Characterization of antibody response to SARS-CoV-2 Orf8 from three waves of COVID-19 outbreak in Thailand.

A dynamic of virus adaptation and a mass vaccination campaign could significantly reduce the severity of clinical manifestations of COVID-19 and transmission. Hence, COVID-19 may become an endemic disease globally. Moreover, mass infection as the COVID-19 pandemic progressed affected the serology of the patients as a result of virus mutation and vaccination. Therefore, a need exists to acquire accurate serological testing to monitor the emergence of new outbreaks of COVID-19 to promptly prevent and control the disease spreading. In this study, the anti-Orf8 antibodies among samples collected in Thailand's first, fourth, and fifth waves of COVID-19 outbreaks compared with pre-epidemic sera were determined by indirect ELISA. The diagnostic sensitivity and specificity of the anti-Orf8 IgG ELISA for COVID-19 samples from the first, fourth, and fifth waves of outbreaks was found to be 100% compared with pre-epidemic sera. However, the diagnostic sensitivity and specificity of the anti-Orf8 IgG ELISA for a larger number of patient samples and controls from the fifth wave of outbreaks which were collected on day 7 and 14 after an RT-PCR positive result were 58.79 and 58.44% and 89.19 and 58.44%, respectively. Our data indicated that some of the controls might have antibodies from natural past infections. Our study highlighted the potential utility of anti-Orf8 IgG antibody testing for seroprevalence surveys but still warrants further investigations.

Simultaneous detection of influenza A, B and respiratory syncytial virus in wastewater samples by one-step multiplex RT-ddPCR assay.

BACKGROUND: After the occurrence of the COVID-19 pandemic, detection of other disseminated respiratory viruses using highly sensitive molecular methods was declared essential for monitoring the spread of health-threatening viruses in communities. The development of multiplex molecular assays are essential for the simultaneous detection of such viruses even at low concentrations. In the present study, a highly sensitive and specific multiplex one-step droplet digital PCR (RT-ddPCR) assay was developed for the simultaneous detection and absolute quantification of influenza A (IAV), influenza B (IBV), respiratory syncytial virus (RSV), and beta-2-microglobulin transcript as an endogenous internal control (IC B2M). RESULTS: The assay was first evaluated for analytical sensitivity and specificity, linearity, reproducibility, and recovery rates with excellent performance characteristics and then applied to 37 wastewater samples previously evaluated with commercially available and in-house quantitative real-time reverse transcription PCR (RT-qPCR) assays. IAV was detected in 16/37 (43%), IBV in 19/37 (51%), and RSV in 10/37 (27%) of the wastewater samples. Direct comparison of the developed assay with real-time RT-qPCR assays showed statistically significant high agreement in the detection of IAV (kappa Cohen's correlation coefficient: 0.834, p = 0.001) and RSV (kappa: 0.773, p = 0.001) viruses between the two assays, while the results for the detection of IBV (kappa: 0.355, p = 0.27) showed good agreement without statistical significance. CONCLUSIONS: Overall, the developed one-step multiplex ddPCR assay is cost-effective, highly sensitive and specific, and can simultaneously detect three common respiratory viruses in the complex matrix of wastewater samples even at low concentrations. Due to its high sensitivity and resistance to PCR inhibitors, the developed assay could be further used as an early warning system for wastewater monitoring.

Diagnostic accuracy of digital technologies compared with 12-lead ECG in the diagnosis of atrial fibrillation in adults: A protocol for a systematic review.

