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Viral outbreaks can cause widespread disruption, creating the need for diagnostic tools that provide high performance and sample versatility at the point of use with moderate complexity. Current gold standards such as PCR and rapid antigen tests fall short in one or more of these aspects. Here, we report a label-free and amplification-free nanopore sensor platform that overcomes these challenges via direct detection and quantification of viral RNA in clinical samples from a variety of biological fluids. The assay uses an optofluidic chip that combines optical waveguides with a fluidic channel and integrates a solid-state nanopore for sensing of individual biomolecules upon translocation through the pore. High specificity and low limit of detection are ensured by capturing RNA targets on microbeads and collecting them by optical trapping at the nanopore location where targets are released and rapidly detected. We use this device for longitudinal studies of the viral load progression for Zika and Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections in marmoset and baboon animal models, respectively. The up to million-fold trapping-based target concentration enhancement enables amplification-free RNA quantification across the clinically relevant concentration range down to the assay limit of RT-qPCR as well as cases in which PCR failed. The assay operates across all relevant biofluids, including semen, urine, and whole blood for Zika and nasopharyngeal and throat swab, rectal swab, and bronchoalveolar lavage for SARS-CoV-2. The versatility, performance, simplicity, and potential for full microfluidic integration of the amplification-free nanopore assay points toward a unique approach to molecular diagnostics for nucleic acids, proteins, and other targets.

Interactive graphical genome browsers are essential tools in genomics, but they do not contain all the recent genome assemblies. We create Genome Archive (GenArk) collection of UCSC Genome Browsers from NCBI assemblies. Built on our established track hub system, this enables fast visualization of annotations. Assemblies come with gene models, repeat masks, BLAT, and in silico PCR. Users can add annotations via track hubs and custom tracks. We can bulk-import third-party resources, demonstrated with TOGA and Ensembl gene models for hundreds of assemblies.Three thousand two hundred sixty-nine GenArk assemblies are listed at https://hgdownload.soe.ucsc.edu/hubs/ and can be searched for on the Genome Browser gateway page.

Objective The aim of this study was to compare the performance of direct amplification of viral nucleic acid from transport medium to extracted nucleic acid for polymerase chain reaction (PCR), sequencing, and genotyping applications. Methods XpressAmp lysate and extracted total nucleic acid from viral transport medium containing nasopharyngeal specimens were evaluated across different molecular applications to determine performance characteristics. Results SARS-CoV-2 quantitative PCR and angiotensin-converting enzyme (ACE) genotyping assays worked well with XpressAmp lysate, almost equal with or better than extracted nucleic acid in some specimens. However, XpressAmp completely failed to perform in next-generation sequencing for strain typing. Both protocols failed to detect ACE2 expression in viral transport medium. Conclusion Direct amplification of viral nucleic acid from viral transport medium containing nasopharyngeal specimen works well for molecular assays with low thresholds of quality; however, it does have limitations with assays that require high quality nucleic acid for input. Use of the XpressAmp protocol significantly improves turnaround time and allows for easy ramp-up of PCR and genotyping assays. [ABSTRACT FROM AUTHOR]

Nucleic acid amplification tests (NAAT) are considered the gold standard for COVID-19 diagnosis. These tests require professional manpower and equipment, long processing and swab sampling which is unpleasant to the patients. Several volatile organic compounds (VOCs) have been identified in the breath of COVID-19 patients. Detection of these VOCs using a breath test could help rapidly identify COVID-19 patients. Assess the accuracy of 'Breath of Health' (BOH) COVID-19 Fourier-transform infra-red (FTIR) Spectroscopy-based breath test. Breath samples from patients with or without symptoms suggestive for COVID-19 who had NAAT results were collected using Tedlar bags and were blindly analysed using BOH FTIR spectroscopy. BOH Measures several VOCs simultaneously and differentiating positive and negative results. BOH results were compared to NAAT results as gold standard. Breath samples from 531 patients were analysed. The sensitivity of BOH breath test was found to be 79.5% and specificity was 87.2%. Positive predictive value (PPV) was 74.7% and negative predictive value (NPV) 90.0%. Calculated accuracy rate was 84.8% and area under the curve 0.834. Subgroup analysis revealed that the NPV of patients without respiratory symptoms was superior over the NPV of symptomatic patients (94.7% vs 80.7%, P-value < 0.0001) and PPV of patients with respiratory symptoms outranks the PPV of individuals without symptoms (85.3% vs 69.2%, P-value 0.0196) We found BOH COVID-19 breath test to be a patient-friendly, rapid, non-invasive diagnostic test with high accuracy rate and NPV that could efficiently rule out COVID-19 especially among individuals with low pre-test probability. [ABSTRACT FROM AUTHOR]

Background: Recent studies suggest the transplantation of Hepatitis C (HCV) hearts from viremic donors is associated with comparable 1 year survival to nonviremic donors. Though HCV viremia is a known risk factor for accelerated atherosclerosis, data on cardiac allograft vasculopathy (CAV) outcomes are limited. We compared the incidence of CAV in heart transplant recipients from HCV viremic donors (nucleic acid amplification test positive; NAT+) compared to non‐HCV infected donors (NAT−). Methods: We retrospectively reviewed annual coronary angiograms with intravascular ultrasound from April 2017 to August 2020 at two large cardiac transplant centers. CAV was graded according to ISHLT guidelines. Maximal intimal thickness (MIT) ≥ 0.5 mm was considered significant for subclinical disease. Results: Among 270 heart transplant recipients (mean age 54; 77% male), 62 patients were transplanted from NAT+ donors. CAV ≥ grade 1 was present in 8.8% of the NAT+ versus 16.8% of the NAT‐ group at 1 year, 20% versus 28.8% at 2 years, and 33.3% versus 41.5% at 3 years. After adjusting for donor age, donor smoking history, recipient BMI, recipient, hypertension, and recipient diabetes, NAT+ status did not confer increased risk of CAV (HR.80; 95% CI.45–1.40, p = 0.43) or subclinical IVUS disease (HR.87; 95% CI.58–1.30, p = 0.49). Additionally, there was no difference in the presence of rapidly progressive lesions on IVUS. Conclusion: Our data show that NAT+ donors conferred no increased risk for early CAV or subclinical IVUS disease following transplantation in a cohort of heart transplant patients who were treated for HCV, suggesting the short‐term safety of this strategy to maximize the pool of available donor hearts. [ABSTRACT FROM AUTHOR]

Background and Objectives: Nucleic acid amplification testing (NAT), in blood services context, is used for the detection of viral and parasite nucleic acids to reduce transfusion‐transmitted infections. This project reviewed NAT for screening blood donations globally. Materials and Methods: A survey on NAT usage, developed by the International Society of Blood Transfusion Working Party on Transfusion‐transmitted Infectious Diseases (ISBT WP‐TTID), was distributed through ISBT WP‐TTID members. Data were analysed using descriptive statistics. Results: Forty‐three responses were received from 32 countries. Increased adoption of blood donation viral screening by NAT was observed over the past decade. NAT‐positive donations were detected for all viruses tested in 2019 (proportion of donations positive by NAT were 0.0099% for human immunodeficiency virus [HIV], 0.0063% for hepatitis C virus [HCV], 0.0247% for hepatitis B virus [HBV], 0.0323% for hepatitis E virus [HEV], 0.0014% for West Nile virus [WNV] and 0.00005% for Zika virus [ZIKV]). Globally, over 3100 NAT‐positive donations were identified as NAT yield or solely by NAT in 2019 and over 22,000 since the introduction of NAT, with HBV accounting for over half. NAT‐positivity rate was higher in first‐time donors for all viruses tested except WNV. During 2019, a small number of participants performed NAT for parasites (Trypanosoma cruzi, Babesia spp., Plasmodium spp.). Conclusion: This survey captures current use of blood donation NAT globally. There has been increased NAT usage over the last decade. It is clear that NAT contributes to improving blood transfusion safety globally; however, there is a need to overcome economic barriers for regions/countries not performing NAT. [ABSTRACT FROM AUTHOR]

Background: Tuberculosis and diabetes both diseases are present in large numbers in the country and we are major contributors to both globally. With the objective to understand the various traits of patients having both tuberculosis and diabetes and to ascertain various possible predictors for such occurrence based on the public health database we carried out this study. We seek answers to questions like they have any effects? Are they having any additive role to play? Methods: One-year data from the NIKSHAY portal of both districts were analyzed to look for possible associations and other variable traits. Data were analyzed using standard methods to express data in frequency and percentage. Chi-square test was used to establish association, while step-wise approach was used to calculate univariate and multivariate logistic regression analysis for knowing various predictors. P-value of <0.05 was considered statistically significant. Results: Concurrent diabetes in tuberculosis patients was close to 294 (6%) in the 4933 individuals. In total, 65.2% of the study population were male. Diagnosis of tuberculosis was made most of the time by chest X-ray (49.4%) followed by Microscopy ZN staining and cartridge-based nucleic acid amplification test (CBNAAT). Death was more among diabetics (4.4%) as compared to nondiabetics (3.5%). Conclusion: Diabetes is increasing in tuberculosis patients; improvement in data quality is needed. More research is required to reveal various other reasons that make tuberculosis patients more prone to develop diabetes. [ABSTRACT FROM AUTHOR]

