Many countries have applied the wastewater surveillance of the COVID-19 pandemic to their national public health monitoring measures. The most used methods for detecting SARS-CoV-2 in wastewater are quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and reverse transcriptase-droplet digital polymerase chain reaction (RT-ddPCR). Previous comparison studies have produced conflicting results, thus more research on the subject is required.
This study aims to compare RT-qPCR and RT-ddPCR for detecting SARS-CoV-2 in wastewater. It also aimed to investigate the effect of changes in the analytical pipeline, including the RNA extraction kit, RT-PCR kit, and target gene assay, on the results. Another aim was to find a detection method for low-resource settings.
We compared 2 RT-qPCR kits, TaqMan RT-qPCR and QuantiTect RT-qPCR, and RT-ddPCR based on sensitivity, positivity rates, variability, and correlation of SARS-CoV-2 gene copy numbers in wastewater to the incidence of COVID-19. Furthermore, we compared 2 RNA extraction methods, column- and magnetic-bead-based. In addition, we assessed 2 target gene assays for RT-qPCR, N1 and N2, and 2 target gene assays for ddPCR N1 and E. Reverse transcription strand invasion-based amplification (RT-SIBA) was used to detect SARS-CoV-2 from wastewater qualitatively.
Our results indicated that the most sensitive method to detect SARS-CoV-2 in wastewater was RT-ddPCR. It had the highest positivity rate (26/30), and its limit of detection was the lowest (0.06 gene copies/µL). However, we obtained the best correlation between COVID-19 incidence and SARS-CoV-2 gene copy number in wastewater using TaqMan RT-qPCR (correlation coefficient [CC]=0.697, P<.001). We found a significant difference in sensitivity between the TaqMan RT-qPCR kit and the QuantiTect RT-qPCR kit, the first having a significantly lower limit of detection and a higher positivity rate than the latter. Furthermore, the N1 target gene assay was the most sensitive for both RT-qPCR kits, while no significant difference was found between the gene targets using RT-ddPCR. In addition, the use of different RNA extraction kits affected the result when the TaqMan RT-qPCR kit was used. RT-SIBA was able to detect SARS-CoV-2 RNA in wastewater.
As our study, as well as most of the previous studies, has shown RT-ddPCR to be more sensitive than RT-qPCR, its use in the wastewater surveillance of SARS-CoV-2 should be considered, especially if the amount of SARS-CoV-2 circulating in the population was low. All the analysis steps must be optimized for wastewater surveillance as our study showed that all the analysis steps including the compatibility of the RNA extraction, the RT-PCR kit, and the target gene assay influence the results. In addition, our study showed that RT-SIBA could be used to detect SARS-CoV-2 in wastewater if a qualitative result is sufficient.