BACKGROUND: Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia in the world. AF increases the risk of stroke 5-fold, though the risk can be reduced with appropriate treatment. Therefore, early diagnosis is imperative but remains a global challenge. In low-and middle-income countries (LMICs), a lack of diagnostic equipment and under-resourced healthcare systems generate further barriers. The rapid development of digital technologies that are capable of diagnosing AF remotely and cost-effectively could prove beneficial for LMICs. However, evidence is lacking on what digital technologies exist and how they compare in regards to diagnostic accuracy. We aim to systematically review the diagnostic accuracy of all digital technologies capable of AF diagnosis. METHODS: MEDLINE, Embase and Web of Science will be searched for eligible studies. Free text terms will be combined with corresponding index terms where available and searches will not be limited by language nor time of publication. Cohort or cross-sectional studies comprising adult (≥18 years) participants will be included. Only studies that use a 12-lead ECG as the reference test (comparator) and report outcomes of sensitivity, specificity, the diagnostic odds ratio (DOR) or the positive and negative predictive value (PPV and NPV) will be included (or if they provide sufficient data to calculate these outcomes). Two reviewers will independently assess articles for inclusion, extract data using a piloted tool and assess risk of bias using the QUADAS-2 tool. The feasibility of a meta-analysis will be determined by assessing heterogeneity across the studies, grouped by index device, diagnostic threshold and setting. If a meta-analysis is feasible for any index device, pooled sensitivity and specificity will be calculated using a random effect model and presented in forest plots. DISCUSSION: The findings of our review will provide a comprehensive synthesis of the diagnostic accuracy of available digital technologies capable for diagnosing AF. Thus, this review will aid in the identification of which devices could be further trialed and implemented, particularly in a LMIC setting, to improve the early diagnosis of AF. TRIAL REGISTRATION: Systematic review registration: PROSPERO registration number is CRD42021290542. https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021290542.

Analysis of the diagnostic performance of PAX1/SOX1 gene methylation in cervical precancerous lesions and its role in triage diagnosis.

Methylation panels, tools for investigating epigenetic changes associated with diseases like cancer, can identify DNA methylation patterns indicative of disease, providing diagnostic or prognostic insights. However, the application of methylation panels focusing on the sex-determining region Y-box 1 (SOX1) and paired box gene 1 (PAX1) genes for diagnosing cervical lesions is under-researched. This study aims to examine the diagnostic performance of PAX1/SOX1 gene methylation as a marker for cervical precancerous lesions and its potential application in triage diagnosis. From September 2022 to April 2023, 181 patients with abnormal HPV-DNA tests or cytological exam results requiring colposcopy were studied at Hubei Maternal and Child Health Hospital, China. Data were collected from colposcopy, cytology, HPV-DNA tests, and PAX1/SOX1 methylation detection. Patients were categorized as control, cervical intraepithelial neoplasia Grade 1 (CIN1), Grade 2 (CIN2), Grade 3 (CIN3), and cervical cancer (CC) groups based on histopathology. We performed HPV testing, liquid-based cytology, and PAX1/SOX1 gene methylation testing. We evaluated the diagnostic value of methylation detection in cervical cancer using DNA methylation positivity rate, sensitivity, specificity, and area under the curve (AUC), and explored its potential for triage diagnosis. PAX1/SOX1 methylation positivity rates were: control 17.1%, CIN1 22.5%, CIN2 100.0%, CIN3 90.0%, and CC 100.0%. The AUC values for PAX1 gene methylation detection in diagnosing CIN1+, CIN2+, and CIN3+ were 0.52 (95% confidence interval [CI]: 0.43-0.62), 0.88 (95% CI: 0.80-0.97), and 0.88 (95% CI: 0.75-1.00), respectively. Corresponding AUC values for SOX1 gene methylation detection were 0.47 (95% CI: 0.40-0.58), 0.80 (95% CI: 0.68-0.93), and 0.92 (95% CI: 0.811-1.00), respectively. In HPV16/18-negative patients, methylation detection showed sensitivity of 32.4% and specificity of 83.7% for CIN1+. For CIN2+ and CIN3+, sensitivity was all 100%, with specificities of 83.0% and 81.1%. Among the patients who underwent colposcopy examination, 166 cases had cytological examination results ≤ASCUS, of which 37 cases were positive for methylation, and the colposcopy referral rate was 22.29%. PAX1/SOX1 gene methylation detection exhibits strong diagnostic efficacy for cervical precancerous lesions and holds significant value in triage diagnosis.

