Asymptomatic enteric pathogen carriage and its association with proton pump inhibitors use in men who have sex with men in Taiwan, 2019-2022.

OBJECTIVES: Currently recognized risk factors for sexually transmitted enteric infections (STEIs) among men who have sex with men (MSM) include oroanal sex, multiple sexual partners, and chemsex. This study aimed to investigate the prevalence of the asymptomatic carriage of enteric pathogens in men who have sex with men (MSM) and to identify the associated risk factors. METHODS: Questionnaires were completed by 375 MSM in Taiwan from December 2019 to November 2022. Fecal samples were analyzed by multiplex PCR to determine whether seven enteric pathogens, including Entamoeba histolytica, Giardia duodenalis, Shiga toxin-producing Escherichia coli, Cryptosporidium, Campylobacter, Salmonella, and Shigella species, were present. RESULTS: Among 375 fecal samples from asymptomatic MSM, 27 (7.2%) fecal samples tested positive for at least one enteric pathogen. The recent use of proton pump inhibitors (PPIs) was significantly associated with asymptomatic fecal carriage (22.2% vs. 2.0%, P < 0.001). G. duodenalis (2.1%, 8 cases), E. histolytica (1.6%, 6 cases), and Shigella species (1.3%, 5 cases) were commonly detected. Oroanal sex and PPI use were associated with the asymptomatic carriage of enteric pathogens. Specifically, Shigella, Salmonella, or Campylobacter carriage was significantly correlated with PPI use. In contrast, rectal gonorrhea was associated with multiple sexual partners and prior syphilis. CONCLUSIONS: Recent use of PPIs was associated with the asymptomatic carriage of enteric pathogens. Therefore, targeted education about the appropriate use of PPIs is necessary to mitigate the risk of STEIs among MSM.

Innovative and versatile surface-enhanced Raman spectroscopy-inspired approaches for viral detection leading to clinical applications: A review.

The evolution of analytical techniques has opened the possibilities of accurate analyte detection through a straightforward method and short acquisition time, leading towards their applicability to identify medical conditions. Surface-enhanced Raman spectroscopy (SERS) has long been proven effective for rapid detection and relies on SERS spectra that are unique to each specific analyte. However, the complexity of viruses poses challenges to SERS and hinders further progress in its practical applications. The principle of SERS revolves around the interaction among substrate, analyte, and Raman laser, but most studies only emphasize the substrate, especially label-free methods, and the synergy among these factors is often ignored. Therefore, issues related to reproducibility and consistency of results, which are crucial for medical diagnosis and are the main highlights of this review, can be understood and largely addressed when considering these interactions. Viruses are composed of multiple surface components and can be detected by label-free SERS, but the presence of non-target molecules in clinical samples interferes with the detection process. Appropriate spectral data processing workflow also plays an important role in the interpretation of results. Furthermore, integrating machine learning into data processing can account for changes brought about by the presence of non-target molecules when analyzing spectral features to accurately group the data, for example, whether the sample corresponds to a positive or negative patient, and whether a virus variant or multiple viruses are present in the sample. Subsequently, advances in interdisciplinary fields can bring SERS closer to practical applications.

Focused ion beam-fabricated nanorod substrate for label-free surface-enhanced Raman spectroscopy and enabling dual virus detection.

The COVID-19 pandemic presents global challenges, notably with co-infections in respiratory tract involving SARS-CoV-2 variants and influenza strains. Detecting multiple viruses simultaneously is crucial for accurate diagnosis, effective tracking infectious sources, and containment of the epidemic. This study uses a label-free surface-enhanced Raman spectroscopy (SERS) method using Au NPs/pZrO2 (250) and FIB-made Au NRs (100) to detect dual viruses, including SARS-CoV-2 Delta variant (D) and influenza A (A) or B (B) virus. Results demonstrate distinct peaks facilitating virus differentiation, especially between D and A or B, with clear disparities between substrates; specific peaks at 950 and 1337 cm-1 are pivotal for discerning viruses using Au NPs/pZrO2 (250), while those at 1050, 1394, and 1450 cm-1 and 1033, 1165, 1337, and 1378 cm-1 are key for validation using Au NRs (100). Differences in substrate surface morphology and spatial disposition of accommodating viruses significantly influence hotspot formation and Raman signal amplification efficiency, thereby affecting the ability to distinguish various viruses. Furthermore, both substrates offer insights, even in the presence of oxymetazoline hydrochloride (an interfering substance), with practical implications in viral diagnosis. The customized design and reproducibility underscore efficient Raman signal amplification, even in challenging environments, highlighting potential for widespread virus detection.

