Diagnostic Microbiology and Infectious Disease

Sequencing the respiratory tract transcriptome has the potential to provide insights into infectious pathogens and the hosts immune response. While DNA-based sequencing is more standard in clinical laboratories due to its stability, RNA assays offer unique advantages. RNA reflects dynamic physiological changes, and for RNA viruses, viral RNA particles directly represent copies of the viral genome, enabling greater diagnostic sensitivity. However, RNAs susceptibility to degradation remains a significant challenge, particularly in RNase-rich specimens like saliva. To address this, we conducted a systematic, combinatorial evaluation of 24 distinct mNGS workflows, crossing eight nucleic acid extraction methods with three RNA-Seq library preparation protocols. Remnant saliva samples (n = 6) were pooled and spiked with MS2 phage as a control. The SARS-CoV-2 virus was spiked into half of the samples, which were extracted using the eight different extraction methods (n = 3) and compared using RNA Integrity Number equivalent (RINe) scores and RNA concentration. The extracted RNA was then processed across the three library construction methods and subjected to short-read sequencing to assess all 24 combinations head-to-head. We compared methods based on viral read recovery and found that RINe and concentration did not correlate with viral detection. The Zymo Quick-RNA Magbead kit and the Tecan Revelo RNA-Seq High-Sensitivity RNA library kit were the extraction and library-preparation kits that yielded the most SARS-CoV-2 reads, respectively. Importantly, our combinatorial analysis revealed that any small variability attributable to different nucleic acid extraction methods was heavily overshadowed by differences in quality attributable to the RNA-Seq library preparation methods. These findings challenge the reliance on conventional RNA quality metrics for clinical metagenomics and underscore the need to redefine extraction quality standards for mNGS applications. IMPORTANCEmNGS is a powerful and unbiased approach towards pathogen detection that has mostly been applied to blood and cerebrospinal fluid samples. However mNGS has recently been applied to more areas including the respiratory pathogen detection space, with potential applications in both in-patient diagnostics and public health surveillance. Saliva samples are an ideal sample type for these use cases since they can be collected non-invasively. However, saliva is also a challenging sample type due to its high RNase activity and often yields low-quality nucleic acid. This study explores the feasibility of using saliva specimens in mNGS with contrived SARS-CoV-2 samples to optimize the combination of two factors: nucleic acid extraction and RNA-seq library preparation. Exploration in this area could enhance the sensitivity of saliva-based mNGS assays, with the goal of future expansion of this specimen type in clinical diagnostics and public health surveillance. Key PointsO_LIThe choice of RNA-Seq library preparation kit has a greater impact on pathogen detection than the nucleic acid extraction method. C_LIO_LIThe combination of Zymo Quick-RNA Magbead extraction kit and TECAN Revelo RNA-Seq High Sensitivity RNA library kit recovered the highest percentage of total SARS-CoV-2 reads. C_LIO_LIRNA quantity and RINe score do not correlate with viral read capture, indicating a need for an alternative metric to assess RNA quality for downstream mNGS clinical diagnostics. C_LI

Respiratory syncytial virus (RSV), an approximately 15.2 kb negative sense RNA virus, causes acute respiratory infections in infants and older adults. Its two subtypes, RSV/A and RSV/B, evolve rapidly, making ongoing monitoring of circulating strains essential. The Georgia Public Health Laboratory (GPHL) developed and evaluated an amplicon-based whole-genome sequencing (WGS) assay for RSV surveillance. A total of 214 deidentified remnant clinical specimens (102 RSV/A; 112 RSV/B) with RT PCR Ct values <31 were included. RSV genomes were amplified using ARTIC style and custom primer sets, with the ARTIC set showing superior performance. Libraries were prepared using a modified Illumina COVIDSeq protocol, sequenced on NextSeq 1000/2000 instruments, and analyzed using the GPHL-RSV-PIPE bioinformatics pipeline. Among genomes meeting validation criteria, sequencing depth was slightly higher for RSV/A (median 53,433x; mean 51,076x) than RSV/B (median 49,699x; mean 46,945x), whereas genomic coverage was slightly lower for RSV/A (median 97.5%; mean 96.6%) than RSV/B (median 98.3%; mean 97.6%). Predominant lineages were A.D.3.1 and A.D.5.2 for RSV/A and B.D.E.1 for RSV/B. For RSV/A, the assay showed 92.8% accuracy, 96.2% sensitivity, 87.2% specificity, 92.6% positive predictive value, and 93.2% negative predictive value. Intra and inter run precision assessed using 16 and 53-57 genomes, respectively, showed nearly 100% consensus genome identity with 0 to 5 nucleotide differences. Specificity testing of 31 non-RSV specimens produced no false-positive detections. These results demonstrate that the ARTIC-based RSV WGS assay enables near real time surveillance and strengthens data driven public health responses to future outbreaks.