HPV genotyping in clinical samples using long-read single-molecule real-time sequencing.

Human papillomavirus (HPV) genotyping is widely used, particularly in combination with high-risk (HR) HPV tests for cervical cancer screening. We developed a genotyping method using sequences of approximately 800 bp in the E6/E7 region obtained by PacBio single molecule real-time sequencing (SMRT) and evaluated its performance against MY09-11 L1 sequencing and after the APTIMA HPV genotyping assay. The levels of concordance of PacBio E6/E7 SMRT sequencing with MY09-11 L1 sequencing and APTIMA HPV genotyping were 100% and 90.8%, respectively. The sensitivity of PacBio E6/EA7 SMRT was slightly greater than that of L1 sequencing and, as expected, lower than that of HR-HPV tests. In the context of cervical cancer screening, PacBio E6/E7 SMRT is then best used after a positive HPV test. PacBio E6/E7 SMRT genotyping is an attractive alternative for HR and LR-HPV genotyping of clinical samples. PacBio SMRT sequencing provides unbiased genotyping and can detect multiple HPV infections and haplotypes within a genotype.

Establishment of a Multiplex Detection Method for Common Bacteria in Blood Based on Human Mannan-Binding Lectin Protein-Conjugated Magnetic Bead Enrichment Combined with Recombinase-Aided PCR Technology.

Objective: Recombinase-aided polymerase chain reaction (RAP) is a sensitive, single-tube, two-stage nucleic acid amplification method. This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus (SA), Pseudomonas aeruginosa (PA), and Acinetobacter baumannii (AB) in the bloodstream based on recombinant human mannan-binding lectin protein (M1 protein)-conjugated magnetic bead (M1 bead) enrichment of pathogens combined with RAP. Methods: Recombinant plasmids were used to evaluate the assay sensitivity. Common blood influenza bacteria were used for the specific detection. Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR (M-RAP) and quantitative PCR (qPCR) assays. Kappa analysis was used to evaluate the consistency between the two assays. Results: The M-RAP method had sensitivity rates of 1, 10, and 1 copies/µL for the detection of SA, PA, and AB plasmids, respectively, without cross-reaction to other bacterial species. The M-RAP assay obtained results for < 10 CFU/mL pathogens in the blood within 4 h, with higher sensitivity than qPCR. M-RAP and qPCR for SA, PA, and AB yielded Kappa values of 0.839, 0.815, and 0.856, respectively ( P < 0.05). Conclusion: An M-RAP assay for SA, PA, and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.

The Suitable Population for Opportunistic Low Bone Mineral Density Screening Using Computed Tomography.

Objective: To explore the suitable population of CT value for predicting low bone mineral density (low-BMD). Methods: A total of 1268 patients who underwent chest CT examination and DXA within one-month period retrospectively analyzed. The CT attenuation values of trabecular bone were measured in mid-sagittal plane from thoracic vertebra 7 (T7). Receiver operating characteristic (ROC) curves were used to evaluate the ability to diagnose low-BMD. Results: The AUC for diagnosing low BMD was larger in women than in men (0.894 vs 0.744, p < 0.05). The AUC increased gradually with the increase of age but decreased gradually with the increase in height and weight (p < 0.05). In females, when specificity was adjusted to approximately 90%, a threshold of 140.25 HU has a sensitivity of 69.3%, which is higher than the sensitivity of 36.5% in males for distinguishing low-BMD from normal. At the age of 70 or more, when specificity was adjusted to approximately 90%, a threshold of 126.31 HU has a sensitivity of 76.1%, which was higher than that of other age groups. Conclusion: For patients who had completed chest CTs, the CT values were more effective in predicting low-BMD in female, elderly, lower height, and lower weight patients.