Background: Mycoplasmoides genitalium remains a difficult sexually-transmitted infection (STI) to manage due to its potential for antimicrobial resistance and post-infection sequelae. University students are especially vulnerable, as this demographic has the highest rate of STI in the United States. As a result, investigating prevalence rates and therapeutic outcomes in this population is essential to minimize future impact of M. genitalium. The purpose of this study was to investigate a university student population for M. genitalium distribution and treatment outcome. Design: Retrospective chart-review of university health clinic attendees, augmented by laboratory detection of M. genitalium following therapeutic intervention. Methods: A total of 1617 student encounters at a midwestern United States university health clinic over a 28-month interval from November 2017 through February 2020 were analyzed for M. genitalium and Chlamydia trachomatis positivity rates and prevalence. Detection of these sexually-transmitted pathogens occurred by commercial RNA amplification testing. Chart review was focused on participant outcomes following initial M. genitalium detection and therapeutic intervention. Results: C. trachomatis positivity and prevalence rates were 7.05% and 9.00%, respectively, while analogous rates for M. genitalium were 7.05% and 6.51 %, respectively. An average of 1.83 positive results was generated from participants infected with M. genitalium at any time, with an average of 1.17 positive results for C. trachomatis (P < 0.0002). For students treated with azithromycin, 30.3% generated a negative M. genitalium result upon follow-up, with 1g daily and 2-day 500mg dosing regimens demonstrating less efficacy than a 4-day 250mg regimen or moxifloxacin. Conclusion: Data indicate a need for molecular M. genitalium macrolide resistance determination from primary specimens in the university setting. [ABSTRACT FROM AUTHOR]

Trichomoniasis is the most common non-viral sexually transmitted infection (STI) in the world. The estimated global prevalence in 2016 was 156 million adults aged 15–49. However, these data are underestimated, since the most used diagnostic method is the wet mount, which has low sensitivity, the information regarding the estimated duration of infection is limited and there is evidence of undiagnosed asymptomatic cases in both sexes. Currently 80% of cases—including both sexes—are asymptomatic, which makes the disease silent and chronic in course, leading to complications. The aim of this review was to discuss the diagnostic methods for T. vaginalis detection that are currently available and applicable in the clinical laboratory routine. Overall, nucleic acid amplification techniques are the best option for T. vaginalis detection, with higher sensitivity and specificity than other tests. Although these techniques present higher cost, their implementation should be supported to ensure correct trichomoniasis diagnosis and treatment beyond contributing to questions on epidemiology and control. [ABSTRACT FROM AUTHOR]

Background: A novel coronavirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV- 2) that emerged in China in December 2019 has spread rapidly globally to many countries including India and World Health Organization declared it as a pandemic on March 11th, 2020. Aims and Objectives: The current study endeavors to determine the SARS-CoV-2 positivity rate as well as epidemiological, clinical, and diagnostic profiles from the second wave. Materials and Methods: We performed a retrospective analysis of all suspected COVID-19 cases from January 2021 to October 2021 presenting at a large testing center for SARS-CoV-2 infection by real-time polymerase chain reaction (RT-PCR). Descriptive analysis has been performed for profiling of clinical-epidemiological aspects of suspected cases. Results: A total of 694427 participants were enrolled during the study from January 2021 to October 2021. Overall RT-PCR positivity rate was found to be 1.7% in the year 2021 and the positivity was maximum in April 2021 which represents the second wave of COVID-19 infection in India. In the study population, more than half (57.07%) of the persons screened for COVID-19 infection were between 21 and 40 years of age, and about two-thirds (65.20%) of the persons screened were male followed by 34.79% were female. Conclusions: SARS-CoV-2 poses a high burden of infections in the community. Males had a higher RT-PCR detection rate as compared to females. The younger age group (<20 years) expressed the least RT-PCR positivity rate and the elderly population (>80 years) expressed the highest positivity rate. [ABSTRACT FROM AUTHOR]

Background: Transfusion‐transmitted malaria (TTM) is a public health problem in endemic and nonendemic areas. The Brazilian Ministry of Health (MH) requested the development of a nucleic acid amplification test (NAT) for the detection of Plasmodium spp. in public blood centers to increase blood safety. Study Design and Methods: The new Brazilian NAT kit named NAT PLUS HIV/HBV/HCV/Malaria Bio‐Manguinhos was first implemented in HEMORIO, a public blood center in the city of Rio de Janeiro. Since October 1, 2022, this blood center has been testing all its blood donations for malaria in a pool of six plasma samples to detect Plasmodium spp. by real‐time polymerase chain reaction (PCR). Results: Since the implementation of the NAT PLUS platform until February 2023, HEMORIO has successfully received and tested 200,277 donations. The platform detected two asymptomatic donors in the city of Rio de Janeiro, which is a nonendemic region for malaria. Our analyses suggested a malaria from the Amazon region caused by Plasmodium vivax, in the first case, while an autochthonous transmission case by Plasmodium malariae was identified in the rural area of Rio de Janeiro state. Discussion: The NAT PLUS platform detects Plasmodium spp. in plasma samples with sensitivity capable of detecting subpatent infections. This is the first time worldwide that a group developed and implemented molecular diagnosis for Plasmodium spp. to be used by public blood centers to avoid TTM. [ABSTRACT FROM AUTHOR]

The urgent need for accurate COVID-19 diagnostics has led to the development of various SARS-CoV-2 detection technologies. Real-time reverse transcriptase polymerase chain reaction (RT-qPCR) remains a reliable viral gene detection technique, while other molecular methods, including nucleic acid amplification techniques (NAATs) and isothermal amplification techniques, provide diverse and effective approaches. Serological assays, detecting antibodies in response to viral infection, are crucial for disease surveillance. Saliva-based immunoassays show promise for surveillance purposes. The efficiency of SARS-CoV-2 antibody detection varies, with IgM indicating recent exposure and IgG offering prolonged detectability. Various rapid tests, including lateral-flow immunoassays, present opportunities for quick diagnosis, but their clinical significance requires validation through further studies. Challenges include variations in specificity and sensitivity among testing platforms and evolving assay sensitivities over time. SARS-CoV-2 antigens, particularly the N and S proteins, play a crucial role in diagnostic methods. Innovative approaches, such as nanozyme-based assays and specific nucleotide aptamers, offer enhanced sensitivity and flexibility. In conclusion, ongoing advancements in SARS-CoV-2 detection methods contribute to the global effort in combating the COVID-19 pandemic. [ABSTRACT FROM AUTHOR]

Active regular surveillance testing of asymptomatic and symptomatic individuals can reduce infection and onward transmission rates, as demonstrated for SARS-CoV-2. Cost–benefit analysis based on real-world data. Two different surveillance-testing strategies using nucleic acid amplification tests (NAATs) performed in 14,177 hospital employees were compared for their costs and their effectiveness in preventing secondary infections. Compared to not testing, NAAT-based testing twice a week accompanied by contact tracing or testing five times a week without tracing of contacts were more effective in preventing infections through early identification of infected individuals. While expansion of the test frequency from two to five times per week increased the initial costs, importantly, a 49.6 % higher inhibitory effect on infection growth with a 11.1-fold reduction of potentially averted infections and resulting workforce loss was observed, demonstrating a substantial cost–benefit of the 5-tests-per-week strategy. Adaptation of the test frequency of SARS-CoV-2 and possibly of other pathogens with epidemic potential according to the prevailing incidences and reproduction rates in high-prevalence situations may not only be beneficial in averting potential infections in hospital employees and, subsequently, on a population level but may also represent the most cost-effective method. [ABSTRACT FROM AUTHOR]

Purpose: Group B Streptococcus (GBS) is the leading cause of invasive infections in newborns. The prevention of GBS neonatal disease relies on the administration of an intrapartum antibiotic prophylaxis to GBS-colonized women. In recent years, rapid intrapartum detection of GBS vaginal colonization using real-time nucleic acid amplification tests (NAATs) emerged as an alternative to antenatal culture screening methods. Methods: We compared the performances of two loop-mediated isothermal amplification (LAMP) tests, the Ampliflash® GBS and the PlusLife® GBS tests, to standard culture for GBS detection in vaginal specimens from pregnant women. The study was conducted from April to July 2023 in a French hospital of the Paris area. Results: A total of 303 samples were analyzed, including 85 culture-positive samples (28.1%). The Ampliflash® GBS test and the PlusLife® GBS tests gave a result for 100% and 96.3% tests, respectively. The performances of the tests were as follows: sensitivity 87.1% (95% confidence interval (CI) 78.3–92.6) and 98.7% (95% CI 93.0-99.8), specificity 99.1% (95% CI 96.7–99.8), and 91.9% (95% CI 87.3–95.0), respectively. False negative results of the Ampliflash® GBS test correlated with low-density GBS cultures. Time-to-results correlated with GBS culture density only for the PlusLife® GBS test (p < 0.001). Conclusion: Both techniques provide excellent analytical performances with high sensitivity and specificity together with a short turnaround time and results available in 10 to 35 min. Their potential to further reduce the burden of GBS neonatal disease compared with antenatal culture screening needs to be assessed in future clinical studies. [ABSTRACT FROM AUTHOR]