The molecular epidemiology of a dengue virus outbreak in Taiwan: population wide versus infrapopulation mutation analysis.

Dengue virus (DENV) causes approximately 390 million dengue infections worldwide every year. There were 22,777 reported DENV infections in Tainan, Taiwan in 2015. In this study, we sequenced the C-prM-E genes from 45 DENV 2015 strains, and phylogenetic analysis based on C-prM-E genes revealed that all strains were classified as DENV serotype 2 Cosmopolitan genotype. Sequence analysis comparing different DENV-2 genotypes and Cosmopolitan DENV-2 sequences prior to 2015 showed a clade replacement event in the DENV-2 Cosmopolitan genotype. Additionally, a major substitution C-A314G (K73R) was found in the capsid region which may have contributed to the clade replacement event. Reverse genetics virus rgC-A314G (K73R) showed slower replication in BHK-21 and C6/36 cells compared to wildtype virus, as well as a decrease in NS1 production in BHK-21-infected cells. After a series of passaging, the C-A314G (K73R) mutation reverted to wildtype and was thus considered to be unstable. Next generation sequencing (NGS) of three sera collected from a single DENV2-infected patient at 1-, 2-, and 5-days post-admission was employed to examine the genetic diversity over-time and mutations that may work in conjunction with C-A314G (K73R). Results showed that the number of haplotypes decreased with time in the DENV-infected patient. On the fifth day after admission, two new haplotypes emerged, and a single non-synonymous NS4A-L115I mutation was identified. Therefore, we have identified a persistent mutation C-A314G (K73R) in all of the DENV-2 isolates, and during the course of an infection, a single new non-synonymous mutation in the NS4A region appears in the virus population within a single host. The C-A314G (K73R) thus may have played a role in the DENV-2 2015 outbreak while the NS4A-L115I may be advantageous during DENV infection within the host.

Antiviral and immunoregulatory effects of curcumin on coxsackievirus B3-infected hepatitis.

Fulminant hepatitis is a life-threatening complication of coxsackievirus B (CVB) 3 infections. The condition may deteriorate to disseminated intravascular coagulopathy with markedly increased liver enzymes, inflammatory cytokines, and chemokines, which significantly induce local and systemic inflammation. Curcumin exhibits anti-inflammatory and antiviral characteristics in inflammatory and infectious diseases. Here we determined effects of curcumin on viral replications, cytokine and chemokine expressions, and liver damage in CVB3-infected Huh-7 cells. The mouse-adapted CVB3 strain was used to investigate the antiviral and anti-inflammatory effects of curcumin on CVB3-induced hepatitis in a mouse model. In vitro studies showed that curcumin reduced viral protein and titer levels and increased cell viability. Curcumin enhanced the heme oxygenase-1 (HO-1) protein level and decreased the levels of cleaved caspase-3 protein and mRNA of gene encoding C-X-C motif chemokine 10 in infected cells. In vivo studies showed that curcumin improved the survival rate and clinical scores in mice and reduced the viral titer in the liver during CVB3 infection. Moreover, the HO-1 levels were increased, and the cleaved caspase-3 levels were diminished in the CVB3-infected liver. Curcumin reduced the levels of interferon (IFN)-γ and monokine induced by IFN-γ in liver and levels of interleukin (IL)-8 in serum, but increased levels of regulated activation, normal T cell expression in liver and levels of IL-10 in serum of CVB3-infected mice. In summary, curcumin presents antiviral and anti-inflammation efficacies in CVB3 infection in vitro and in vivo; these results provide potential evidence on the feasibility of curcumin for clinical treatment.

Acyclovir-resistant HSV-1 isolates among immunocompromised patients in southern Taiwan: Low prevalence and novel mutations.