Usefulness of the qSOFA score for predicting hospital mortality in cancer patients.

BACKGROUND: The quick Sequential Sepsis-related Organ Failure Assessment (qSOFA) is a score that has been proposed to quickly identify patients at higher risk of death. OBJECTIVE: To describe the usefulness of the qSOFA score to predict in-hospital mortality in cancer patients. MATERIAL AND METHODS: Cross-sectional study carried out between January 2021 and December 2022. Hospital mortality was the dependent variable. The area under the ROC curve (AUC) was calculated to determine the discriminative ability of qSOFA to predict in-hospital mortality. RESULTS: A total of 587 cancer patients were included. A qSOFA score higher than 1 obtained a sensitivity of 57.2%, specificity of 78.5%, a positive predictive value of 55.4% and negative predictive value of 79.7%. The AUC of qSOFA for predicting in-hospital mortality was 0.70. In-hospital mortality of patients with qSOFA scores of 2 and 3 points was 52.7 and 64.4%, respectively. In-hospital mortality was 31.9% (187/587). CONCLUSION: qSOFA showed acceptable discriminative ability for predicting in-hospital mortality in cancer patients.

ANTECEDENTES: El quick Sequential Sepsis-related Organ Failure Assessment (qSOFA) es una puntuación propuesta para identificar de forma rápida a pacientes con mayor probabilidad de morir. OBJETIVO: Describir la utilidad de la puntuación qSOFA para predecir mortalidad hospitalaria en pacientes con cáncer. MATERIAL Y MÉTODOS: Estudio transversal realizado entre enero de 2021 y diciembre de 2022. La mortalidad hospitalaria fue la variable dependiente. Se calculó el área bajo la curva ROC (ABC) para determinar la capacidad discriminativa de qSOFA para predecir mortalidad hospitalaria. RESULTADOS: Se incluyeron 587 pacientes con cáncer. La puntuación qSOFA < 1 obtuvo una sensibilidad de 57.2 %, una especificidad de 78.5 %, un valor predictivo positivo de 55.4 % y un valor predictivo negativo de 79.7 %. El ABC de qSOFA para predecir mortalidad hospitalaria fue de 0.70. La mortalidad hospitalaria de los pacientes con qSOFA de 2 y 3 puntos fue de 52.7 y 64.4 %, respectivamente. La mortalidad hospitalaria fue de 31.9 % (187/587). CONCLUSIÓN: qSOFA mostró capacidad discriminativa aceptable para predecir mortalidad hospitalaria en pacientes con cáncer.

Infrared thermography for normal endothelial function screening.

BACKGROUND: Endothelial dysfunction (ED) suspicion will allow to prevent accelerated atherosclerosis and premature death. OBJECTIVE: To establish the usefulness of thermography for endothelial function screening in adults with cardiovascular risk factors. MATERIAL AND METHODS: Cross-sectional, analytical diagnostic test. A brachial arterial diameter (BAD) increase < 11% at one-minute post-ischemia meant probable ED and was confirmed if BAD was ≥ 11% post-sublingual nitroglycerin. Thermographic photographs of the palmar region were obtained at one minute. Descriptive statistics, ROC curve, Mann-Whitney's U-test, chi-square test, or Fisher's exact test were used. RESULTS: Thirty-eight subjects with a median age of 50 years, and with 624 thermographic measurements were included. Nine had ED (flow-mediated vasodilation [FMV]: 2.5%). The best cutoff point for normal endothelial function in subjects with cardiovascular risk factors was ≥ 36 °C at one minute of ischemia, with 85% sensitivity, 70% specificity, positive and negative predictive values of 78 and 77%, area under the curve of 0.796, LR+ 2.82, LR- 0.22. CONCLUSION: An infrared thermography-measured temperature in the palmar region greater than or equal to 36 °C after one minute of ischemia is practical, non-invasive, and inexpensive for normal endothelial function screening in adults with cardiovascular risk factors.