Mycobacteria can be one of the main contaminants of biological products, and their presence can have serious consequences on patients' health. For this reason, the European Pharmacopoeia mandates the specific testing of biological products for mycobacteria, a critical regulatory requirement aimed at ensuring the safety of these products before they are released to the market. The current pharmacopeial reference, i.e., microbial culture method, cannot ensure an exhaustive detection of mycobacteria due to their growth characteristics. Additionally, the method is time consuming and requires a continuous supply of culture media, posing logistical challenges. Thus, to overcome these issues, pharmaceutical industries need to consider alternative non-microbiological techniques to detect these fastidious, slow-growing contaminating agents. This review provides an overview of alternative methods, which could be applied within a quality control environment for biological products and underlines their advantages and limitations. Nucleic acid amplification techniques or direct measurement of mycobacteria stand out as the most suitable alternatives for mycobacterial testing in biological products. [ABSTRACT FROM AUTHOR]

Background: Prompt malarial treatment and surveillance is crucial for accurate diagnosis of Plasmodium Sp. Gold standard microscopic examination has been widely applied for diagnosis of malaria in most part of the endemic areas. But in case of submicroscopic and asymptomatic microscopic diagnosis is questioned. The study aims to develop a simple, cost effective & robust nucleic acid amplification technique for the detection of malaria parasite. Methods: Study population included 50 clinically diagnosed positive malaria patient samples from various pathological laboratories. Microscopy by preparing thick film was carried out of every sample for primary screening in the available facility of Surat Raktadan Kendra & Research Centre- Blood Bank. The conventional PCR (Polymerase Chain Reaction) was applied for genus-specific amplification targeting the 18 S rRNA gene of Plasmodium. Agarose gel electrophoresis was used to separate and analyze the amplified PCR product using 2% Agarose gel. Results and Conclusion: The study shows that nested PCR not only detected all microscopic positive samples, but also detected submicroscopic infections that were missed or misread by microscopy. Hence, the sensitivity of molecular based detection technique is proved to be more compared to microscopic examination. [ABSTRACT FROM AUTHOR]

The rapid and accurate identification of parasites is crucial for prompt therapeutic intervention in parasitosis and effective epidemiological surveillance. For accurate and effective clinical diagnosis, it is imperative to develop a nucleic-acid-based diagnostic tool that combines the sensitivity and specificity of nucleic acid amplification tests (NAATs) with the speed, cost-effectiveness, and convenience of isothermal amplification methods. A new nucleic acid detection method, utilizing the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, holds promise in point-of-care testing (POCT). CRISPR/Cas12a is presently employed for the detection of Plasmodium falciparum, Toxoplasma gondii, Schistosoma haematobium, and other parasites in blood, urine, or feces. Compared to traditional assays, the CRISPR assay has demonstrated notable advantages, including comparable sensitivity and specificity, simple observation of reaction results, easy and stable transportation conditions, and low equipment dependence. However, a common issue arises as both amplification and cis-cleavage compete in one-pot assays, leading to an extended reaction time. The use of suboptimal crRNA, light-activated crRNA, and spatial separation can potentially weaken or entirely eliminate the competition between amplification and cis-cleavage. This could lead to enhanced sensitivity and reduced reaction times in one-pot assays. Nevertheless, higher costs and complex pre-test genome extraction have hindered the popularization of CRISPR/Cas12a in POCT. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, exposed a pressing need for new public health tools for pathogen detection, disease diagnosis, and viral genotyping. REVEALR (RNA-encoded viral nucleic acid analyte reporter) is an isothermal DNAzyme-based point-of-care diagnostic that functions with a detection limit of ∼10 copies/μL when coupled with a preamplification step and can be utilized for viral genotyping of SARS-CoV-2 variants of concern through base pair mismatch recognition in a competitive binding format. Here, we describe an advanced REVEALR platform, termed digital droplet REVEALR (ddREVEALR), that can achieve direct viral detection and absolute sample quantitation utilizing a signal amplification strategy that relies on chemical modifications, DNAzyme multiplexing, and volume compression. Using an AI-assisted image-based readout, ddREVEALR was found to achieve 95% positive predictive agreement from a set of 20 nasal pharyngeal swabs collected at UCI Medical Center in Orange, California. We propose that the combination of amplification-free and protein-free analysis makes ddREVEALR a promising application for direct viral RNA detection of clinical samples.

OBJECTIVE: Most health care personnel (HCP) reporting symptoms consistent with COVID-19 illness are assessed by high-accuracy SARS-CoV-2 assays performed in clinical laboratories, but the results of such assays typically are not available until the following day. METHODS: This is an observational study over 16 weeks of a rapid nucleic acid amplification test (NAAT) performed at point of contact. The benchmark for comparison was a simultaneously obtained specimen assayed by a routine NAAT assay performed in a clinical laboratory. RESULTS: There were 577 paired rapid and routine NAAT results. Rapid test positive predictive value was 90.0% (95% confidence interval = 88.8%-91.2%), and negative predictive value was 95.2% (95% confidence interval = 93.5%-96.9%). The rapid test avoided an estimated 160 to 184 lost work shifts over 4 months. CONCLUSIONS: A rapid NAAT test-based strategy proved effective in safely clearing symptomatic employees without infection for earlier return to work.

Background: Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infections have increased globally. Asymptomatic infections represent a significant risk of long-term complications. Men who have sex with men (MSM) are disproportionally affected, underscoring the need to offer screening programmes to this population. CT/NG Point of Care Testing (POCT) constitutes a strategic tool to improve the continuum of STI care, however extensive real-life evaluations amongst at risk populations are lacking. The aim of this study is to estimate the GeneXpert CT/NG assay performance and usability for CT and NG at genital and extragenital sites for screening amongst MSM. Methods: This study was a multi-site sexual health clinic-based evaluation (Italy, Malta and Peru) with consecutive enrolment. A first void urine sample (divided in two aliquots), two oropharyngeal and two anorectal swabs were collected for each study participant. One specimen set (one for each anatomical site) was tested with the dual index test (Cepheid) at the clinics by the healthcare staff, the other set with FDA/CE approved Nucleic Acid Amplification Tests (NAATs) at the laboratory. Clinical sites and reference laboratories participated in an internal and external quality control programme. Sensitivity, specificity, positive and negative likelihood ratios, positive and negative predictive values for each anatomical site were estimated using a meta-analytic approach. Results: One thousand seven hundred two MSM were recruited across all clinical sites for a total of 5049 biological specimens. NG and CT were respectively detected in 274 and 287 of samples. Overall, the NG POCT sensitivity and specificity was 91.43% and 99.75% in urine (LR + 372.80, LR- 0.09), 89.68% and 99.55% in rectal specimens (LR + 197.30, LR- 0.10) and 75.87% and 98.77% at the pharynx respectively (LR + 61.94, LR- 0.24). The CT component of the POCT sensitivity was 84.82% and specificity 99.63% in urine (LR + 228.68, LR- 0.15), 78.07% and 99.19% respectively on rectal site (LR + 96.23, LR-0.22), 67.79% and 99.88% respectively at pharyngeal site (LR + 554.89, LR- 0.32). 95.95% of MSM reported to be willing to wait for POCT results and no provider reported difficulties in terms of performance or interpretation of the results of the Xpert CT/NG. Conclusion: Rapid turnaround time, ease of use and high acceptability make the Xpert CT/NG testing system a strategic tool for increasing testing frequency, reaching those not yet tested and offering the possibility of immediate treatment if needed. The assay showed good negative likelihood ratios and confirms its use to rule out CT/NG infections. Sensitivity varied across sites and pathogens. Periodic staff training at the testing sites should be mandatory. [ABSTRACT FROM AUTHOR]