Herpes simplex virus type 1 (HSV-1) can establish latency in humans and easily relapse in immunocompromised patients, with significant mortality. Treatment with acyclovir (ACV) can result in the emergence of HSV resistance. A total of 440 frozen HSV-1 isolates collected from 318 patients from January 2014 to July 2019 were obtained from National Cheng Kung University Hospital in southern Taiwan. These 440 isolates were subjected to phenotypic studies for ACV-resistance by initial screening with the plaque reduction assay (PRA) and further validation by the DNA reduction assay (DRA). The ACV-resistant strains were further investigated by Sanger sequencing for the full-length UL23 and UL30 genes, which encode thymidine kinase and DNA polymerase, respectively. Hematological malignancies or hematopoietic stem-cell transplantation patients accounted for 56.9% (124/218) among the immunocompromised patients (218/318) in this study. Repeated sampling for HSV testing was 50% (109/218) in immunocompromised patients. Only 1.38% (3/218) of immunocompromised patients and 0.9% (3/318) of all patients developed ACV-resistant HSV-1 as measured by phenotypic screening assays. It is noteworthy that a novel Y248D mutation in the UL23 gene from an immunocompromised patient was found by both PRA and DRA. In 3D protein predicting analysis, uncharged Y248 was located at an alpha-helix and substituted by negative-charged D248, which may alter the function of viral thymidine kinase. Besides, three unreported mutations related to natural polymorphism were found in virus isolates from two immunocompetent patients, including 683-688 deletion, R227H, and A351D in the UL30 gene. These data show that the prevalence of ACV-resistant HSV-1 among immunocompromised patients in southern Taiwan is low. These results will be helpful for the clinical management and treatment of HSV infections.

Highly-specific aptamer targeting SARS-CoV-2 S1 protein screened on an automatic integrated microfluidic system for COVID-19 diagnosis.

Variants of the severe acute respiratory syndrome coronavirus (SARS-CoV-2) have evolved such that it may be challenging for diagnosis and clinical treatment of the pandemic coronavirus disease-19 (COVID-19). Compared with developed SARS-CoV-2 diagnostic tools recently, aptamers may exhibit some advantages, including high specificity/affinity, longer shelf life (vs. antibodies), and could be easily prepared. Herein an integrated microfluidic system was developed to automatically carry out one novel screening process based on the systematic evolution of ligands by exponential enrichment (SELEX) for screening aptamers specific with SARS-CoV-2. The new screening process started with five rounds of positive selection (with the S1 protein of SARS-CoV-2). In addition, including non-target viruses (influenza A and B), human respiratory tract-related cancer cells (adenocarcinoma human alveolar basal epithelial cells and dysplastic oral keratinocytes), and upper respiratory tract-related infectious bacteria (including methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae), and human saliva were involved to increase the specificity of the screened aptamer during the negative selection. Totally, all 10 rounds could be completed within 20 h. The dissociation constant of the selected aptamer was determined to be 63.0 nM with S1 protein. Limits of detection for Wuhan and Omicron clinical strains were found to be satisfactory for clinical applications (i.e. 4.80 × 101 and 1.95 × 102 copies/mL, respectively). Moreover, the developed aptamer was verified to be capable of capturing inactivated SARS-CoV-2 viruses, eight SARS-CoV-2 pseudo-viruses, and clinical isolates of SARS-CoV-2 viruses. For high-variable emerging viruses, this developed integrated microfluidic system can be used to rapidly select highly-specific aptamers based on the novel SELEX methods to deal with infectious diseases in the future.

Novel Software for High-level Virological Testing: Self-Designed Immersive Virtual Reality Training Approach.

BACKGROUND: To ensure the timely diagnosis of emerging infectious diseases, high-tech molecular biotechnology is often used to detect pathogens and has gradually become the gold standard for virological testing. However, beginners and students are often unable to practice their skills due to the higher costs associated with high-level virological testing, the increasing complexity of the equipment, and the limited number of specimens from patients. Therefore, a new training program is necessary to increase training and reduce the risk of test failure. OBJECTIVE: The aim of the study is to (1) develop and implement a virtual reality (VR) software for simulated and interactive high-level virological testing that can be applied in clinical practice and skills building or training settings and (2) evaluate the VR simulation's effectiveness on reaction, learning, and behavior of the students (trainees). METHODS: Viral nucleic acid tests on a BD MAX instrument were selected for our VR project because it is a high-tech automatic detection system. There was cooperation between teachers of medical technology and biomedical engineering. Medical technology teachers were responsible for designing the lesson plan, and the biomedical engineering personnel developed the VR software. We designed a novel VR teaching software to simulate cognitive learning via various procedure scenarios and interactive models. The VR software contains 2D VR "cognitive test and learning" lessons and 3D VR "practical skills training" lessons. We evaluated students' learning effectiveness pre- and posttraining and then recorded their behavior patterns when answering questions, performing repeated exercises, and engaging in clinical practice. RESULTS: The results showed that the use of the VR software met participants' needs and enhanced their interest in learning. The average posttraining scores of participants exposed to 2D and 3D VR training were significantly higher than participants who were exposed solely to traditional demonstration teaching (P<.001). Behavioral assessments of students pre- and posttraining showed that students exposed to VR-based training to acquire relevant knowledge of advanced virological testing exhibited significantly improved knowledge of specific items posttraining (P<.01). A higher participant score led to fewer attempts when responding to each item in a matching task. Thus, VR can enhance students' understanding of difficult topics. CONCLUSIONS: The VR program designed for this study can reduce the costs associated with virological testing training, thus, increasing their accessibility for students and beginners. It can also reduce the risk of viral infections particularly during disease outbreaks (eg, the COVID-19 pandemic) and also enhance students' learning motivation to strengthen their practical skills.