BACKGROUND: The dynamics of viral shedding and the specific humoral response against monkeypox virus (MPXV) have not been well characterized in patients across their disease course during hospitalisation. The aim of this study was to determine the viral load and the levels of antibodies against MPXV using longitudinal paired-collected samples from hospitalized patients.
METHODS: Patients who were hospitalised with mpox were recruited at Beijing Ditan Hospital Capital Medical University in China between June 2 and September 23, 2023. Paired samples, including samples from skin lesions, the oropharynx, saliva, faeces, urine, plasma, and serum, were serially collected at days 1, 3, 7, and 14 after admission until discharge. Not all of the patients had samples obtained at all of the timepoints. All the samples were analysed via quantitative PCR. Virus isolation was performed by using clinical samples and Vero cells. The presence of IgM, IgA, IgG, and neutralising antibodies (NAbs) against MPXV was evaluated. The first collected plasma sample was taken when the patient was hospitalised, and the levels of cytokines and chemokines were measured in the sample. The demographic data, smallpox vaccination status, history of known exposure to MPVX, HIV status and other clinical data were collected using a standard case report form.
RESULTS: A total of 510 specimens were serially collected from 39 recruited people with mpox. Among all the samples, the skin lesions had the highest viral DNA detection rates and viral loads, and the saliva samples had the second highest rates and viral loads. One day before discharge, 85% of the dry scrabs (median Ct 28.2, range 19.0-38.3) and 70% of the saliva samples (median Ct 32.4, range 24.5-38.1) were positive for viral DNA, Of which, 23.1% of dry scrabs were positive in viral culture. The rate of viral DNA detection in the oropharyngeal, saliva, and faecal samples decreased with time, while the rates in the plasma, serum, and urine samples increased quickly before 10 days post symptom onset (PSO). The median days of appearance of MPXV-IgM, MPXV-IgA, MPXV-IgG, and NAb were at 8 (interquartile range [IQR] 7-9), 9 (7-10), 12 (9-15), and 12 (9-15) PSO, respectively. The IgM, IgA, IgG, and NAb titres increased with time. Between days 11 and 21 PSO, the NAb titres were lower in people living with HIV (PWH) than in people living without HIV (PWOH). Increased NAb titres were associated with decreased viral loads in the saliva (r = 0.28, p = 0.025), faeces (r = 0.35, p = 0.021), plasma (r = 0.30, p = 0.0044), and serum samples (r = 0.37, p = 0.001). Compared with PWOH, PWH had higher plasma levels of MIP-1α, MIP-1β, G-CSF, IL-4, and FGF-basic.
CONCLUSIONS: The high positive viral culture rate of clinical samples of patients when they are discharged from the hospital indicates that effective public health management strategies are needed for people with mpox. The low NAb titres and high levels of cytokines in PWH shows that earlier treatment is needed to control inflammation in high-risk populations.
BACKGROUND: National Natural Science Foundation of China, Chinese Academy of Medical Sciences, Fundamental Research Funds for the Central Universities for Peking Union Medical College, National Key R&D Program of China.

Distinct circulating bile acid (BA) subtypes may play roles in regulating lipid homeostasis and atherosclerosis.
We investigated whether changes in circulating BA subtypes induced by weight-loss dietary interventions were associated with improved lipid profiles and atherosclerotic cardiovascular disease (ASCVD) risk estimates.
This study included adults with overweight or obesity (n = 536) who participated in a randomized weight-loss dietary intervention trial. Circulating primary and secondary unconjugated BAs and their taurine-/glycine-conjugates were measured at baseline and 6 mo after the weight-loss diet intervention. The ASCVD risk estimates were calculated using the validated equations.
At baseline, higher concentrations of specific BA subtypes were related to higher concentrations of atherogenic very low-density lipoprotein lipid subtypes and ASCVD risk estimates. Weight-loss diet-induced decreases in primary BAs were related to larger reductions in triglycerides and total cholesterol [every 1 standard deviation (SD) decrease of glycocholate, glycochenodeoxycholate, or taurochenodeoxycholate was related to β (standard error) -3.3 (1.3), -3.4 (1.3), or -3.8 (1.3) mg/dL, respectively; PFDR < 0.05 for all]. Greater decreases in specific secondary BA subtypes were also associated with improved lipid metabolism at 6 mo; there was β -4.0 (1.1) mg/dL per 1-SD decrease of glycoursodeoxycholate (PFDR =0.003) for changes in low-density lipoprotein cholesterol. We found significant interactions (P-interaction < 0.05) between dietary fat intake and changes in BA subtypes on changes in ASCVD risk estimates; decreases in primary and secondary BAs (such as conjugated cholate or deoxycholate) were significantly associated with improved ASCVD risk after consuming a high-fat diet, but not after consuming a low-fat diet.
Decreases in distinct BA subtypes were associated with improved lipid profiles and ASCVD risk estimates, highlighting the importance of changes in circulating BA subtypes as significant factors linked to improved lipid metabolism and ASCVD risk estimates in response to weight-loss dietary interventions. Habitual dietary fat intake may modify the associations of changes in BAs with ASCVD risk. This trial was registered at clinicaltrials.gov as NCT00072995.