Introduction: Herpes simplex virus type 1 (HSV-1) is highly prevalent worldwide, making accurate serological testing essential for both clinical diagnosis and epidemiological surveillance. Automated chemiluminescent immunoassays (CLIAs) offer operational advantages over enzyme-linked immunosorbent assays (ELISAs); however, their diagnostic performance relative to Western blot (WB) confirmation in high-prevalence settings remains insufficiently characterized. Hypothesis/Gap Statement: The comparative diagnostic accuracy of CLIA- and ELISA-based assays for HSV-1 IgG detection, when benchmarked against a WB reference standard in endemic populations, remains unclear. Aim: This study aimed to evaluate HSV-1 IgG seroprevalence and diagnostic performance of one CLIA and two ELISA platforms using Western blot as the reference method. Methodology: Four hundred archived serum samples from adult male craft and manual workers in Qatar were tested using the Mindray CL-900i CLIA, HerpeSelect ELISA, NovaLisa ELISA, and Euroimmun Western blot. Seroprevalence, diagnostic accuracy, and interassay agreement were assessed using WB as the reference standard, with equivocal and indeterminate results excluded from analysis. Results: HSV-1 IgG seroprevalence estimates were comparable across assays: HerpeSelect 72.5%, Mindray 70.5%, NovaLisa 66.3%, and Western blot 66.5%, with no statistically significant differences (all p > 0.05). The Mindray CLIA demonstrated the highest diagnostic performance (sensitivity 95.7%, specificity 88.9%, accuracy 93.4%) and strong agreement with Western blot ({kappa} = 0.85). HerpeSelect showed substantial agreement ({kappa} = 0.81), while NovaLisa exhibited lower specificity. Conclusion: CLIA- and ELISA-based assays produced comparable HSV-1 seroprevalence estimates in this high-prevalence population; however, diagnostic accuracy varied across platforms. The CLIA platform demonstrated the strongest agreement with Western blot, supporting its use in high-throughput settings, while confirmatory testing remains important to minimize misclassification.

A multiplex diagnostic test is evaluated for self-reported long COVID associated persistent symptoms and a poor recovery from a SARS-CoV-2 infection. A mass-standardised concentration of total antibodies (AC), high-quality (HQ) antibodies and percentage of HQ antibodies (HQ%) is assessed against a spectrum of spike proteins to the SARS-CoV-2 variants: Wuhan, , {delta}, and the Omicron variants BA.1, BA.2, BA.2.12.1, BA.2.75, BA.5, CH.1.1, BQ.1.1 and XBB.1.5 in three cohorts. A cohort of control patients (n = 46) recovered (CC) and a cohort of self-declared long COVID patients (n = 113) (LCC). A nested Receiver Operating Characteristic (ROC) analysis, performed for the variant with lowest HQ concentration in the spectrum, produced an area under the curve and AUC = 0.61 (0.53-0.70) for the CC vs LCC cohorts. For the LCC cohort, the cut-off thresholds for AC = 0.8 mg/L, HQ = 1.5 mg/L and HQ% of 34% were determined, leading to a 71% sensitivity and 66% specificity derived by the Youden metric. The cohorts may be fully classified based on ROC and outlier analysis to give an incidence of persistent virus 62% (95% CI 52% - 71%), hyperimmune 12% (95% CI 7% - 20%) and unclassified, 26% (95% CI 18% - 35%). The overall diagnostic accuracy for both the hyper and hypo immune is 69%. All clinical interventions can now be tailored for the heterogenous long COVID patient cohort.

