniques, encompassing viral culture, immunological methods, serology, quantitative polymerase chain reaction (qPCR), electron microscopy, and advanced technologies such as artificial intelligence (AI) and the GeneXpert system. qPCR is highlighted as the benchmark for MPXV detection and quantification. These diagnostic advancements have significantly enhanced the precision and efficiency of Mpox diagnosis, facilitating prompt identification and treatment of infected individuals. The study underscores the critical importance of accurate and timely diagnosis, proper handling and transportation of clinical specimens, and the imperative for point-of-care (POC) testing to control the global spread of Mpox.

Design, manufacturing and experimental validation of additive manufacturing cores for bearing seats in carbon fibre reinforced polymer structures.

The integration of carbon-fibre reinforced polymers (CFRP) in structural applications offers significant advantages due to their high strength-to-weight ratio. However, these materials exhibit limitations under out-of-plane loads, particularly in bearing applications. This study explores an innovative approach to enhance the performance of CFRP structures in such scenarios by incorporating annular polyamide inserts manufactured via additive manufacturing (AM). To evaluate the mechanical performance of the AM inserts, a novel ring tensile test is designed to emulate the bearing load conditions. This test also enables the analysis of the impact of several design and manufacturing parameters of the AM inserts, including surface geometry, surface treatment, internal structure, and curing process. These are then compared with specimens made of carbon fibre sheet moulding compounds (CF-SMC), commonly used to support bearing loads. The study reveals that AM inserts provide a viable alternative to state-of-the-art CF-SMC, offering a significant enhancement in mechanical properties under specific bearing loading conditions. The test results indicate a 17.7% improvement in the first failure limit and an 8.6% increase in the ultimate strength for AM inserts compared to CF-SMC. Additionally, the study develops a simplified analytical model to predict stress distributions and potential failure mechanisms, validating its efficacy through experimental data with discrepancies of less than 6%. Economic analysis underscores the cost benefits of AM inserts due to reduced labour and higher repeatability. This research demonstrates the potential of AM inserts to improve the stability and strength of CFRP structures, paving the way for their broader application in demanding load-bearing environments.

Synergy of Xpert (MTB/RIF) and Line probe assay for detection of rifampicin resistant strains of Mycobacterium tuberculosis.

INTRODUCTION: Early diagnosis and successful treatment of drug-resistant tuberculosis (TB) demands rapid, precise, and consistent diagnostic methods to minimise the development of resistance. Therefore, this comparative study was designed to evaluate the diagnostic performance of Xpert (MTB/RIF) and Line probe assay (LPA) for detecting drug-resistant TB. METHODOLOGY: This study comprised 389 (279 pulmonary and 110 extrapulmonary) samples from patients suspected of having TB. All samples were subjected to Xpert (MTB/RIF), LPA, solid culture, and drug-susceptibility testing. Out of 320 samples, only 180 culture (gold standard) positive were included in the final evaluation. The diagnostic characteristics for methods used were determined by calculating diagnostic sensitivity, specificity, and predictive values. The agreement between all methods was determined by calculating the kappa coefficient. RESULTS: The sensitivity and specificity for Xpert (MTB/RIF) for detecting TB were 88.5% and 96.4%, respectively, against the solid culture. On the other hand, LPA showed sensitivity and specificity at 94.3% and 100%, respectively. Xpert (MTB/RIF) showed moderate agreement (kappa 0.65, p < 0.01) - (73.3% sensitivity; 97.6% specificity) for the detection of rifampicin resistance. However, LPA achieved better diagnostic accuracy (kappa 0.80, p < 0.01) - (84.6% sensitivity; 98.4% specificity) against drug-resistant TB. CONCLUSIONS: Xpert (MTB/RIF) and LPA have outstanding diagnostic sensitivity and specificity against RIF resistance with a shorter turnaround time, which could result in a substantial therapeutic outcome. Our findings showed LPA superiority over Xpert (MTB/RIF) for drug resistance. However, due to operational challenges, the requirement of technical expertise and infrastructure issues, LPA cannot be used as point-of-care testing in resource-limited countries.

An at-home and electro-free COVID-19 rapid test based on colorimetric RT-LAMP.

INTRODUCTION: In the fight against virus-caused pandemics like COVID-19, diagnostic tests based on RT-qPCR are essential, but they are sometimes limited by their dependence on expensive, specialized equipment and skilled personnel. Consequently, an alternative nucleic acid detection technique that overcomes these restrictions, called loop-mediated isothermal amplification following reverse transcription (RT-LAMP), has been broadly investigated. Nevertheless, the developed RT-LAMP assays for SARS-CoV-2 detection still require laboratory devices and electrical power, limiting their widespread use as rapid home tests. This work developed a flexible RT-LAMP assay that gets beyond the drawbacks of the available isothermal LAMP-based SARS-CoV-2 detection, establishing a simple and effective at-home diagnostic tool for COVID-19. METHODOLOGY: A multiplex direct RT-LAMP assay, modified from the previously developed test was applied to simultaneously identify the two genes of SARS-CoV-2. We used a colorimetric readout, lyophilized reagents, and benchmarked an electro-free and micropipette-free method that enables sensitive and specific detection of SARS-CoV-2 in home settings. RESULTS: Forty-one nasopharyngeal swab samples were tested using the developed home-testing RT-LAMP (HT-LAMP) assay, showing 100% agreement with the RT-qPCR results. CONCLUSIONS: This is the first electrically independent RT-LAMP assay successfully developed for SARS-CoV-2 detection in a home setting. Our HT-LAMP assay is thus an important development for diagnosing COVID-19 or any other infectious pandemic on a population scale.