Background: Qualified malaria diagnosis competency has contributed to the great achievement of malaria elimination in China. After eliminating malaria, it is still critical to the prevention of re-establishment of malaria transmission in China. This study was aimed to assess the malaria detection competency at national and provincial levels in China at the beginning of malaria post-elimination phase. Methods: In the present study, different competency assessment activities on the laboratory malaria diagnosis were carried out for national and provincial malaria diagnostic laboratories based on the WHO scoring schedules, including malaria microscopy or nucleic acid amplification tests (NAAT), at the beginning of malaria post-elimination phase (2021–2022) in China. Results: A total of 60 slides for malaria microscopy and 10 specimen for NAAT were included into the WHO External Quality Assessments of malaria parasite qualitative detection and species identification, and the scoring rate was 96.6% (microscopy: 171/177) and 85.0% (NAAT: 17/20), respectively. Moreover, 124 samples were included into the national NAAT quality assessment, and an accuracy of 87.9% (109/124) was found without significance among reference laboratories and non-reference laboratories. Conclusions: The findings suggest that there is still a need for sustained strengthening of malaria detection competency, particularly in the areas of parasite counting and detection of low-density parasitemia, to ensure prompt detection of the sources of infection and accurate identification of Plasmodium species, and contribute to case management and focus disposal, thereby effectively preventing the malaria re-establishment. [ABSTRACT FROM AUTHOR]

Background Standalone nucleic acid amplification tests (NAATs) are frequently used to diagnose Clostridioides difficile infections (CDI), although they may be unable to distinguish colonization from disease. A 2-stage algorithm pairing NAATs with toxin immunoassays (Toxin) may improve specificity. We evaluated clinical outcomes of patients who were NAAT+/Toxin+ versus NAAT+/Toxin− and treated versus untreated NAAT+/Toxin− cases through systematic review and meta-analysis. Methods We searched EMBASE and MEDLINE from inception to April 1, 2023 for articles comparing CDI outcomes among symptomatic patients tested by NAAT and Toxin tests. The risk differences (RD) of all-cause mortality and CDI recurrence were computed by random effects meta-analysis between patients who were NAAT+/Toxin+ and NAAT+/Toxin−, as well as between patients who were NAAT+/Toxin− and treated or untreated. Results Twenty-six observational studies comprising 12 737 patients were included. The 30-day all-cause mortality was not significantly different between those who were NAAT+/Toxin+ (8.4%) and NAAT+/Toxin− (6.7%) (RD = 0.41%, 95% confidence interval [CI] = −.67, 1.49). Recurrence at 60 days was significantly higher among patients who were NAAT+/Toxin+ (19.8%) versus NAAT+/Toxin− (11.0%) (RD = 7.65%, 95% CI = 4.60, 10.71). Among treated compared to untreated NAAT+/Toxin- cases, the all-cause 30-day mortalities were 5.0% and 12.7%, respectively (RD = −7.45%, 95% CI = −12.29, −2.60), but 60-day recurrence was not significantly different (11.6% vs 7.0%, respectively; RD = 5.25%, 95% CI −1.71, 12.22). Conclusions Treatment of patients who were NAAT+/Toxin− was associated with reduced all-cause mortality but not recurrence. Although subject to the inherent limitations of observational studies, these results suggest that some patients who are NAAT+/Toxin− may benefit from treatment. [ABSTRACT FROM AUTHOR]

Endobronchial tuberculosis (EBTB) is characterized by tuberculosis infection of the tracheobronchial tree. It has variable presentation but tumorous growth-like presentation in bronchus is very rare. The clinical and radiological features are non-specific, which creates a diagnostic dilemma. Bronchoscopy and biopsy of the lesion are mandatory to confirm the diagnosis. In this case series, we are presenting three unique cases of endobronchial growth diagnosed as EBTB after biopsy and evaluation of bronchoalveolar lavage (BAL) with cartridge-based nucleic acid amplification test (CBNAAT) and other ancillary investigations for tuberculosis. Four patients presented to the outpatient department with non-specific symptoms of fever, cough, hoarseness of voice, and hemoptysis. They were evaluated with chest radiograph (CXR), contrast-enhanced computed tomography (CECT) thorax, and bronchoscopy. Bronchoscopy revealed growth in the bronchus in all three cases. A biopsy was taken and BAL was performed. All cases turned out to be EBTB in histopathological examination and BAL CBNAAT. They were treated with anti-tubercular drugs and all responded well to treatment. Endobronchial tuberculosis presenting as tumorous growth in the tracheobronchial tree is rare. There should be a high index of suspicion while dealing with patients with non-specific clinical and radiological features of tuberculosis. EBTB can be misdiagnosed as malignancy in most cases. Therefore, it should be kept as a differential diagnosis while encountering a mass lesion in the trachea or bronchus during bronchoscopy. [ABSTRACT FROM AUTHOR]

Background: Based on the theme of World Tuberculosis (TB) Day 2018—"Wanted Leaders for TB free world," various studies and country-level project have witnessed how training leaders on TB has helped enhance TB awareness in the community. This study was conducted to evaluate the effectiveness of Advocacy Communication and Social Mobilization (ACSM) intervention strategy through key community leaders, using Reach, Effectiveness, Adoption, Implementation, Maintenance (RE-AIM) framework. Materials and Methods: This study was undertaken by the Department of Community Medicine, Sri Manakula Vinayagar Medical College and Hospital (SMVMCH), Puducherry, in the field practice area of Primary Health Centre (PHC), Thirubuvanai. The STOP TB, ENGAGE-TB, and National TB Elimination Program (NTEP) guidelines were used for interventions. Key community leaders (KCL) list (N = 117) was obtained from Mannadipet commune panchayat and PHC. The study was conducted in four phases: phase 1 (baseline): quantitative data: proportion of presumptive TB cases from notification register; phase 2 (intervention): training program for staff nurses, auxiliary nurse midwife (ANM), Accredited Social Health Activist (ASHA), and Anganwadi workers (group activity), participatory rural appraisal (PRA) activity for self-help group (SHG) leaders, and health education session for religious leaders and ex-panchayat leaders; phase 3: strengthening of ongoing activity: health education session and active case finding for TB; and phase 4: end line: outcome of the intervention, evaluated using the RE-AIM framework. Results: As an outcome of the intervention, there was a marginal increase (21 cases) in the proportion of presumptive TB cases when comparing before (2018) and after (2019) intervention. The proportion of presumptive TB cases identified and referred by KCL was 312. A total of 77 presumptive TB cases were identified through community-based active case finding (ACF). Two sputum samples (on the spot early morning) were collected and sent for Cartridge-based nucleic acid amplification testing (CBNAAT), of which three cases were positive. Conclusion: This study was community led and involved leaders from diverse backgrounds, optimizing the chance of success. For sustainability, we have initiated TB support group. [ABSTRACT FROM AUTHOR]

Background Computer-aided detection (CAD) may be a useful screening tool for tuberculosis (TB). However, there are limited data about its utility in active case finding (ACF) in a community-based setting, and particularly in an HIV-endemic setting where performance may be compromised. Methods We performed a systematic review and evaluated articles published between January 2012 and February 2023 that included CAD as a screening tool to detect pulmonary TB against a microbiological reference standard (sputum culture and/or nucleic acid amplification test [NAAT]). We collected and summarized data on study characteristics and diagnostic accuracy measures. Two reviewers independently extracted data and assessed methodological quality against Quality Assessment of Diagnostic Accuracy Studies–2 criteria. Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Diagnostic Test Accuracy Studies (PRISMA-DTA) guidelines were followed. Results Of 1748 articles reviewed, 5 met with the eligibility criteria and were included in this review. A meta-analysis revealed pooled sensitivity of 0.87 (95% CI, 0.78–0.96) and specificity of 0.74 (95% CI, 0.55–0.93), just below the World Health Organization (WHO)–recommended target product profile (TPP) for a screening test (sensitivity ≥0.90 and specificity ≥0.70). We found a high risk of bias and applicability concerns across all studies. Subgroup analyses, including the impact of HIV and previous TB, were not possible due to the nature of the reporting within the included studies. Conclusions This review provides evidence, specifically in the context of ACF, for CAD as a potentially useful and cost-effective screening tool for TB in a resource-poor HIV-endemic African setting. However, given methodological concerns, caution is required with regards to applicability and generalizability. [ABSTRACT FROM AUTHOR]

Background: More than 45 cases of transfusion‐transmitted hepatitis E virus infection (TT‐HEV) have been reported in Japan. Therefore, in 2020, universal individual donation nucleic acid amplification testing (ID‐NAT) was implemented for HEV. Study Design and Methods: We characterized HEV NAT‐positive blood donors. The number of new HEV infections and the asymptomatic infection rate were estimated using the HEV NAT‐positive rate. HEV RNA quantitation, phylogenetic analysis, and antibody tests were performed, and the residual risk of TT‐HEV was assessed based on the lookback study results. Results: A total of 5,075,100 blood donations were screened with ID‐NAT during the first year of implementation, among which 2804 (0.055%; males: 0.060%, females: 0.043%) were NAT‐positive with regional differences. Approximately 270,000 new HEV infection cases were estimated to occur annually in Japan, with an asymptomatic infection rate of 99.9%. The median HEV RNA concentration, excluding cases below the limit of quantification, was 205 IU/mL. Among the 1113 cases where the genotype could be determined, HEV‐3 and HEV‐4 accounted for 98.8% (1100) and 1.2% (13), respectively. The maximum duration of HEV viremia, including the pre‐ and post‐ID‐NAT window periods, was estimated to be 88.2 days. Within the 3 years since ID‐NAT implementation, no confirmed cases of breakthrough TT‐HEV were observed. Discussion: Multiple indigenous HEV strains are prevalent in Japan, infecting a significant number of individuals. However, since the implementation of ID‐NAT, TT‐HEV has been prevented due to the test's high sensitivity. [ABSTRACT FROM AUTHOR]