Detection of live SARS-CoV-2 virus and its variants by specially designed SERS-active substrates and spectroscopic analyses.

A method using label-free surface enhanced Raman spectroscopy (SERS) based on substrate design is provided for an early detection and differentiation of spike glycoprotein mutation sites in live SARS-CoV-2 variants. Two SERS-active substrates, Au nanocavities (Au NCs) and Au NPs on porous ZrO2 (Au NPs/pZrO2), were used to identify specific peaks of A.3, Alpha, and Delta variants at different concentrations and demonstrated the ability to provide their SERS spectra with detection limits of 0.1-1.0% (or 104-5 copies/mL). Variant identification can be achieved by cross-examining reference spectra and analyzing the substrate-analyte relationship between the suitability of the analyte upon the hotspot(s) formed at high concentrations and the effective detection distance at low concentrations. Mutation sites on the S1 chain of the spike glycoprotein for each variant may be related and distinguishable. This method does not require sample preprocessing and therefore allows for fast screening, which is of high value for more comprehensive and specific studies to distinguish upcoming variants.

Characteristics of rectal chlamydia among men who have sex with men in southern Taiwan, 2020-2022: An emerging threat of rectal lymphogranuloma venereum L2b.

BACKGROUND: The prevalence of rectal chlamydia among men who have sex with men (MSM) without human deficiency virus infection (non-HIV) remains uncertain in Taiwan, and rectal lymphogranuloma venereum (LGV) among MSM has never been reported in the Far East. MATERIAL AND METHODS: From January 2020 to April 2022, MSM coming for anonymous voluntary counseling and testing, for pre-exposure prophylaxis, and for antiretroviral therapy were enrolled. All participants submitted his fecal samples and completed a QR-code questionnaire. Medical records of those who took regular medical visits for HIV were recorded. Multiplex polymerase chain reaction (PCR) was performed for all fecal samples, and ompA gene sequencing was therefore performed for each Chlamydia-positive fecal sample. RESULTS: Among 341 MSM during 2020-2022 in southern Taiwan, 21 (6.2%) had rectal chlamydia infection. Risk factors of rectal chlamydia included co-infection with rectal gonorrhea (adjusted odds ratio [AOR] 6.78, 95% confidence interval [CI] 1.44-31.91, P = 0.015) and multiple sexual partners (AOR 1.373, 95% CI 1.002-1.882, P = 0.048). Further ompA gene sequencing from 19 Chlamydia-positive fecal samples revealed that the prevalent genotypes or genovariants were Da (26.3%) and L2b (26.3%), followed by B (21.1%), J (14.3%), and G (9.5%). All cases of rectal LGV genovariant L2b presented as acute proctitis with diarrhea, anal pain, or discharge and were treated successfully with prolonged treatment of doxycycline. CONCLUSIONS: Rectal gonorrhea and multiple sexual partners are risk factors for rectal chlamydia. Clinicians in Taiwan should be aware of the emerging threat of rectal LGV among MSM with acute proctitis.

Therapeutics for fulminant hepatitis caused by enteroviruses in neonates.