ANTECEDENTES: La sospecha de disfunción endotelial (DE) permitirá prevenir la aterosclerosis acelerada y la muerte prematura. OBJETIVO: Establecer la utilidad de la termografía en el cribado de la función endotelial en adultos con factores de riesgo cardiovascular. MATERIAL Y MÉTODOS: Estudio transversal analítico de prueba diagnóstica. El incremento del diámetro de la arteria braquial < 11 % a un minuto posisquemia significó probable DE, confirmada si el diámetro fue ≥ 11 % posnitroglicerina sublingual. Se obtuvieron fotografías termográficas al minuto de la región palmar. Se aplicó estadística descriptiva, curva ROC, pruebas U de Mann-Whitney, chi cuadrada o exacta de Fisher. RESULTADOS: Se incluyeron 38 sujetos, mediana de edad de 50 años, con 624 mediciones termográficas; nueve presentaron DE (vasodilatación mediada por flujo de 2.5 %). El mejor punto de corte para la función endotelial normal en sujetos con factores de riesgo cardiovascular fue ≥ 36 °C al minuto de isquemia, con sensibilidad de 85%, especificidad de 70%, valores predictivos positivo y negativo de 78 y 77%, área bajo la curva de 0.796, razón de verisimilitud positiva de 2.82 y razón de verisimilitud negativa de 0.22. CONCLUSIÓN: La medición de la temperatura en la región palmar mediante termografía infrarroja ≥ 36 °C tras un minuto de isquemia es práctica, no invasiva y económica para el cribado de la función endotelial normal en adultos con factores de riesgo cardiovascular.

Classification of presence of missing teeth in each quadrant using deep learning artificial intelligence on panoramic radiographs of pediatric patients.

Early tooth loss in pediatric patients can lead to various complications, making quick and accurate diagnosis essential. This study aimed to develop a novel deep learning model for classification of missing teeth on panoramic radiographs in pediatric patients and to assess the accuracy. The study included patients aged 8-16 years who visited the Pusan National University Dental Hospital and underwent panoramic radiography. A total of 806 panoramic radiographs were retrospectively analyzed to determine the presence or absence of missing teeth for each tooth number. Moreover, each panoramic radiograph was divided into four quadrants, each of a smaller size, containing both primary and permanent teeth, generating 3224 data. Quadrants with missing teeth (n = 1457) were set as the experimental group, and quadrants without missing teeth (n = 1767) were set as the control group. The data were split into training and validation sets in a 4:1 ratio, and a 5-fold cross-validation was conducted. A gradient-weighted class activation map was used to visualize the deep learning model. The average values of sensitivity, specificity, accuracy, precision, recall and F1-score of this deep learning model were 0.635, 0.814, 0.738, 0.730, 0.732 and 0.731, respectively. In the experimental group, the accuracy was the highest for missing canines and premolars, and the lowest for molars. The deep learning model exhibited a moderate to good distinguishing power with a classification performance of 0.730. This deep learning model and the newly defined small sized region of interest proved adequate for classifying the presence of missing teeth.

Is increased activator protein 1 in cerebrospinal fluid as a potential biomarker that distinguishes idiopathic intracranial hypertension from multiple sclerosis?

OBJECTIVES: To distinguish whether idiopathic intracranial hypertension (IIH) is a condition predisposing to multiple sclerosis (MS) or an isolated disease, the current gene transcription factor Activator Protein-1 (AP-1) was evaluated with its potential to differentiate both diseases. BACKGROUND: The aim of this study was to investigate the use of AP-1 as biomarkers for the discrimination of IIH and MS. METHODS: AP-1, TNF-α, and IL-6 protein values in the CSF of the cases were evaluated by the ELISA method. The numerical measures of the groups and the ability of AP-1 to distinguish the groups were analyzed with the ROC curve. RESULTS: There was no difference between the groups in CSF TNF-α, IL-6, CSF, and serum biochemistry analyses. However, it was determined that the AP-1 concentration (pg/ml) was significantly higher in the IIH group, the sensitivity of AP-1 in separating those with IIH was 75%, and the specificity in separating those with MS was 60% in those with an AP-1 concentration of 606.5 and above. CONCLUSION: According to our results, the fact that CSF TNF-α and IL-6 values did not differ in IIH compared to MS revealed that IIH could not methodologically control MS, and AP-1 was a supportive parameter in differentiating both diseases (Tab. 2, Fig. 1, Ref. 31).