Background: Mucopolysaccharidosis type IIIA (MPS IIIA; Sanfilippo syndrome) is a fatal neurodegenerative lysosomal storage disorder caused by impaired degradation of heparan sulfate (HS). Despite rapid advances in gene and enzyme therapies, there remains a critical need for an analytically validated, quantitative biomarker that accurately reflects central nervous system (CNS) substrate burden. Such biomarker would be a valuable tool in assessing disease progression and monitoring therapeutic efficacy. Objective: This study describes the method development, fit for purpose validation, and preliminary clinical application of a quantitative liquid chromatography-mass spectrometry (LC-MS/MS) assay for the HS-derived disaccharide N-sulfoglucosamine-glucuronic acid (GlcNS-GlcUA) in human cerebrospinal fluid (CSF), a critical biomarker for diagnosis, disease monitoring, and regulatory evaluation of emerging MPS IIIA therapies. Methods: A structurally defined GlcNS-GlcUA reference standard and its [13C6]-labeled internal standard were used in a derivatization and detection workflow employing 1-phenyl-3-methyl-5-pyrazolone labeling, and LC-MS/MS. Results: The method exhibited acceptable linearity across 0.005-0.500 nmol/mL (r[&ge;]0.9976), with intra- and inter-assay imprecision [&le;]3.5%CV and accuracy within 95%-110% of nominal concentrations. No matrix or hemolysis interference or carryover was observed, and the analyte remained stable during freeze-thaw storage conditions. Application of the method to 12 CSF samples from patients with MPS IIIA demonstrated quantifiable GlcNS-GlcUA levels ranging from 0.0054 to 0.106 nmol/mL, confirming suitability for clinical and regulatory use. Comparison of the MPS IIIA sample results between the development laboratory and the contract research organization laboratory support robust inter-lab assay transfer. Conclusions: This validated LC-MS/MS method establishes a regulatory-grade quantitative assay for measurement of CSF HS in MPS IIIA. Its high analytical sensitivity and reproducibility enable reliable assessment of CNS substrate reduction and pharmacodynamic response, supporting biomarker-driven therapeutic development and accelerated approval pathways for neuronopathic mucopolysaccharidoses.

Background: Sub-Saharan Africa (SSA) still experiences a high burden of micronutrient deficiencies. For monitoring of micronutrient status among young children in SSA, non-invasive alternatives to blood-based biomarkers are desirable. Handheld Raman spectrophotometry appears to offer this alternative to quantify intracellular stores of micronutrients. In rural Burkina Faso and Kenya, we validated the Cell-/SO-Check device (ZellCheck(R)) against conventional laboratory-based methods. Methods: For this validation study, we recruited children aged [&ge;]24 months attending routine clinics within the Health and Demographic Surveillance Systems (HDSS) in Siaya and Nouna. Anthropometric measurements and venous blood samples were taken. Plasma ferritin, soluble transferrin receptor (sTfR) and C-reactive protein (CRP) were measured by ELISA, and plasma zinc by atom absorption. The spectrometer was used to quantify zinc and iron. For continuous outcomes, we generated Bland Altman plots and calculated bias and limits of agreement (LoA). For binary outcomes, we produced Receiver Operator Characteristic (ROC) areas under the curve (AUC), and estimated sensitivity, specificity and predictive values. Results: We analysed data of 48 children from Burkina Faso and 54 children from Kenya (male: 53%; age range: 24-66 months). According to spectrophotometry, the proportions of iron deficiency and zinc deficiency were 16.7% and 25.5%, respectively. The median concentrations were for ferritin 24.0 {micro}g/L (range: 2.0-330.0), for sTfR 5.7 mg/L (2.8-51.0), and for zinc 9.9 {micro}mol/L (5.2-25.0). The corresponding bias for iron levels by spectrophotometry was 42.4 with LoA: -18.7, 103.6. The bias for zinc levels was 7.5 with LoA: -49.3, 64.2. For the classification of deficiency, the ROC-AUC, sensitivity, and specificity for spectrophotometry vs. biomarker-based diagnosis were for iron deficiency 0.62, 68% and 55%, respectively, and for zinc deficiency 0.55, 33% and 91%, respectively. Conclusions: The Cell-/SO-Check device may be used to rank children in population-based studies in SSA according to their zinc status, but not iron status. The method should not replace the standard laboratory measurements for clinical diagnoses of zinc and iron deficiencies.