Neonatal infection with nonpolio enteroviruses (EVs) causes nonspecific febrile illnesses and even life-threatening multiorgan failure. Hepatitis, which often results in hepatic necrosis followed by disseminated intravascular coagulopathy, is one of the most severe and frequent fatal neonatal EV infection complications. Coxsackievirus B (CVB) 1-5 and many echoviruses have been most commonly identified. Neonatal EV infection treatment has usually involved initial supportive care. Studies for CVB and echovirus infection treatments were developed for more than thirty years. Intravenous immunoglobulin and pleconaril therapy was performed in some clinical trials. Additionally, other studies demonstrated antiviral and/or anti-inflammatory pathogenesis mechanisms of neonatal EV hepatitis in in vitro or in vivo models. These treatments represented promising options for the clinical practice of neonatal EV hepatitis. However, further investigation is needed to elucidate the whole therapeutic potential and safety problems.

Genetic and Cross Neutralization Analyses of Coxsackievirus A16 Circulating in Taiwan from 1998 to 2021 Suggest Dominant Genotype B1 can Serve as Vaccine Candidate.

Coxsackievirus A16 (CVA16) is well known for causing hand-foot-and-mouth disease (HFMD) and outbreaks were frequently reported in Taiwan in the past twenty years. The epidemiology and genetic variations of CVA16 in Taiwan from 1998 to 2021 were analyzed in this study. CVA16 infections usually occurred in early summer and early winter, and showed increased incidence in 1998, 2000-2003, 2005, 2007-2008, and 2010 in Taiwan. Little or no CVA16 was detected from 2017 to 2021. CVA16 infection was prevalent in patients between 1 to 3 years old. A total of 69 isolates were sequenced. Phylogenetic analysis based on the VP1 region showed that CVA16 subgenotype B1 was dominantly isolated in Taiwan from 1998 to 2019, and B2 was identified only from isolates collected in 1999 and 2000. There was a high frequency of synonymous mutations in the amino acid sequences of the VP1 region among CVA16 isolates, with the exception of position 145 which showed positive selection. The recombination analysis of the whole genome of CVA16 isolates indicated that the 5'-untranslated region and the non-structural protein region of CVA16 subgenotype B1 were recombined with Coxsackievirus A4 (CVA4) and enterovirus A71 (EVA71) genotype A, respectively. The recombination pattern of subgenotype B2 was similar to B1, however, the 3D region was similar to EVA71 genotype B. Cross-neutralization among CVA16 showed that mouse antisera from various subgenotypes viruses can cross-neutralize different genotype with high neutralizing antibody titers. These results suggest that the dominant CVA16 genotype B1 can serve as a vaccine candidate for CVA16.

Outbreak of respiratory syncytial virus subtype ON1 among children during COVID-19 pandemic in Southern Taiwan.

BACKGROUND: The regional respiratory syncytial virus (RSV) outbreak in southern Taiwan in late 2020 followed the surge of RSV cases in the national surveillance data and displayed distinct clinical features. This study investigated RSV epidemiology in the most recent five years and compared the clinical manifestations of this outbreak with non-outbreak period. METHODS: Medical records of RSV-infected children at the National Cheng Kung University Hospital from January 2016 to December 2020 were retrospectively retrieved from hospital-based electronic medical database. Cases of RSV infection were identified by RSV antigen positive and/or RSV isolated from respiratory specimens. The demographic, clinical presentations, and laboratory data were recorded. The RSV isolates in 2020 was sequenced for phylogenetic analysis. RESULTS: Overall, 442 RSV-infected cases were retrieved and 42.1% (186 cases) clustered in late 2020. The 2020 outbreak started in September, peaked in November, and lasted for 3 months. 2020 RSV-infected children were older (2.3 ± 2.2 years vs. 1.0 ± 1.0 years), more likely to be diagnosed with bronchopneumonia (57.5% vs. 31.6%), but also had a lower hospitalization rate, shorter hospital stay, less oxygen use, and less respiratory distress than those in 2016-2019 (all p value < 0.05). The RSV isolates in 2020 belonged to RSV-A subtype ON1 but were phylogenetically distinct from the ON1 strains prevalent in Taiwan previously. CONCLUSION: The 2020 RSV outbreak was led by the novel RSV-A subtype ON1 variant with clinical manifestations distinct from previous years. Continuous surveillance of new emerging variants of respiratory viruses in the post-pandemic era is warranted.

Impact of Intrahost NS5 Nucleotide Variations on Dengue Virus Replication.

