Explore the vast range of titles available.

Extensive testing is essential to break the transmission of SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Here, we present a CRISPR-based diagnostic assay that is robust to viral genome mutations and temperature, produces results fast, can be applied directly on nasopharyngeal (NP) specimens without RNA purification, and incorporates a human internal control within the same reaction. Specifically, we show that the use of an engineered AsCas12a enzyme enables detection of wildtype and mutated SARS-CoV-2 and allows us to perform the detection step with loop-mediated isothermal amplification (LAMP) at 60-65 °C. We also find that the use of hybrid DNA-RNA guides increases the rate of reaction, enabling our test to be completed within 30 minutes. Utilizing clinical samples from 72 patients with COVID-19 infection and 57 healthy individuals, we demonstrate that our test exhibits a specificity and positive predictive value of 100% with a sensitivity of 50 and 1000 copies per reaction (or 2 and 40 copies per microliter) for purified RNA samples and unpurified NP specimens respectively. As the COVID-19 pandemic continues, variants of the virus are emerging. Here the authors present a diagnostic assay that can detect wildtype and known variants using engineered Cas12a.

The transition from Sanger to next-generation sequencing (NGS) for HIV-1 drug resistance testing offers enhanced sensitivity but also introduces bioinformatic variability. This study evaluated four strategies: the commercial Exatype platform, the academic Stanford HIVdb-NGS, the open-source Quasitools (HyDRA) suite, and a custom de novo assembly workflow, iLunaR. Using 85 clinical HIV-1 pol MiSeq sequencing datasets, concordance was assessed at a 2% mutation detection threshold. A majority consensus standard defined true presence if a mutation was detected by at least three pipelines and supported by Sanger sequencing. While the datasets were successfully processed by all pipelines, discordances emerged in detecting low-abundance mutations and a specific case of structural mutation. iLunaR achieved perfect agreement (Cohen’s kappa = 1.000; 95% CI: 1.000–1.000). Quasitools demonstrated the lowest agreement (Cohen’s kappa = 0.901; 95% CI: 0.807–0.995) due to consistent reporting of mutations at lower abundance levels and aligner-induced reference bias misclassifying a deletion as a point mutation. Exatype (Cohen’s kappa = 0.951; 95% CI: 0.884–1.000) and Stanford (Cohen’s kappa = 0.926; 95% CI: 0.846–1.000) exhibited specific failures, including an omitted integrase mutation and codon translation errors, respectively. These findings confirm that bioinformatic algorithm choice remains a critical clinical variable despite NGS advancements in HIV-1 drug resistance testing.

Coronavirus disease (COVID-19) and tuberculosis (TB) developed in 4 foreign workers living in dormitories in Singapore during April-May 2020. Clinical manifestations and atypical radiographic features of COVID-19 led to the diagnosis of TB through positive interferon-gamma release assay and culture results. During the COVID-19 pandemic, TB should not be overlooked.

Where dengue virus infections are endemic, acute febrile illness is often managed as dengue fever (DF) without diagnostic testing. In a prospective study of 140 patients with clinical features of DF, 3 (2.1%) had acute HIV infection (AHI). We recommend testing for AHI in dengue-like febrile illness.

The viral-inflammatory hypothesis of Alzheimer’s disease offers a new paradigm, yet interventions like antivirals and vaccination present a paradox that challenge therapeutic development. This perspective examines the critical research gap concerning cerebrospinal fluid (CSF) synaptic biomarkers in immunomodulatory therapy trials. Following decades of partially successful amyloid-centric trials, focus has shifted to upstream triggers including viral infections like Herpes Simplex Virus Type 1, Varicella Zoster Virus, and Severe Acute Respiratory Syndrome Coronavirus 2. While large observational and quasi-experimental studies suggest antivirals and vaccines reduce long-term dementia risk, the first major antiviral randomized controlled trial (Valacyclovir for Alzheimer’s Disease) was negative. This perspective posits that this paradox arises from a fundamental flaw in trial design: the absence of synaptic integrity biomarkers. Synaptic loss, not amyloid or tau burden, is the strongest correlate of cognitive decline. Therefore, CSF synaptic protein biomarkers such as the prognostic YWHAG: NPTX2 ratio, postsynaptic Neurogranin (Ng), and presynaptic Growth-Associated Protein 43 (GAP-43) are the most clinically relevant endpoints. The paradoxical trial results may arise from omitting these synaptic measures, creating a mechanistic “black box” obscuring their true biological effects. A strategic framework is proposed, centered on the mandatory inclusion of CSF synaptic biomarkers and relevant co-pathology markers like TAR DNA-Binding Protein 43 (TDP-43; a proteinopathy linked to viral triggers) in all antiviral and vaccine trials. This approach is critical to resolve existing paradoxes, elucidate mechanisms of neuroprotection, and accelerate developing effective therapies that preserve synaptic integrity to prevent and treat dementia.