Diagnostic performance of acoustic radiation force impulse for acute pancreatitis: A meta-analysis.

OBJECTIVE: The objective of this meta-analysis is to evaluate the diagnostic performance of acoustic radiation force impulse (ARFI) in acute pancreatitis (AP) patients. METHODS: PubMed, Web of Science, Embase, Wanfang, Chinese Biological Medicine databases, and Chinese Biomedical Literature Service System were searched for relevant studies to explore the potential diagnostic performance of ARFI in AP from inception to November 2023. STATA 14.0 was used to analyze the standardized mean difference (SMD) with 95% confidence interval (CI), pooled sensitivity, specificity, area under the curve, meta-regression analysis, sensitivity analysis, and publication bias. RESULTS: Nine studies, involving 533 AP patients and 585 healthy controls, were included. AP patients had significantly higher ARFI levels than healthy controls (SMD: 3.13, 95% CI: 1.88-4.39, P = .001). The area under the curve of ARFI for diagnosing AP was 0.99 (95% CI: 0.98-1.00), with 98% sensitivity and 94% specificity. Meta-regression identified the study region and study period as the sources of heterogeneity. Sensitivity analysis showed that the exclusion of any single study did not materially alter the overall combined effect. No evidence of publication bias was observed in the included studies. CONCLUSION: This meta-analysis demonstrated that ARFI exerted satisfactory diagnostic performance in AP.

Seroprevalence of the novel swine acute diarrhea syndrome coronavirus in China assessed by enzyme-linked immunosorbent assay.

The endemic outbreak of SADS-CoV has resulted in economic losses and potentially threatened the safety of China's pig industry. The molecular epidemiology of SADS-CoV in pig herds has been investigated in many provinces in China. However, there are no data over a long-time span, and there is a lack of extensive serological surveys to assess the prevalence of SADS-CoV in Chinese swine herds since the discovery of SADS-CoV. In this study, an indirect anti-SADS-CoV IgG enzyme-linked immunosorbent assay (ELISA) based on the SADS-CoV S1 protein was established to investigate the seroprevalence of SADS-CoV in Chinese swine herds. Cross-reactivity assays, indirect immunofluorescence, and western blotting assays showed that the developed ELISA had excellent SADS-CoV specificity. In total, 12,978 pig serum samples from 29 provinces/municipalities/autonomous regions in China were tested from 2022 to 2023. The results showed that the general seroprevalence of SADS-CoV in China was 59.97%, with seroprevalence ranging from 16.7% to 77.12% in different provinces and from 42.61% to 68.45% in different months. SADS-CoV is widely prevalent in China, and its seroprevalence was higher in Northeast China, North China, and Central China than in other regions. Among the four seasons, the prevalence of SADS-CoV was the highest in spring and the lowest in autumn. The results of this study provide the general seroprevalence profile of SADS-CoV in China, facilitating the understanding of the prevalence of SADS-CoV in pigs. More importantly, this study is beneficial in formulating preventive and control measures for SADS-CoV and may provide directions for vaccine development.

Racial Differences in Diagnostic Accuracy of Retinal Nerve Fiber Layer Thickness in Primary Open-Angle Glaucoma.