Due to the nature of RNA viruses, their high mutation rates produce a population of closely related but genetically diverse viruses, termed quasispecies. To determine the role of quasispecies in DENV disease severity, 22 isolates (10 from mild cases, 12 from fatal cases) were obtained, amplified, and sequenced with Next Generation Sequencing using the Illumina MiSeq platform. Using variation calling, unique wildtype nucleotide positions were selected and analyzed for variant nucleotides between mild and fatal cases. The analysis of variant nucleotides between mild and fatal cases showed 6 positions with a significant difference of p < 0.05 with 1 position in the structural region, and 5 positions in the non-structural (NS) regions. All variations were found to have a higher percentage in fatal cases. To further investigate the genetic changes that affect the virus's properties, reverse genetics (rg) viruses containing substitutions with the variations were generated and viral growth properties were examined. We found that the virus variant rgNS5-T7812G (G81G) had higher replication rates in both Baby hamster kidney cells (BHK-21) and Vero cells while rgNS5-C9420A (A617A) had a higher replication rate only in BHK-21 cells compared to wildtype virus. Both variants were considered temperature sensitive whereby the viral titers of the variants were relatively lower at 39°C, but was higher at 35 and 37°C. Additionally, the variants were thermally stable compared to wildtype at temperatures of 29, 37, and 39°C. In conclusion, viral quasispecies found in isolates from the 2015 DENV epidemic, resulted in variations with significant difference between mild and fatal cases. These variations, NS5-T7812G (G81G) and NS5-C9420A (A617A), affect viral properties which may play a role in the virulence of DENV.

Electromagnetically-driven integrated microfluidic platform using reverse transcription loop-mediated isothermal amplification for detection of severe acute respiratory syndrome coronavirus 2.

Rapid, sensitive and accurate diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of great need for effective quarantining and treatment. Real-time reverse-transcription polymerase chain reaction requiring thermocyling has been commonly used for diagnosis of SARS-CoV-2 though it may take two to 4 h before lengthy sample pretreatment process and require bulky apparatus and well-trained personnel. Since multiple reverse transcription loop-mediated isothermal amplification (multiple RT-LAMP) process without thermocycling is sensitive, specific and fast, an electromagnetically-driven microfluidic chip (EMC) was developed herein to lyse SARS-CoV-2 viruses, extract their RNAs, and perform qualitative analysis of three marker genes by on-chip multiple RT-LAMP in an automatic format within 82 min at a limit of detection of only ∼5000 copies per reaction (i.e. 200 virus/ µL). This compact EMC may be especially promising for SARS-CoV-2 diagnostics in resource-limited countries.

Assessment of the Risk of Severe Dengue Using Intrahost Viral Population in Dengue Virus Serotype 2 Patients via Machine Learning.

Dengue virus, a positive-sense single-stranded RNA virus, continuously threatens human health. Although several criteria for evaluation of severe dengue have been recently established, the ability to prognose the risk of severe outcomes for dengue patients remains limited. Mutant spectra of RNA viruses, including single nucleotide variants (SNVs) and defective virus genomes (DVGs), contribute to viral virulence and growth. Here, we determine the potency of intrahost viral population in dengue patients with primary infection that progresses into severe dengue. A total of 65 dengue virus serotype 2 infected patients in primary infection including 17 severe cases were enrolled. We utilized deep sequencing to directly define the frequency of SNVs and detection times of DVGs in sera of dengue patients and analyzed their associations with severe dengue. Among the detected SNVs and DVGs, the frequencies of 9 SNVs and the detection time of 1 DVG exhibited statistically significant differences between patients with dengue fever and those with severe dengue. By utilizing the detected frequencies/times of the selected SNVs/DVG as features, the machine learning model showed high average with a value of area under the receiver operating characteristic curve (AUROC, 0.966 ± 0.064). The elevation of the frequency of SNVs at E (nucleotide position 995 and 2216), NS2A (nucleotide position 4105), NS3 (nucleotide position 4536, 4606), and NS5 protein (nucleotide position 7643 and 10067) and the detection times of the selected DVG that had a deletion junction in the E protein region (nucleotide positions of the junction: between 969 and 1022) increased the possibility of dengue patients for severe dengue. In summary, we demonstrated the detected frequencies/times of SNVs/DVG in dengue patients associated with severe disease and successfully utilized them to discriminate severe patients using machine learning algorithm. The identified SNVs and DVGs that are associated with severe dengue will expand our understanding of intrahost viral population in dengue pathogenesis.