Introduction: Tuberculosis (TB) is a disease that causes severe mortality and morbidity worldwide, including India. Endobronchial Tuberculosis (EBTB) is an uncommon form of TB, which is often underdiagnosed due to the difficulty in diagnosis. This form of TB often has a poor prognosis and long-lasting sequelae. Aim: To study the proportion of EBTB in clinically diagnosed pulmonary TB and to study the clinicoradiological and bronchoscopic profile of EBTB. Materials and Methods: This study was a hospital-based recordbased cross-sectional study that included patients with sputum smear negative for Acid-Fast Bacilli (AFB) and Mycobacterium tuberculosis (M. tuberculosis) not detected on Nucleic Acid Amplification Test (NAAT), who were clinically diagnosed with TB. Bronchoscopy was performed on such patients, and samples were sent for investigations, including histopathology and NAAT. Demographic characteristics, bronchoscopy and radiology findings, and microbiology results were documented. Quantitative variables were summarised as means, and categorical variables were presented as percentages. Results: A total of 198 underwent bronchoscopy, of which 20 (10%) were diagnosed with EBTB. The mean age was 22 years, and 65% were females. The most common clinical feature in these patients was fever, the most common radiological presentation was lobar collapse, and the most common bronchoscopy feature was a tumorous lesion. NAAT detected M. tuberculosis in the bronchial wash in 50% of patients diagnosed with EBTB. A total of 45% of patients had TB which was diagnosed on histopathology. Conclusion: EBTB was diagnosed in 10% of smear-negative, NAAT-negative cases. The use of NAAT on bronchoscopy wash samples resulted in a high yield in diagnosis. [ABSTRACT FROM AUTHOR]

To evaluate the efficacy and tolerability of a single dose of oral cefixime 800 mg plus oral doxycycline 100 mg twice a day for 7 days, compared with a recommended single dose of ceftriaxone plus single dose of oral azithromycin, for treatment of uncomplicated urogenital, rectal, or pharyngeal gonorrhoea. A noninferiority, open-label, multicentre randomized controlled trial was conducted in Prague, Czech Republic. Some 161 patients, 18–65 years of age diagnosed with uncomplicated urogenital, rectal, or pharyngeal gonorrhoea by nucleic acid amplification test (NAAT) were randomized to treatment with single dose of cefixime 800 mg plus doxycycline 100 mg twice a day for 1 week or a single dose of ceftriaxone 1 g intramuscularly plus single dose of azithromycin 2 g. The primary outcome was the number of participants with negative culture and NAAT at 1 week and 3 weeks, respectively, after treatment initiation. In all, 161 patients were randomized and 152 were included in per-protocol analyses. All 76 (100%; 95% CI, 0.95–1.00) patients treated with ceftriaxone plus azithromycin achieved negative cultures and NAAT after treatment. In the cefixime plus doxycycline arm at week 1, culture was negative in all 76 (100%) patients; at week 3, culture was negative in 70 of the 76 patients (92%; 95% CI, 0.84–0.97) and NAAT negative in 66 of the 76 patients (87%; 95% CI, 0.77–0.94). At week 3, culture and NAAT were negative in 65 of the 76 patients (86%; 95% CI, 0.76–0.93). Per-protocol risk difference was 14.5%; 95% CI, 6.56–22.38. All treatment failures observed in the cefixime arm were pharyngeal gonorrhoea cases. The combination of cefixime and doxycycline did not achieve noninferiority to ceftriaxone and azithromycin for treatment of gonorrhoea when including pharyngeal gonorrhoea. It did, however, show high efficacy for urogenital and rectal gonorrhoea. [ABSTRACT FROM AUTHOR]

A two-step strategy combining assisted benchmark testing (entry controls) and External Quality Assessments (EQAs) with blinded simulated clinical specimens to enhance and maintain the quality of nucleic acid amplification testing was developed. This strategy was successfully applied to 71 diagnostic laboratories in The Netherlands when upscaling the national diagnostic capacity during the SARS-CoV-2 pandemic. The availability of benchmark testing in combination with advice for improvement substantially enhanced the quality of the laboratory testing procedures for SARS-CoV-2 detection. The three subsequent EQA rounds demonstrated high quality testing with regard to specificity (99.6% correctly identified) and sensitivity (93.3% correctly identified). Even with the implementation of novel assays, changing workflows using diverse equipment and a high degree of assay heterogeneity, the overall high quality was maintained using this two-step strategy. We show that in contrast to the limited value of Cq value for absolute proxies of viral load, these Cq values can, in combination with metadata on strategies and techniques, provide valuable information for laboratories to improve their procedures. In conclusion, our two-step strategy (preparation phase followed by a series of EQAs) is a rapid and flexible system capable of scaling, improving, and maintaining high quality diagnostics even in a rapidly evolving (e.g. pandemic) situation. [ABSTRACT FROM AUTHOR]

Background For persons entering congregate settings, optimal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) arrival surveillance screening method, nucleic acid amplification test (NAAT) versus rapid antigen detection test (RADT), is debated. To aid this, we sought to determine the risk of secondary symptomatic coronavirus disease 19 (COVID-19) among military trainees with negative arrival NAAT or RADT screening. Methods Individuals who arrived for US Air Force basic military training from 1 January–31 August 2021 were placed into training groups and screened for SARS-CoV-2 via NAAT or RADT. Secondary symptomatic COVID-19 cases within 2 weeks of training were then measured. A case cluster was defined as ≥5 individual symptomatic COVID-19 cases. Results 406 (1.6%) of 24 601 trainees screened positive upon arrival. The rate of positive screen was greater for those tested with NAAT versus RADT (2.5% vs 0.4%; RR: 5.4; 95% CI: 4.0–7.3; P <.001). The proportion of training groups with ≥1 positive individual screen was greater in groups screened via NAAT (57.5% vs 10.8%; RR: 5.31; 95% CI: 3.65–7.72; P <.001). However, NAAT versus RADT screening was not associated with a difference in number of training groups to develop a secondary symptomatic case (20.3% vs 22.5%; RR:.9; 95% CI:.66–1.23; P =.53) or case cluster of COVID-19 (4% vs 6.6%; RR:.61; 95% CI:.3–1.22; P =.16). Conclusions NAAT versus RADT arrival surveillance screening method impacted individual transmission of COVID-19 but had no effect on number of training groups developing a secondary symptomatic case or case cluster. This study provides consideration for RADT arrival screening in congregate settings. [ABSTRACT FROM AUTHOR]

There is great enthusiasm for using loop-mediated isothermal amplification (LAMP) in point-of-care nucleic acid amplification tests (POC NAATs), as an alternative to PCR. While isothermal amplification techniques like LAMP eliminate the need for rapid temperature cycling in a portable format, these systems are still plagued by requirements for dedicated optical detection apparatus for analysis and manual off-chip sample processing. Here, we developed a new microfluidic system for LAMP-based POC NAATs to address these limitations. The new system combines digital microfluidics (DMF) with distance-based detection (DBD) for direct signal readout. This is the first report of the use of (i) LAMP or (ii) DMF with DBD – thus, we describe a number of characterization steps taken to determine optimal combinations of reagents, materials, and processes for reliable operation. For example, DBD was found to be quite sensitive to background signals from low molecular weight LAMP products; thus, a Capto™ adhere bead-based clean-up procedure was developed to isolate the desirable high-molecular-weight products for analysis. The new method was validated by application to detection of SARS-CoV-2 in saliva. The method was able to distinguish between saliva containing no virus, saliva containing a low viral load (104 genome copies per mL), and saliva containing a high viral load (108 copies per mL), all in an automated system that does not require detection apparatus for analysis. We propose that the combination of DMF with distance-based detection may be a powerful one for implementing a variety of POC NAATs or for other applications in the future. [ABSTRACT FROM AUTHOR]