Purpose: To evaluate the diagnostic accuracy of retinal nerve fiber layer thickness (RNFLT) by spectral-domain optical coherence tomography (OCT) in primary open-angle glaucoma (POAG) in eyes of African (AD) and European descent (ED). Design: Comparative diagnostic accuracy analysis by race. Participants: 379 healthy eyes (125 AD and 254 ED) and 442 glaucomatous eyes (226 AD and 216 ED) from the Diagnostic Innovations in Glaucoma Study and the African Descent and Glaucoma Evaluation Study. Methods: Spectralis (Heidelberg Engineering GmbH) and Cirrus (Carl Zeiss Meditec) OCT scans were taken within one year from each other. Main Outcome Measures: Diagnostic accuracy of RNFLT measurements. Results: Diagnostic accuracy for Spectralis-RNFLT was significantly lower in eyes of AD compared to those of ED (area under the receiver operating curve [AUROC]: 0.85 and 0.91, respectively, P=0.04). Results for Cirrus-RNFLT were similar but did not reach statistical significance (AUROC: 0.86 and 0.90 in AD and ED, respectively, P =0.33). Adjustments for age, central corneal thickness, axial length, disc area, visual field mean deviation, and intraocular pressure yielded similar results. Conclusions: OCT-RNFLT has lower diagnostic accuracy in eyes of AD compared to those of ED. This finding was generally robust across two OCT instruments and remained after adjustment for many potential confounders. Further studies are needed to explore the potential sources of this difference.

A novel indirect ELISA for serodiagnosis of mucormycosis using antigens from Rhizopus arrhizus.

BACKGROUND: Due to a delay in diagnosis by conventional techniques and high mortality, the development of a standardised and rapid non-culture-based technique is an unmet need in pulmonary, gastrointestinal, and disseminated forms of mucormycosis. Though limited studies have been conducted for molecular diagnosis, there are no established serologic tests for this highly fatal infection. OBJECTIVE: To develop and evaluate an indirect in-house enzyme-linked immunosorbent assay (ELISA) utilising antigens of Rhizopus arrhizus for detecting anti-Rhizopus antibodies (IgG and IgM) in sera of patients with mucormycosis. METHODS: We extracted both secretory and mycelial Rhizopus antigens using standardised protocols. Bradford assay was used for protein quantification. We then standardised an indirect ELISA using R. arrhizus mycelial and secretory antigens (10.0 µg/mL in bicarbonate buffer pH 9.2) for detecting anti-Rhizopus IgG and IgM antibodies in patient sera. We included patients with mucormycosis, other fungal infections, and healthy controls. Antibody index value (E-value) was calculated for each patient sample. RESULTS: Asparagine broth culture filtrate utilising 85% ammonium sulphate salt fractionation and mycelial homogenate grown in yeast extract peptone dextrose (YPD) broth precipitated with trichloroacetic acid (TCA) yielded a large amount of good-quality protein for the assay. We included 55 patients with mucormycosis (rhino-orbito-cerebral mucormycosis [ROCM, n = 39], pulmonary [n = 15], gastrointestinal [n = 1]), 24 with other fungal infections (probable aspergillosis [n = 14], candidiasis [n = 10]), and healthy controls (n = 16). The sensitivity of the antibody test for diagnosing mucormycosis ranged from 83.6-92.7% for IgG and 72.7-87.3% for IgM, with a specificity of 91.7-92.5% for IgG and 80-82.5% for IgM. The sera from patients with other fungal infections and healthy individuals did not show significant cross-reactivity. CONCLUSION: The detection of anti-Rhizopus IgG antibody performed significantly better in comparison to IgM-based ELISA for diagnosing both ROCM (sensitivity of 84.6% vs. 69.2%) and pulmonary cases (86.6% vs. 80.0%). More extensive studies are required to confirm our findings.

A multiplex digital PCR assay for detection and quantitation of porcine circovirus type 2 and type 3.