Dual co-infection with both HSV-1 and HSV-2 is rare, with few cases reported in the literature. In this case report, we describe the successful use of unbiased metagenomic next-generation sequencing (mNGS) as a rapid and alternative method for confirming dual genital herpes co-infection. Our case involves a 74-year-old woman who presented with genital lesions and initially tested positive for both HSV-1 and HSV-2 via the Luminex ARIES HSV 1&2 assay. The entire mNGS process, from nucleic acid extraction to result analysis, was completed in less than 48 h. Using mNGS, we identified mapped reads specific to either HSV-1 or HSV-2 and screened the sequences to rule out mis-genotyping by the Luminex ARIES assay. Notably, the generated sequences can reveal sequence variations within multiple gene regions, demonstrating the potential of mNGS for identifying novel HSV-1 and HSV-2 variants. Our findings suggest that mNGS can serve as a rapid and reliable alternative confirmatory method for dual genital herpes infections, providing valuable information to guide appropriate treatment options for patients. By eliminating the need for prior knowledge of causative agents, mNGS offers an unbiased approach for detecting and characterizing viral co-infections.

The HIV genotypic resistance test (GRT) is a standard of care for the clinical management of HIV/AIDS patients. In recent decades, population or Sanger sequencing has been the foundation for drug resistance monitoring in clinical settings. However, the advent of high-throughput or next-generation sequencing has caused a paradigm shift towards the detection and characterization of low-abundance covert mutations that would otherwise be missed by population sequencing. This is clinically significant, as these mutations can potentially compromise the efficacy of antiretroviral therapy, causing poor virologic suppression. Therefore, it is important to develop a more sensitive method so as to reliably detect clinically actionable drug-resistant mutations (DRMs). Here, we evaluated the diagnostic performance of a laboratory-developed, high-throughput, sequencing-based GRT using 103 archived clinical samples that were previously tested for drug resistance using population sequencing. As expected, high-throughput sequencing found all the DRMs that were detectable by population sequencing. Significantly, 78 additional DRMs were identified only by high-throughput sequencing, which is statistically significant based on McNemar’s test. Overall, our results complement previous studies, supporting the notion that the two methods are well correlated, and the high-throughput sequencing method appears to be an excellent alternative for drug resistance testing in a clinical setting.

Pyrexia of unknown origin (PUO) is defined as a temperature of >38.3°C that lasts for >3 weeks, where no cause can be found despite appropriate investigation. Existing protocols for the work-up of PUO can be extensive and costly, motivating the application of recent advances in molecular diagnostics to pathogen testing. There have been many reports describing various analytical methods and performance of metagenomic pathogen testing in clinical samples but the economics of it has been less well studied. This study pragmatically evaluates the feasibility of introducing metagenomic testing in this setting by assessing the relative cost of clinically-relevant strategies employing this investigative tool under various cost and performance scenarios using Singapore as a demonstration case, and assessing the price and performance benchmarks, which would need to be achieved for metagenomic testing to be potentially considered financially viable relative to the current diagnostic standard. This study has some important limitations: we examined only impact of introducing the metagenomic test to the overall diagnostic cost and excluded costs associated with hospitalization and makes assumptions about the performance of the routine diagnostic tests, limiting the cost of metagenomic test, and the lack of further work-up after positive pathogen detection by the metagenomic test. However, these assumptions were necessary to keep the model within reasonable limits. In spite of these, the simplified presentation lends itself to the illustration of the key insights of our paper. In general, we find the use of metagenomic testing as second-line investigation is effectively dominated, and that use of metagenomic testing at first-line would typically require higher rates of detection or lower cost than currently available in order to be justifiable purely as a cost-saving measure. We conclude that current conditions do not warrant a widespread rush to deploy metagenomic testing to resolve any and all uncertainty, but rather as a front-line technology that should be used in specific contexts, as a supplement to rather than a replacement for careful clinical judgement.