Background Emergency departments (EDs) in high-income countries face rising demand, workforce shortages and crowding. We investigated whether prehospital point-of-care blood gas analysis (BGA), used by emergency physicians, is associated with higher ambulatory treatment rates and improved patient selection for hospital admission. Methods We retrospectively analysed routinely collected data from a pilot implementation of a mobile blood gas analyser in physician-staffed emergency medical services (EMS) in Jena, Germany (July 2023 to May 2024). Adult emergency patients receiving prehospital BGA were compared with propensity score-matched EMS controls without BGA. Primary outcomes were the proportion treated on scene and, among transported patients, the hospital admission rate. Secondary outcomes were 30-day safety among ambulatory patients and associations between BGA parameters and disposition. We used standardised mean differences to assess balance and receiver operating characteristic analysis for lactate thresholds. Results Of 109 patients receiving prehospital BGA, 98 met inclusion criteria after excluding 9 patients with missing NACA scores, 1 on-scene death and 1 invalid age record; these were matched to 390 controls (total n = 488). Baseline demographics, severity and vital signs were well balanced. Ambulatory treatment was markedly higher in the BGA cohort compared with matched controls (27.6% vs 8.7%; OR 3.98, 95% CI 2.26 to 7.01; p<0.001). No ambulatory BGA patient required ED re-attendance or repeat EMS contact within 30 days. Among transported patients, 58% in the BGA cohort were admitted to hospital, compared with an overall regional ED conversion rate of approximately 30%. Lactate [&ge;]2.6 mmol/L was the most influential parameter for disposition decisions, with elevated lactate and acid-base disturbances strongly associated with transport and admission. Conclusion Prehospital BGA was associated with fourfold higher ambulatory treatment rates (27.6%) and a twofold higher ED conversion rate among the patients who were transported (58%), indicating improved risk stratification and resource allocation. These findings suggest that integrating objective biochemical data into prehospital assessment may enhance treat-and-refer decision-making and support more efficient use of limited emergency care capacity.

Religion has contributed to societal divides regarding COVID-19 mRNA vaccines. In this study, we conducted a secondary analysis of a survey of U.S. adults (N=4939) focused on how religious affiliations, beliefs, and practices impact attitudes toward genetic and genomic activities, one of which was mRNA vaccines. The dataset included large samples of participants from six religious groups in the U.S. (Black Protestant, Catholic, Evangelical Protestant, Jewish, Mainline Protestant, and Muslim), as well as individuals who were atheist, agnostic, or spiritual. ANCOVA results indicated that Evangelical Protestant participants showed significantly less support for mRNA vaccines than other groups, while atheist participants were the most supportive. Muslim participants had the highest concerns, whereas atheist participants had the lowest. Regression analyses indicated the strongest predictors of support for mRNA vaccines were more spiritual community support for community health, followed by higher acceptance of evolution, more liberal political orientation, less distrust toward the healthcare system, higher frequency of attending religious activities, higher income, lower fundamentalist religious beliefs, and more spiritual community support for liberal reproductive and end of life views. The strongest predictors of concerns about mRNA vaccines were more distrust toward the healthcare system and more conservative political orientation, followed by less spiritual community support for community health, stronger beliefs about God in the body, more fundamentalist religious beliefs, and lower knowledge of genetics. The large sample size, and examination of a broad array of religious variables alongside distrust and political orientation offer new insights. These findings add to the literature on the culture wars surrounding mRNA vaccines, and can perhaps aid in future efforts to build trust and relationships between public health and religious communities.

The Klebsiella pneumoniae species complex (KpSC) is a clinically important group of closely related pathogens associated with invasive infections. The complex comprises seven closely related members, which are often reported as K. pneumoniae, particularly in resource-limited settings. Accurate differentiation of KpSC members remains challenging because routine laboratory methods lack sufficient resolution, and approaches like mass spectrometry and whole genome sequencing (WGS) are not widely available. Consequently, the epidemiology and clinical significance of non-K. pneumoniae members of the KpSC remain underrecognized. We developed a conventional multiplex mismatch amplification mutation assay (MAMA) PCR targeting species- and subspecies-specific single-nucleotide polymorphisms in the housekeeping gene rpoB, with six primer sets for differentiation of common KpSC members. The assay was validated against 49 genomically characterized clinical isolates, after which 179 wastewater-derived isolates provisionally identified as Klebsiella spp. by standard microbiological methods were tested. Of these, 174 were assigned to specific KpSC members by the assay, while 5 produced inconclusive amplification patterns. A subset of 16 environmental isolates was selected for WGS, including four of the five inconclusive isolates. All environmental isolates with interpretable MAMA PCR patterns were concordant with WGS. The four inconclusive environmental isolates were identified as Enterobacter spp. Overall, comparison of MAMA PCR with WGS showed 100% sensitivity and 100% specificity for all tested targets, and the total cost was approximately US$1. This rpoB-based multiplex MAMA PCR provides a simple, accurate, and low-cost approach for differentiation of KpSC members in routine laboratories and may support improved identification and surveillance in resource-limited settings. ImportanceThe Klebsiella pneumoniae species complex (KpSC) has seven members but is often reported as a single organism in routine laboratories, masking clinically and epidemiologically important diversity. As a result, the contribution of non-K. pneumoniae KpSC members to human and environmental microbiology remains poorly defined, especially in low-resource settings. We developed a conventional multiplex mismatch amplification mutation assay (MAMA) PCR based on discriminatory rpoB single nucleotide polymorphisms for differentiation of common KpSC members using standard PCR and agarose gel electrophoresis. The assay demonstrated 100% sensitivity and 100% specificity against whole-genome sequencing and excluded non-Klebsiella environmental isolates initially identified as Klebsiella pneumoniae using standard microbiological procedures. With an estimated per-test cost of about US$1, this method offers an affordable and scalable option for laboratories seeking more accurate KpSC identification and improved surveillance.