Background: Early diagnosis of pediatric tuberculosis (TB) still remains a challenge due to the paucibacillary nature of the disease. Objectives: To assess the role of CB-NAAT in diagnosing pulmonary and extrapulmonary tuberculosis as compared to AFB smear and Mycobacterium tuberculosis culture in clinically diagnosed pediatric tuberculosis cases. Study design: Hospital-based prospective study. Methods: A total of142 patients with strong clinical suspicion of pulmonary or extra-pulmonary tuberculosiswere enrolled over a period of two yearsand then results of CB-NAAT were compared to conventional methods (culture/ AFB smear) on follow-up. Results: In subjects who had clinically diagnosed tuberculosis, culture was positive in 18.9% of cases, AFB smear was positive in only 12.1% whereas CB-NAAT was positive in 55.4% of subjects. The sensitivity, specificity, PPV, and NPV of CB-NAAT in patients with culture proven tuberculosis were100%, 83.3%, 47.5%, and 100% respectively with a diagnostic accuracy of 93.3%. and 60%, 100%, 100%, and 65.8% respectively with a diagnostic accuracy of 80.2% in clinically diagnosed pulmonary cases. Conclusion:CB-NAAT has true diagnostic potential with good sensitivity in confirmed cases and significantly better sensitivity than conventional methods in clinically diagnosed cases. [ABSTRACT FROM AUTHOR]

Background: SARS-CoV-2 as the cause of novel coronavirus disease (COVID-19) is a member of the family Coronaviridea that has generated an emerging global health concern. Controlling and preventing the spread of the disease requires a simple, portable, and rapid diagnostic method. Today, a standard method for detecting SARS-CoV-2 is quantitative real-time reverse transcription PCR, which is time-consuming and needs an advanced device. The aim of this study was to evaluate a faster and more cost-effective field-based testing method at the point of risk. We utilized a one-step RT-LAMP assay and developed, for the first time, a simple and rapid screening detection assay targeting the Envelope (E) gene, using specific primers. Methods: For this, the total RNA was extracted from respiratory samples of COVID-19 infected patients and applied to one-step a RT-LAMP reaction. The LAMP products were visualized using green fluorescence (SYBR Green I). Sensitivity testing was conducted using different concentrations of the designed recombinant plasmid (TA-E) as positive control constructs. Additionally, selectivity testing was performed using the influenza H1N1 genome. Finally, the results were compared using with conventional real time RT-PCR. Results: It was shown that the RT-LAMP assay has a sensitivity of approximately 15 ng for the E gene of SARS-CoV-2 when using extracted total RNA. Additionally, a sensitivity of 112 pg was achieved when using an artificially prepared TA-E plasmid. Accordingly, for the detection of SARS-CoV-2 infection, the RT-LAMP had high sensitivity and specificity and also could be an alternative method for real-time RT-PCR. Conclusion: Overall, this method can be used as a portable, rapid, and easy method for detecting SARS-CoV-2 in the field and clinical laboratories. [ABSTRACT FROM AUTHOR]

Introduction: Parvovirus B19 transmitted by umbilical cord blood (UCB) products may cause severe disease in allogenic hematopoietic stem cell transplant recipients. Thus, commercially available nucleic acid test (NAT) assays for highly sensitive detection of parvovirus B19 DNA validated for the specimen cord blood plasma (CBP) are required to avoid parvovirus B19 transmission by umbilical hematopoietic stem cell preparations. Methods: The multiplex cobas DPX NAT assay was validated for detection of parvovirus B19 DNA in CBP derived from citrate anticoagulated UCB units which have been processed by the Rubinstein method. In total, 363 retained CBP samples pretested negative for parvovirus B19 DNA were prepared for analyzing sensitivity, specificity, and interference of that NAT assay. The 3rd WHO International Standard for parvovirus B19 DNA was used for determining the 95% limit of detection (LOD95) by probit analysis. Results: The validation of the parvovirus B19 NAT assay for CBP demonstrated high sensitivity, specificity, intra- and inter-assay precision. Dilution series and replicate analyses showed a high linearity of the assay with a coefficient of determination above 0.99 and revealed a LOD95 of 17 International Units (IU)/mL (95% confidence interval, 14–44 IU/mL) for parvovirus B19 DNA in CBP samples. Conclusion: The validation of a commercially available parvovirus B19 NAT assay for the specimen CBP demonstrated a high assay performance fulfilling German guidelines and international regulations. [ABSTRACT FROM AUTHOR]

Simple Summary: Approximately 10–15% of patients with epithelial ovarian cancer have an inherited (germline) BRCA1 or BRCA2 mutation. Following a diagnosis of epithelial ovarian cancer, patients are routinely tested for germline and/or somatic (tumour) BRCA1/2 mutations. Our study shows that if germline BRCA1/2 testing is only performed for patients with a positive tumour BRCA1/2 test result, and tumour testing is performed using Myriad's myChoice® companion diagnostic, a proportion of germline BRCA1/2 large rearrangements could be missed. If paired germline-tumour DNA testing is not possible for all patients, our data shows that it would be appropriate to test all patients with epithelial ovarian cancer aged < 79 years old for germline BRCA1/2 mutations, regardless of the tumour BRCA1/2 result, whilst only needing to test patients aged ≥ 80 years old for a germline BRCA1/2 mutation if they have a positive tumour BRCA1/2 result. Patients diagnosed with epithelial ovarian cancer may undergo reflex tumour BRCA1/2 testing followed by germline BRCA1/2 testing in patients with a positive tumour test result. This testing model relies on tumour BRCA1/2 tests being able to detect all types of pathogenic variant. We analysed germline and tumour BRCA1/2 test results from patients treated for epithelial ovarian cancer at our specialist oncological referral centre. Tumour BRCA1/2 testing was performed using the next-generation sequencing (NGS)-based myChoice® companion diagnostic (CDx; Myriad Genetics, Inc.). Germline BRCA1/2 testing was performed in the North West Genomic Laboratory Hub using NGS and multiplex ligation-dependent probe amplification. Between 11 April 2021 and 11 October 2023, 382 patients were successfully tested for tumour BRCA1 and BRCA2 variants. Of these, 367 (96.1%) patients were tested for germline BRCA1/2 variants. In those patients who underwent tumour and germline testing, 15.3% (56/367) had a BRCA1/2 pathogenic variant (36 germline and 20 somatic). All germline BRCA1/2 pathogenic small sequencing variants were detected in tumour DNA. By contrast, 3 out of 8 germline BRCA1/2 pathogenic large rearrangements were not reported in tumour DNA. The overall concordance of germline BRCA1/2 pathogenic variants detected in germline and tumour DNA was clinically acceptable at 91.7% (33/36). The myChoice® CDx was able to detect most germline BRCA1/2 pathogenic variants in tumour DNA, although a proportion of pathogenic large rearrangements were not reported. If Myriad's myChoice® CDx is used for tumour BRCA1/2 testing, our data supports a testing strategy of germline and tumour BRCA1/2 testing in all patients diagnosed with epithelial ovarian cancer aged < 79 years old, with germline BRCA1/2 testing only necessary for patients aged ≥ 80 years old with a tumour BRCA1/2 pathogenic variant. [ABSTRACT FROM AUTHOR]

The failures in Trichomonas vaginalis (TV) infection diagnosis leave more than half of cases unidentified. In this report, urine and vaginal discharge samples were analyzed by wet mount, culture examination, and real-time PCR by Allplex™ (Seegene®) kit, in a population assisted by the Brazilian Public Health System. From 747 samples, 2.81% were positive for TV in wet mount and culture, and 3.88% by Allplex™. Samples kept at − 80 ºC for 22 months did not impair the PCR technique. The sensitivity for wet mount, culture, and Allplex™ was 72, 100, and 100%, respectively. Allplex™ technique showed highest detection of TV. [ABSTRACT FROM AUTHOR]

The incidence of gonorrhea has increased significantly in recent years in the United States, especially among sexually active twenty-year-olds. Although the incidence of gonorrhea has decreased in Korea since the early 2000s, it is still common among people in their twenties. Nucleic acid amplification test (NAAT) is the most sensitive diagnostic test for detecting gonococcal infection. Gram-staining is a simple and useful laboratory test for diagnosing symptomatic male gonococcal urethritis. Although bacterial culture can be used to detect antimicrobial susceptibility, its sensitivity is lower than that of NAAT. Treatment for uncomplicated gonorrhea infection is a single intramuscular injection of ceftriaxone 500 mg. Doxycycline (100 mg twice daily for 7 days) is added if there is a possibility of co-infection with chlamydia. If ceftriaxone is difficult to use, spectinomycin 2 g can be injected intramuscularly in Korea. Patients with gonorrhea should have repeated examinations within three months at the exposure site because of a high risk of re-infection. A person diagnosed with gonorrhea should discuss the nature of the infection, the importance of informing partners, when sexual activity can resume, and how to reduce the risk of sexually transmitted infections. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is an important and leading cause of foodborne diseases worldwide. Prompt detection and recall of contaminated foods are crucial to prevent untoward health consequences caused by S. aureus. Helix loop-mediated isothermal amplification (HAMP) is an exciting recent addition to the array of available isothermal-based nucleic acid amplification techniques. This study aimed to develop and evaluate a HAMP assay for detecting S. aureus in milk and milk products. The assay is completed in 75 minutes of isothermal temperature incubation (64 ˚C) and dye-based visual interpretation of results based on colour change. The specificity of the developed assay was ascertained using 27 S. aureus and 17 non S. aureus bacterial strains. The analytical sensitivity of the developed HAMP assay was 9.7 fg/µL of pure S. aureus DNA. The detection limit of the HAMP assay in milk (86 CFU/mL) was 1000x greater than the routinely used endpoint PCR (86 × 103 CFU/mL). The practicality of applying the HAMP assay was also assessed by analysing milk and milk product samples (n = 95) obtained from different dairy farms and retail outlets. The developed test is a more rapid, sensitive, and user-friendly method for the high-throughput screening of S. aureus in food samples and may therefore be suitable for field laboratories. To our knowledge, this is the first study to develop and evaluate the HAMP platform for detecting S. aureus. [ABSTRACT FROM AUTHOR]