Porcine circovirus (PCV) has become a major pathogen, causing major economic losses in the global pig industry, and PCV type 2 (PCV2) and 3 (PCV3) are distributed worldwide. We designed specific primer and probe sequences targeting PCV2 Cap and PCV3 Rap and developed a multiplex crystal digital PCR (cdPCR) method after optimizing the primer concentration, probe concentration, and annealing temperature. The multiplex cdPCR assay permits precise and differential detection of PCV2 and PCV3, with a limit of detection of 1.39 × 101 and 1.27 × 101 copies/reaction, respectively, and no cross-reaction with other porcine viruses was observed. The intra-assay and interassay coefficients of variation (CVs) were less than 8.75%, indicating good repeatability and reproducibility. To evaluate the practical value of this assay, 40 tissue samples and 70 feed samples were tested for both PCV2 and PCV3 by cdPCR and quantitative PCR (qPCR). Using multiplex cdPCR, the rates of PCV2 infection, PCV3 infection, and coinfection were 28.45%, 1.72%, and 12.93%, respectively, and using multiplex qPCR, they were 25.00%, 0.86%, and 4.31%, respectively This highly specific and sensitive multiplex cdPCR thus allows accurate simultaneous detection of PCV2 and PCV3, and it is particularly well suited for applications that require the detection of small amounts of input nucleic acid or samples with intensive processing and complex matrices.

Raising the bar: genus-specific nested PCR improves detection and lineage identification of avian haemosporidian parasites.

Avian haemosporidian parasites are useful model organisms to study the ecology and evolution of parasite-host interactions due to their global distribution and extensive biodiversity. Detection of these parasites has evolved from microscopic examination to PCR-based methods, with the mitochondrial cytochrome b gene serving as barcoding region. However, standard PCR protocols used for screening and identification purposes have limitations in detecting mixed infections and generating phylogenetically informative data due to short amplicon lengths. To address these issues, we developed a novel genus-specific nested PCR protocol targeting avian haemosporidian parasites. The protocol underwent rigorous testing utilizing a large dataset comprising blood samples from Malagasy birds of three distinct Passeriformes families. Furthermore, validation was done by examining smaller datasets in two other laboratories employing divergent master mixes and different bird species. Comparative analyses were conducted between the outcomes of the novel PCR protocol and those obtained through the widely used standard nested PCR method. The novel protocol enables specific identification of Plasmodium, Haemoproteus (Parahaemoproteus), and Leucocytozoon parasites. The analyses demonstrated comparable sensitivity to the standard nested PCR with notable improvements in detecting mixed infections. In addition, phylogenetic resolution is improved by amplification of longer fragments, leading to a better understanding of the haemosporidian biodiversity and evolution. Overall, the novel protocol represents a valuable addition to avian haemosporidian detection methodologies, facilitating comprehensive studies on parasite ecology, epidemiology, and evolution.

The Future Role of Abdominal US in Hepatocellular Carcinoma Surveillance.

Abdominal US is currently the best-validated surveillance strategy for hepatocellular carcinoma (HCC) in at-risk patients. It is the only modality shown to have completed all five phases of validation and can achieve high sensitivity and specificity for HCC detection, especially when conducted by expert sonographers in high-volume centers. However, US also has limitations, including operator dependency and varying sensitivity in clinical practice. Further, the sensitivity of US for early-stage HCC detection is lower in patients with obesity or nonviral liver disease, increasingly common populations undergoing surveillance. Imaging-based and blood-based surveillance strategies, including abbreviated MRI and biomarker panels, may overcome some limitations of US-based surveillance. Both strategies have promising test performance in phase II and phase III biomarker studies and are undergoing prospective validation. Considering the variation in HCC risk and test performance between patients, there will likely be a shift away from a one-size-fits-all approach and toward precision screening, in which the "best" test is selected based on individual patient characteristics. In this upcoming era of precision HCC screening among patients with cirrhosis, US will likely continue to have an important, albeit reduced, surveillance role.