Background: Mucormycosis is a rapidly progressive and often fatal invasive fungal infection caused by moulds in the order, Mucorales. Early diagnosis is essential for effective clinical management; however, conventional diagnostic approaches such as culture and histopathology are slow, insensitive, and require specialist mycological expertise. Although molecular methods are available for disease detection, they are not widely accessible. At present, no enzyme immunoassay (EIA) exists for the detection of mucormycosis. Methods: A murine IgG1 monoclonal antibody (mAb), FH12, was generated against extracellular polysaccharides (EPSs) produced by Mucorales pathogens during active growth. The antibody was characterised for specificity, epitope stability, and antigen localisation using ELISA, immunoblotting, and immunofluorescence techniques. The mAb was incorporated into a Sandwich-ELISA and evaluated using culture filtrates, purified EPSs spiked into human serum, and tissue homogenates from a patient with cutaneous mucormycosis caused by Lichtheimia ramosa. Results: mAb FH12 demonstrated pan-Mucorales specificity and no cross-reactivity with other clinically relevant yeasts and moulds. The epitope recognised by FH12 is periodate-insensitive and moderately heat-stable. The Sandwich-ELISA detected EPS antigens in human serum with limits of detection ranging from pg/mL to low ng/mL levels, and successfully identified the EPS biomarker in patient tissue homogenates. Conclusion: The FH12-based Sandwich-ELISA shows high sensitivity and specificity, and has the potential to be used as a laboratory-based adjunct diagnostic test for the detection of mucormycosis in humans.

Currently there is a lack of high-throughput, low material-input methods to screen early-stage product quality of viral and non-viral gene therapy products. Here we propose using multiplex droplet digital PCR (dPCR) to screen and characterize vector sequences. We describe the adaptation of a Poisson-multinomial model to quantitate integrity of any combination of 4 targets in multiplexed ddPCR. We show the success and limitations of model employment and provide some suggested best practices.

Background: External Quality Assurance (EQA) is an essential component of modern laboratory medicine. Current scientific evidence on EQA focuses primarily on the analyses carried out by EQA providers while relatively little research has been conducted in individual clinical laboratories. Methods: In this retrospective single-center observational study in a clinical laboratory, EQA results were analyzed over a period of four years (2021-2024). The evaluation was based on EQA action reports documented in the institutes internal quality management system. Deviations were classified according to department, type of discrepancy, root cause category (analytical, preanalytical, systemic, unidentifiable), and measures taken. Results: A total of 7226 EQA participations were evaluated during the observation period. The overall error rate remained consistently low, ranging between 0.8% and 1.6%, with no significant change over time (p = 0.87). Most deviations occurred in the departments of clinical chemistry and immuno/autoimmune diagnostics (p < 0.001). These were predominantly quantitative discrepancies (false low/false negative or false high/false positive). Root cause analysis showed a clear dominance of analytical causes (p < 0.001), while preanalytical and systemic causes were identified less frequently. In most cases, corrective measures, such as re-analyses, recalibrations, process adjustments, or staff training, were implemented promptly. Hard structural measures, such as changing methods or discontinuing tests, were rarely necessary. Conclusion: In a clinical laboratory, EQA is an important tool for structured error analysis and continuous quality improvement. Consistent processing of deviating EQA results goes hand in hand with stable analytical performance and a low error rate.