Introduction: Tuberculosis is an infectious disease caused by the Mycobacterium tuberculosis which typically involve lung but can affect other sites called extra pulmonary tuberculosis (EPTB). Common disease manifestations of EPTB include meningitis, lymphadenitis, ocular, oral, pleuritis, pericarditis, peritonitis, cutaneous, musculoskeletal, abdominal, genitourinary, and miliary forms of tuberculosis. EPTB can be either primary (at the site of initial infection) or secondary (disseminated), which usually occurs due to haematogenous or lymphatic spread of bacteria from the primary organ, reactivation of Latent TB Infection (LTBI), ingestion of infected sputum, or spread locally from adjacent organs. EPTB cases accounted for 16% of the 7.5 million incident cases worldwide in 2019. Cartridge based nucleic acid amplification test (CBNAAT) have high sensitivity in both respiratory and non-respiratory specimens. Materials and Methods: A laboratory based retrospective observational study. The study was conducted in the department of Microbiology, Narendra Modi medical college, Ahmedabad, Gujarat over a period of one year i.e. from January 2022 to December 2022. The main Aim of study to determine the rate of rifampicin resistant M. tuberculosis among EPTB and to determine the age and gender dependent preponderance of EPTB. A total of 1523 EPTB specimens were received from various clinical sites suspected of EPTB infections. All received EPTB specimens were processed in CBNAAT laboratory with aseptic precautions and tested in Gene Xpert MTB / RIF, according to NTEP guidelines. Results: Overall isolation rate of Mycobacterium tuberculosis causing EPTB infection was 208 (13.7 %). Among that rifampicin resistant were 23 (11%). EPTB infections were found in various specimens like pleural fluids 57(15%), FNACs 52 (29 %), pus 50 (54%), gastric aspirates 34 (5%), CSF 7 (8 %), ascitic fluids 5 (7%), other body fluids 2 (10%) and synovial fluid 1 (25%). In present study rifampicin resistant were seen in females 19 (83%) as compared to male 4 (17%). Age group of 11-20 years - 11 (24%) showed highest rifampicin resistance followed by 21-30 years - 08 (16 %). Rifampicin resistance was highest in "medium" level EPTB load - 10 (21%) specimens of EPTB. Conclusions: The study shows a high proportion of rifampicin resistance at tertiary care hospital, Ahmedabad. The review showed that the incidence of extra pulmonary Rifampicin Resistance Tuberculosis has continued to become a serious public health problem. Hope thus, this finding could help the programmatic management of the disease within the context of the National TB Control program (NTEP). [ABSTRACT FROM AUTHOR]

Simple Summary: Colorectal cancer (CRC) is the second leading cause of cancer-related death globally. Developing liquid biopsies that aid in the detection and monitoring of recurrent CRC may improve clinical management and patient outcomes. In this study, we investigated a serially sampled blood assay of methylated DNA markers (MDMs) in combination with carcinoembryonic antigen as a clinical tool to surveil and monitor recurrent cancer in a prospective cohort of patients who completed curative-intent therapy for CRC. We demonstrated that the MDMs detected recurrent CRC before the clinical or radiographic detection of recurrence. In a small number of patients, we further showed that the MDMs may correlate with the tumor burden. These data highlight the importance of incorporating epigenetic signatures into liquid biopsies and support further research to validate the correlations of circulating tumor DNA with the tumor burden. Background: Radiographic surveillance of colorectal cancer (CRC) after curative-intent therapy is costly and unreliable. Methylated DNA markers (MDMs) detected primary CRC and metastatic recurrence with high sensitivity and specificity in cross-sectional studies. This study evaluated using serial MDMs to detect recurrence and monitor the treatment response to anti-cancer therapies. Methods: A nested case-control study was drawn from a prospective cohort of patients with CRC who completed curative-intent therapy for CRC of all stages. Plasma MDMs were assayed vis target enrichment long-probe quantitative-amplified signal assays, normalized to B3GALT6, and analyzed in combination with serum carcinoembryonic antigen to yield an MDM score. Clinical information, including treatment and radiographic measurements of the tumor burden, were longitudinally collected. Results: Of the 35 patients, 18 had recurrence and 17 had no evidence of disease during the study period. The MDM score was positive in 16 out of 18 patients who recurred and only 2 of the 17 patients without recurrence. The MDM score detected recurrence in 12 patients preceding clinical or radiographic detection of recurrent CRC by a median of 106 days (range 90–232 days). Conclusions: Plasma MDMs can detect recurrent CRC prior to radiographic detection; this tumor-agnostic liquid biopsy approach may assist cancer surveillance and monitoring. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is a significant public health challenge, especially in developing nations. Developing a TB eradication strategy is hampered by the global health concern of drug-resistant (DR) TB. Effective patient treatment, preventing TB transfer and avoiding the upsurge of DR strains depend primarily on the timely and accurate identification of DR TB. Due to inadequate sensitivity, the necessity of trained laboratory personnel, the sluggish growth pattern of Mycobacterium bacilli in culture, and the small number of bacilli that are usually found in extrapulmonary TB samples, TB diagnosis is still tricky in clinical practice. Although mycobacterial culture is the gold standard to identify TB and determine drug resistance, it takes 2 to 8 weeks to develop. Despite their high cost, nucleic acid amplification tests (NAATs) and whole-genome sequencing (WGS) are the commonly employed molecular-based methods for diagnosing and identifying TB. The WHO suggested the GeneXpert MTB/RIF to identify TB and detect resistance to rifampicin. In comparison, numerous molecular techniques were developed, including allele-specific PCR (MAS-PCR), solid-phase hybridization, real-time PCR (RT-PCR) and droplet digital PCR-based technique (DDPCR). This manuscript is intended to overview the current approaches for the phenotypic and genotypic diagnosis of TB disease and identifying resistance to antitubercular drugs depending on recently published articles, WHO and CDC reports, and commercially available diagnostic tools. [ABSTRACT FROM AUTHOR]

Graphene oxide (GO) is an oxidized form of graphene accommodating various oxygen-containing functional groups such as hydroxyl, epoxy, and carboxyl groups on its surface. GO has been extensively utilized in various biomedical applications including the delivery of biomolecules and the development of biosensors owing to its beneficial properties such as high surface area, nucleic acid adsorption, and fluorescence quenching through fluorescence resonance energy transfer (FRET). However, despite these favorable properties, the direct utilization of GO in these applications is often limited by low dispersibility in a physiological medium, cytotoxicity, low biocompatibility, and a strong binding affinity of nucleic acids to GO surface. The large surface area of GO and the presence of various functional groups on its surface make it highly amenable to facile surface modifications, offering scope for GO surface functionalization to overcome these limitations. When polyethylene glycol (PEG), which is a biocompatible polymer, is conjugated to GO, the PEGylated GO enhances the biocompatibility and dispersibility, reduces cytotoxicity, and allows controlled drug delivery with controllable binding affinity towards nucleic acid. PEG-engrafted GO retains the beneficial properties of GO while effectively addressing its limitations, rendering it suitable for various biomedical applications. In this review, we present the recent advancements of PEGylated GO in gene/drug delivery and the facilitation of nucleic acid amplification techniques, which aid in the development of therapeutic and diagnostic tools, respectively. [ABSTRACT FROM AUTHOR]

During period of the ongoing COVID-19 pandemic, the unexpected outbreak and worldwide spread of monkeypox has gained global attention. Monkeypox is a viral zoonotic disease caused by MPXV, which is an enveloped, linear, double-stranded DNA virus belonging to the Orthopoxvirus genus, of the Chordopoxvirinaesubfamily, within the Poxviridaefamily. Monkeypox virus (MPXV) is transmitted from human-to-human through direct contact with infectious skin or mucosal skin lesions, respiratory droplets, or indirect contact with contaminated objects or materials, as well as mother-to-child vertical transmission. It is also possibly sexually transmitted through semen/vaginal fluid, and the possibility of community transmission cannot be ruled out. The typical presentation of monkeypox includes prodromal symptoms, followed by a rash that usually begins within13 days of symptom onset, and the skin lesions can last for 24 weeks and then gradually resolve. A definite diagnosis of monkeypox virus infection requires nucleic acid amplification testing via the polymerase chain reaction method. Supportive care is essential along with treatment of complications affecting multiple organs of the body. [ABSTRACT FROM AUTHOR]