An integrated microfluidic platform featuring real-time reverse transcription loop-mediated isothermal amplification for detection of COVID-19.

An integrated microfluidic platform (IMP) utilizing real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) was developed here for detection and quantification of three genes of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; i.e., coronavirus diseases 2019 (COVID-19)): RNA-dependent RNA polymerase, the envelope gene, and the nucleocapsid gene for molecular diagnosis. The IMP comprised a microfluidic chip, a temperature control module, a fluidic control module that collectively carried out viral lysis, RNA extraction, RT-LAMP, and the real-time detection within 90 min in an automatic format. A limit of detection of 5 × 103 copies/reaction for each gene was determined with three samples including synthesized RNAs, inactive viruses, and RNAs extracted from clinical samples; this compact platform could be a useful tool for COVID-19 diagnostics.

Synergistic surface-enhanced Raman scattering effect to distinguish live SARS-CoV-2 S pseudovirus.

The COVID-19 pandemic negatively affected the economy and health security on a global scale, causing a drastic change on lifestyle, calling a need to mitigate further transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. Surface-enhanced Raman spectroscopy (SERS) has shown great potential in the sensitive and rapid detection of various molecules including viruses, through the identification of characteristic peaks of their outer membrane proteins. Accurate detection can be developed through the synergistic integration effect among SERS-active substrate, the appropriate laser wavelength, and the target analyte. In this study, gold nanocavities (Au NC) and Au nanoparticles upon ZrO2 nano-bowls (Au NPs/pZrO2) were tested and used as SERS-active substrates in detecting SARS-CoV-2 pseudovirus containing S protein as a surface capsid glycoprotein (SARS-CoV-2 S pseudovirus) and vesicular stomatitis virus G (VSV-G) pseudo-type lentivirus (VSV-G pseudovirus) to demonstrate their virus detection capability. The optimized Au NCs and Au NPs/pZrO2 substrates were then verified by examining the repetition of measurement, reproducibility, and detection limit. Due to the difference in geometry and composition of the substrates, the characteristic peak-positions of live SARS-CoV-2 S and VSV-G pseudoviruses in the obtained Raman spectra vary, which were also compared with those of inactivated ones. Based on the experimental results, SERS mechanism of each substrate to detect virus is proposed. The formation of hot spots brought by the synergistic integration effect among substrate, analyte, and laser induction may result differences in the obtained SERS spectra.

Morphologic Spectrum of Lymphadenopathy in Drug Reaction With Eosinophilia and Systemic Symptoms Syndrome.

CONTEXT.­: Drug-induced hypersensitivity syndrome/drug reaction with eosinophilia and systemic symptoms (DRESS) is a drug-induced, adverse T-cell-mediated hypersensitivity reaction that most often involves skin. The pathologic findings of DRESS-related lymphadenopathy have been described infrequently in the literature. OBJECTIVE.­: To present a case series of DRESS-related lymphadenopathy with an emphasis on the morphologic spectrum. DESIGN.­: We describe detailed clinical and pathologic findings along with the literature review. We focus on the differential diagnosis between DRESS lymphadenopathy and angioimmunoblastic T-cell lymphoma (AITL). RESULTS.­: There were 4 men and 1 woman with a mean age of 41 years (range, 23-59 years). One patient (20%) died. Three lymph node biopsy specimens showed a pattern reminiscent of AITL (AITL-like pattern) and 2 cases showed necrotizing lymphadenitis (Kikuchi-like pattern), associated with vasculitis in 1 case. The AITL-like morphology of DRESS-related lymphadenopathy may be difficult to distinguish from genuine AITL. The clinical information is important for differential diagnosis, including history of drug exposure, age, and the rarity or absence of AITL-associated manifestations such as hemolytic anemia and hypergammaglobulinemia. Molecular analysis of the T-cell receptor genes is helpful, typically revealing a polyclonal pattern in DRESS-related lymphadenopathy. CONCLUSIONS.­: In the literature, 4 histologic patterns of DRESS lymphadenopathy have been described: reactive lymphoid hyperplasia, necrotizing lymphadenitis, Hodgkin lymphoma-like, and AITL-like. These patterns, particularly those that resemble lymphoma, highlight the importance of correct diagnosis to avoid unnecessary therapies.