Background: Smell testing is increasingly recognized as essential in rhinology practice but remains underutilized because of time constraints and limited clinical resources. This study aimed to evaluate the performance (test-retest reliability, accuracy and test completion time) of a self-administered, digital version of SMELL-RS, a non-semantic test of olfactory resolution (SMELL-R) and sensitivity (SMELL-S). Methodology: We performed a test-retest reliability study in a tertiary care facility. We enrolled 100 subjects with and without smell dysfunction. The primary outcome measures were two replicates of olfactory test scores (SMELL-RS composite score, SMELL-R score, SMELL-S score). The secondary outcome measures were Sniffin Sticks score, test completion time, patient demographics, and other clinical characteristics (clinical symptoms, etiologies). Results: The SMELL-RS composite score was reliable (ICC=0.71; p<0.0001) and correlated with the Sniffin Sticks composite score (r=0.68; p<0.0001). Different etiologies have different magnitudes of smell loss as revealed by the SMELL-RS score. SMELL-S reduces misdiagnosis associated with Sniffin Sticks threshold tests. The average completion time of the olfactory resolution test (SMELL-R) was on average 5.9 minutes (SD=1.9), while the average completion time of the olfactory sensitivity test (SMELL-S) was 5.5 minutes (SD=2.7). This is two to three times faster than the corresponding Sniffin Sticks tests. Conclusions: SMELL-RS is a rapid, fully automated, reliable, and accurate olfactory test suitable for self-administration in a clinical setting.

1.Etiological diagnosis of lower respiratory tract infections (LRTIs) relies on sputum or bronchoalveolar lavage (BAL), which may be difficult to obtain or invasive. Exhaled breath aerosol (XBA) sampling offers a non-invasive alternative for pathogen detection. We evaluated the performance of the AveloMask, a face mask-based device designed to capture XBAs for molecular testing. In this prospective paired-sample study, hospitalized adults with pneumonia at three hospitals in Switzerland and Georgia provided an XBA sample using the AveloMask and a lower respiratory tract (LRT) specimen (sputum or BAL). XBA samples were analyzed by multiplex PCR using the Roche LightMix(R) panel and LRT samples were tested using the BioFire(R) FilmArray(R) Pneumonia Panel. Concordance between XBA and LRT samples was assessed using positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement (OPA). Ninety-three participants were enrolled and 63 participants provided paired samples. AveloMask sampling identified the dominant pathogen (lowest Ct value in the LRT sample) in 40/47 LRT-positive cases (85.1%). Across all targets, PPA was 61% (95%CI, 50-72%), NPA was 100% (95%CI, 99-100%), and OPA was 95% (95% CI, 92-96%). PPA was higher for bacteria than for viruses and lower PPA was largely driven by reduced detection of low-abundance or co-infecting pathogens. In a subset analysis, AveloMask results showed substantial overlap with standard-of-care testing and could have supported antimicrobial de-escalation. Breath aerosol sampling using the AveloMask enabled non-invasive molecular detection of LRT pathogens in pneumonia cases and may complement conventional standard-of-care testing, particularly when sputum is unavailable.

This novel study detected persistent low level infections of Elephant Endotheliotropic Herpesviruses (EEHV), that can cause highly pathogenic Elephant Hemorrhagic Disease (EHD) in Loxodonta and Elephas, and co-infection of presumed less pathogenic Elephant Gammaherpesviruses (EGHV), in skin nodule biopsies, saliva and tissues collected from 43 wild L. africana (savannah elephant) in Botswana, Kenya, South Africa and Zimbabwe; in saliva from 25 wild L. cyclotis (forest elephant) in Gabon; and in saliva collected over seven years from 7 wild-born L.africana at Six Flags Safari Park, USA; and in saliva, blood and tissues from an additional 200 L. africana in USA zoos. DNA from these samples was extracted in our USA laboratories and amplified by conventional polymerase chain reaction using three-round nested primer sets designed specifically to screen for known EEHV and EGHV genes loci and to discover new species and subtypes. Sanger sequencing of purified DNA from nearly all samples yielded unambiguous positive genetic matches to previously known Loxodonta-associated EEHV2, EEHV3A, EEHV3B, EEHV6, EEHV7A, and EGHV1B, EGHV2, EGHV3B, EGHV4B, EGHV5B and discovered novel types EEHV3C-H and EEHV7B and the prototype EGHV1B. Many of the primer sets used could also have detected known Elephas-associated EEHV1A, EEHV1B, EEHV4, and EEHV5 if present in these samples, but they did not. Our extensive library of EEHV and EGHV sequences from wild and zoo Loxodonta, (as well as from 100 zoo Elephas maximus not discussed in this review), is a significant contribution to the elephant virology community, particularly for comparing subtypes types of EEHV found in pathogenic cases of EHD in zoos as well as determining and comparing species and subtypes of EEHV present in existing zoo herds, and in individual elephants being transported between zoos, and for importation of wild elephants into existing zoo herds.