Expanding our global testing capacity is critical to preventing and containing pandemics1-9. Accordingly, accessible and adaptable automated platforms that in decentralized settings perform nucleic acid amplification tests resource-efficiently are required10-14. Pooled testing can be extremely efficient if the pooling strategy is based on local viral prevalence15-20; however, it requires automation, small sample volume handling and feedback not available in current bulky, capital-intensive liquid handling technologies21-29. Here we use a swarm of millimetre-sized magnets as mobile robotic agents ('ferrobots') for precise and robust handling of magnetized sample droplets and high-fidelity delivery of flexible workflows based on nucleic acid amplification tests to overcome these limitations. Within a palm-sized printed circuit board-based programmable platform, we demonstrated the myriad of laboratory-equivalent operations involved in pooled testing. These operations were guided by an introduced square matrix pooled testing algorithm to identify the samples from infected patients, while maximizing the testing efficiency. We applied this automated technology for the loop-mediated isothermal amplification and detection of the SARS-CoV-2 virus in clinical samples, in which the test results completely matched those obtained off-chip. This technology is easily manufacturable and distributable, and its adoption for viral testing could lead to a 10-300-fold reduction in reagent costs (depending on the viral prevalence) and three orders of magnitude reduction in instrumentation cost. Therefore, it is a promising solution to expand our testing capacity for pandemic preparedness and to reimagine the automated clinical laboratory of the future.

The objective of this prospective observational study carried out at China-Uganda Friendship Hospital-Naguru in Kampala, Uganda, was to determine the performance of GeneXpert MTB/RIF Ultra (Xpert Ultra) molecular testing on saliva for active tuberculosis (TB) disease among consecutive adults undergoing TB diagnostic evaluation who were Xpert Ultra positive on sputum. We calculated sensitivity to determine TB diagnostic performance in comparison to a composite reference standard of Mycobacterium tuberculosis liquid and solid cultures on two spot sputum specimens. Xpert Ultra on a single saliva sample had a sensitivity of 90% (95% confidence interval [CI], 81 to 95%) relative to the composite sputum culture-based reference standard, similar to the composite sensitivity of 87% (95% CI, 77 to 94%) for fluorescence microscopy (FM) for acid-fast bacilli on two sputum smears. The sensitivity of salivary Xpert Ultra was 24% lower (95% CI for difference, 2 to 48%; P = 0.003) among persons living with HIV (71%; 95% CI, 44 to 90%) than among persons living without HIV (95%; 95% CI, 86 to 99%) and 46% higher (95% CI, 14 to 77%; P 3 million people go undiagnosed and untreated each year. Sputum has been the mainstay for TB diagnosis for over a century but can be difficult for patients to produce. In addition, the vigorous coughing required during sputum collection can lead to infection of nearby individuals and health workers. In this case-only study, applying the ultra-sensitive GeneXpert MTB/RIF Ultra molecular diagnostic assay to saliva detected 90% of culture-confirmed TB cases among 81 adults who were undergoing TB evaluation at the outpatient department of a general hospital in Uganda and tested sputum GeneXpert MTB/RIF Ultra positive. These results suggest that saliva may be a feasible and sensitive alternative to sputum for TB diagnosis, thereby meeting two key metrics proposed by the World Health Organization in its target performance profile for a nonsputum test for TB.

Respiratory pathogens pose a huge threat to public health, especially the highly mutant RNA viruses. Therefore, reliable, on-site, rapid diagnosis of such pathogens is an urgent need. Traditional assays such as nucleic acid amplification tests (NAATs) have good sensitivity and specificity, but these assays require complex sample pre-treatment and a long test time. Herein, we present an on-site biosensor for rapid and multiplex detection of RNA pathogens. Samples with viruses are first lysed in a lysis buffer containing carrier RNA to release the target RNAs. Then, the lysate is used for amplification by one-step reverse transcription and single-direction isothermal strand displacement amplification (SDA). The yield single-strand DNAs (ssDNAs) are visually detected by a lateral flow biosensor. With a secondary signal amplification system, as low as 20 copies/μL of virus can be detected in this study. This assay avoids the process of nucleic acid purification, making it equipment-independent and easier to operate, so it is more suitable for on-site molecular diagnostic applications. [ABSTRACT FROM AUTHOR]

Clostridioides difficile is the most important cause of healthcare-associated diarrhea in the United States. The high incidence and recurrence rates of C. difficile infection (CDI), associated with high morbidity and mortality, pose a public health challenge. Although antibiotics targeting C. difficile bacteria are the first treatment choice, antibiotics also disrupt the indigenous gut flora and, therefore, create an environment that is favorable for recurrent CDI. The challenge of treating CDI is further exacerbated by the rise of antibiotic-resistant strains of C. difficile, placing it among the top five most urgent antibiotic resistance threats in the USA. The evolution of antibiotic resistance in C. difficile involves the acquisition of new resistance mechanisms, which can be shared among various bacterial species and different C. difficile strains within clinical and community settings. This review provides a summary of commonly used diagnostic tests and antibiotic treatment strategies for CDI. In addition, it discusses antibiotic treatment and its resistance mechanisms. This review aims to enhance our current understanding and pinpoint knowledge gaps in antimicrobial resistance mechanisms in C. difficile, with an emphasis on CDI therapies. [ABSTRACT FROM AUTHOR]

In response to the emergence of the monkeypox virus (MPXV) in Australia in May 2022, we developed and evaluated indirect immunofluorescence assays (IFA) for MPXV and Vaccinia virus (VACV) IgG and IgM antibodies using serum samples from patients with nucleic acid amplification test (NAAT)‐confirmed mpox and uninfected unvaccinated controls. Additionally, 47 healthcare workers receiving two doses of the third‐generation smallpox vaccine Modified Vaccinia Ankara‐Bavarian Nordic (MVA‐BN) undertook serial serum collection to describe the serological response to vaccination. MPXV antibodies were detected in 16/18 individuals with NAAT‐confirmed mpox (sensitivity 0.89, specificity 1.00), and VACV antibodies were detected in 28/29 individuals who received two doses of MVA‐BN vaccine (sensitivity 0.97, specificity 1.00). Detectable antibody in subjects historically vaccinated with early‐generation vaccines against smallpox was found in 7/7 subjects, at a median of 48 years following vaccination. MPXV NAAT‐positive patients with serum samples collected within the first 14 days after rash onset had detectable IgG and IgM in 9/12 and 5/12 of patients, respectively, with maintenance of IgG and disappearance of IgM titers after 60 days. While specificity was high when testing unvaccinated and uninfected subjects, significant cross‐reactivity between MPXV and VACV antibodies was observed. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis (VL) is a potentially fatal disease, with an increasing occurrence in northern Italy, affecting children and both immunocompetent and immunocompromised adults. Methods: This retrospective study conducted at the St. Orsola University Hospital of Bologna, Italy, evaluates the characteristics of 16 children (with a median age of 14.3 months) who were hospitalized between 2013 and 2022 for VL. Results: Seventy-five percent of patients presented with a triad of fever, cytopenia, and splenomegaly. An abdominal ultrasound examination revealed splenomegaly and hypoechoic spleen abnormalities in 93.8% and 73.3% of cases, respectively. Five VL cases were complicated by secondary hemophagocytic lymphohistiocytosis. Eleven patients were treated with a single 10 mg/kg dose of Liposomal Amphotericin B (L-AmB), while five received two doses (total of 20 mg/kg); one of the former groups experienced a recurrence. The fever generally decreased 48 h after the first L-AmB dose, and hemoglobin levels normalized within a month. The splenomegaly resolved in approximately 4.5 months. Conclusions: Pediatricians should consider VL in children with fever of an unknown origin, anemia, cytopenia, and splenomegaly. In our experience, abdominal ultrasounds and molecular tests on peripheral blood contributed to diagnosis without the need for bone marrow aspiration. The short-course therapy with two 10 mg/kg doses of L-AmB is safe and effective. [ABSTRACT FROM AUTHOR]

Infective endocarditis due to Kingella kingae is a rare but serious invasive infection that occurs mostly in children. Recent advances in nucleic acid amplification testing as well as in cardiac imaging have enabled more accurate diagnosis. A good understanding of the epidemiology and virulence factors remains crucial to guide the therapeutic approach. Here, we synthesize the current state of knowledge on epidemiological features, pathophysiological insights, complications, and therapy regarding Kingella kingae endocarditis in children and adults. Finally, throughout this comprehensive review, knowledge gaps and areas for future research are also identified. [ABSTRACT FROM AUTHOR]