PurposeAntibodies to Epstein-Barr virus (EBV) proteins can predict nasopharyngeal carcinoma (NPC) risk. We previously defined a prototype EBNA1 protein panel and multiplex immunoblot assay that distinguishes NPC risk several years pre-diagnosis. Assay throughput and specificity are critical to effectively implement a population-level screening program. Here, we developed a strip test assay - EBNA1 SeroStrip-HT - with an objective to increase throughput and maximize specificity. Experimental DesignEBNA1 full-length (FL) and glycine-alanine repeat deletion mutants (dGAr) were purified from insect and mammalian cells to screen serum IgA/IgG from prospective cohorts in Singapore and Shanghai, China, with known time intervals to NPC diagnosis. Twenty pre-diagnostic sera within 4 years to diagnosis were compared to 96 healthy controls using a nested case-control study design. ResultsIgA to mammalian-derived EBNA1 dGAr achieved 85.0% sensitivity and 94.8% specificity (AUC, 0.939) for NPC status. IgA to insect-derived EBNA1 dGAr showed the same sensitivity (85.0%) and similar specificity (93.8%) (AUC, 0.941). IgA to insect-derived EBNA1 FL had a higher 90% sensitivity, but lower 91.7% specificity (AUC, 0.940). Combining EBNA1 FL and dGAr results showed that subjects positive for both proteins had a 243.67 odds ratio for NPC incidence compared to double-negative scores. ConclusionThis study demonstrated the efficacy of EBNA1 SeroStrip-HT for NPC risk assessment and stratification in high- and intermediate-risk populations, yielding high accuracy and a 12-fold increased throughput over the prototype. The insect system was appropriate for large-scale production of purified EBNA1. Larger, geographically diverse cohorts are warranted to confirm these results, especially in low-incidence populations.

BackgroundART effectively suppresses HIV replication and restores CD4+ T cells; however, long-lived HIV latent reservoirs enable viral persistence. Tuberculosis (TB) co-infection further impacts HIV latency and enhances viral replication. Given the high prevalence of latent TB infection (LTBI) in TB-endemic settings, understanding its impact on HIV biology is critical. Our study aims to investigate the influence of TB co-infection on HIV reservoir dynamics, viral diversity, and drug resistance mutations in ART-naive individuals. MethodologySamples from 90 ART-naive HIV-1C individuals, stratified based on IGRA and TB diagnosis, were used in this study. Plasma and PBMCs were isolated for viral RNA and DNA extraction respectively. Total proviral DNA was quantified using gag PCR. Full-length env and pol genes were amplified, purified and sequenced using ONT and Illumina platforms. Pol sequences were subjected to Drug Resistance Mutation (DRM) analysis via Stanford HIVdb with a minimum threshold mutation frequency of [&ge;]10%. Full length env sequences were used for phylogenetic analysis by aligning with Indian Subtype C reference sequence and phylogenetic tree was generated using ggplot2. ResultProviral load analysis showed no significant differences across HIV+LTBI-, HIV+LTBI+, and HIV+TB+ groups, although a trend toward higher levels was observed in HIV+TB+ individuals. Correlation analysis revealed distinct immune associations, with HIV+LTBI+ individuals showing positive correlations with activation and PD-1 expression. Longitudinal analysis of proviral loads demonstrated a modest decline in proviral load post-ART but remained persistent for up to 18-20 months following initiation of ART accompanied by low level ongoing viral replication. DRM analysis revealed a 33% prevalence in ART-naive individuals, with higher occurrence in HIV+LTBI+ group. Of the identified DRMs, 38% (5/13) and 71% (5/7) in sequences obtained from PBMC and plasma respectively were attributed to polymorphic mutations associated with Integrase strand transfer inhibitors (INSTIs). DRMs within plasma and PBMC derived viruses showed high concordance. Phylogenetic analysis of env sequences indicated overlapping viral populations between the 3 groups, with greater diversity in PBMCs compared to plasma. ConclusionThe study highlights that HIV reservoir dynamics, drug resistance, and viral diversity are significantly influenced by TB co-infection. While proviral loads were comparable, LTBI-associated immune activation and granuloma niches may have driven viral diversification and DRM emergence. High concordance between compartments and presence of transmitted resistance underscore the need for baseline screening, multi-compartment analysis, and sustained surveillance.

BackgroundLower respiratory tract infections (LRTIs) remain diagnostically challenging when culture and molecular assays are negative or delayed. We evaluated ONETest Pathogenome (OT), an automated hybrid-capture metagenomic assay with core-genome enrichment probes, for direct pathogen detection in bronchoalveolar lavage (BAL). MethodsAnalytical performance (LoD, precision, continuity) was assessed using whole-cell spike-ins into culture-negative BAL fluid. Technical performance was assessed in 119 specimens profiled by OT and whole-metagenome shotgun sequencing (WmGS, cohort 1). Clinical accuracy was evaluated in 360 specimens (cohort 2) benchmarked against routine bacterial and acid-fast bacillus (AFB) culture. Laboratory-developed test (LDT) validation included 43 specimens (cohort 3) benchmarked to bacterial and AFB culture. ResultsOT uses 6.2 million probes covering core genomes across 50 microbial families (>250 respiratory pathogens). In BAL specimens, OT increased normalized on-target microbial abundance 26-fold versus that of WmGS while preserving within-sample microbial diversity. In cohort 2, OT achieved species-level sensitivity of 80% and specificity of 99% across culture-confirmed isolates and detected [&ge;]1 culture-confirmed organism in 100/115 culture-positive specimens (87%), while applying species-specific background baselines to mitigate overcalling. Additive yield was 21% (76/360), with 7.5% (27/360) of specimens having [&ge;]1 additional finding supported by orthogonal testing. In LDT validation, OT identified [&ge;]1 culture-confirmed organism in 34/40 culture-positive specimens (85%) with one OT-positive/culture-negative specimen. ConclusionsOT is an assay with a turnaround time <24 h complementary to culture that improves pathogen detection and expands microbiologic findings through additional detections and co-detections, including slow-growing organisms that may require prolonged incubation by conventional methods.

Background As part of Singapore's effort towards precision medicine tailored to Asian diversity, we describe the implementation of a nationwide reproductive carrier screening program. Using a customised 112-gene panel, incorporating population-specific recessive genetic diseases, we outline the overall program design, and initial efforts of community and stakeholder engagement, to inform culturally appropriate implementation. Methods Participants receive culturally tailored online education regarding our reproductive screening program and are provided results with genetic counselling and reproductive options. Community and stakeholder perspectives were assessed through questionnaires and consultations with religious leaders. Results Recruitment is nation-wide, and since initiation of our pilot phase in September 2024, 1,619 couples have registered interest, with 60% uptake of those deemed eligible. Among the 456 couples that have received results to date, four couples (0.9%) were identified to be at increased risk. Community questionnaire responses (n=1002), involving couples who participated in the program as well as the general public, indicated interest is high (59%) across the cohort but awareness, intent to participate and implications for reproductive options differed by sociodemographic factors such as ancestry and religion. Healthcare professional respondents (n=113) acknowledged carrier screening will be routine in medical care, but report limited confidence and resources. Engagement with religious leaders indicated support for the program. Conclusion These early program outcomes and community engagement are guiding the implementation of expanding population-based carrier screening in Singapore, contingent on addressing practical challenges through equitable outreach and professional training.

Background: Pharmacogenomic testing (PGx) can optimise drug efficacy and minimise toxicity, but the extent of prescriber adherence to PGx recommendations remains unclear. We aimed to quantify clinician adherence to international genotype-guided prescribing recommendations in a cohort of paediatric oncology patients. Methods: We reviewed files of children enrolled in the MARVEL-PIC (NCT05667766) randomised control trial, who had PGx recommendations available. Patients were included if 12 weeks had passed since their PGx report was released to clinicians. Prescribing events were identified for actionable PGx recommendations, and classified as "explicitly followed", "inadvertently followed", or "not followed". Adherence was assessed by patient, drug, and recommendation. Results: 2,063 PGx recommendations were available for 216 patients. 64 (3.1%) recommendations were actionable for 44 patients and 10 drugs within the 12-week study period. Recommendations were explicitly followed in 57/288 (19.8%) of prescribing events, inadvertently followed in 145 (50.3%), and not followed in 86 (29.9%). Mercaptopurine demonstrated the highest rate of explicit adherence (87.5%). No significant associations were observed between adherence and age group, cancer type, drug type, or strength of recommendation. Conclusion: Adherence to pharmacogenomic recommendations was very low, highlighting the need to understand barriers to PGx implementation, and consideration of clinical decision supports to facilitate adherence.