Advances in the diagnosis and treatment of pulmonary hypertension (PH) have gradually improved the disease course. This retrospective cohort study aims to explore the diagnostic hemodynamic profile and survival of PH patients and their temporal changes, as well as investigate potential prognostic factors. Overall, 257 adult patients were diagnosed with PH following right heart catheterization (RHC) from January 2008 to June 2023 according to the hemodynamic cut-off values proposed by the corresponding ESC/ERS guidelines at the time RHC was performed. Of these patients, 46.3% were Group 1, 17.8% Group 2, 14.0% Group 3, 18.0% Group 4, and 3.0% Group 5 PH. Temporal improvement in both diagnostic hemodynamic profile and survival of patients with PH and pulmonary arterial hypertension (PAH) was identified after 2013. Survival analysis demonstrated 5-year survival rates of 65% in Group 1 PH (90.3% in idiopathic PAH) and 77% in Group 4 PH. PAH patients being at low risk at diagnosis presented a similar 1-year all-cause mortality rate (12.4%) with high-risk ones (12.8%), primarily due to non-PH-related causes of death (62%), while high-risk patients died mostly due to PH (67%). The observed improvements in diagnostic hemodynamic profiles and overall survival highlight the importance of timely diagnosis and successful treatment strategies in PH.

The global prevalence of insufficient physical activity (PA) was reported to be 27.5% in 2016, and there were stable levels of insufficient PA worldwide between 2001 and 2016. The global target of a 10% reduction in insufficient PA by 2025 will not be met if the trends remain. The relevant data for trends in China were still scarce. This study aimed to determine nationwide temporal trends in insufficient PA among adults in China from 2010 to 2018.
645 903 adults aged 18 years or older were randomly selected from four nationally representative cross-sectional surveys of the China Chronic Disease and Risk Factor Surveillance conducted in 2010, 2013, 2015, and 2018. PA was measured using the Global Physical Activity Questionnaire. Temporal changes in insufficient PA prevalence and participation of domain-specific moderate- to vigorous-intensity PA (MVPA) were analyzed using logistic regression.
From 2010 to 2018, the age-adjusted prevalence of insufficient PA in China increased from 17.9% (95% confidence interval 16.3% to 19.5%) in 2010 to 22.3% (20.9% to 23.8%) in 2018 (P for trend < 0.001). By age group, with a significant increase in insufficient PA in adults aged 18-34 years (P for trend < 0.001), which rose more rapidly than in adults aged ≥ 35 years (P for interaction < 0.001). Insufficient PA has increased significantly among adults engaged in agriculture-related work, non-manual work, and other manual work (all P for trend < 0.05). And among the occupational groups, those engaged in agriculture-related work had the fastest increase (P for interaction = 0.01). The percentage of adults participating in work-related MVPA decreased from 79.6% (77.8% to 81.5%) to 66.8% (64.9% to 68.7%) along with a decrease in time spent on work-related MVPA, while percentages of adults participating in recreation-related MVPA increased from 14.2% (12.5% to 15.9%) to 17.2% (16.0% to 18.4%) (all P for trend < 0.05).
Among Chinese adults, an increasing trend was found in insufficient PA from 2010 to 2018, with more than one-fifth of adults failing to achieve the recommendation of adequate PA. More targeted PA promotion strategies should be developed to improve population health.

OBJECTIVE: To study the optical coherence tomography (OCT) changes in eyes with acute central retinal artery occlusion (CRAO) of different severity and at different disease stages.
METHODS: The study included acute CRAO cases of < 7 days duration, imaged on OCT at various time points. Based on the OCT findings at presentation, cases were classified into three severity groups: mild, moderate, and severe. OCT scans were evaluated and classified into four-time intervals based on symptom duration.
RESULTS: There were 39 eyes from 38 patients with acute CRAO who underwent 96 OCT scans. At presentation, the study had 11, 16, and 12 cases of mild, moderate, and severe CRAO, respectively. Middle retinal layer opacification was more common in mild CRAO cases, which caused inner retinal layer thinning over time. Moderate CRAO cases had total inner retinal layer opacification, which resulted in retinal thinning over time. Prominent middle limiting membrane (p-MLM) sign was seen in mild and moderate CRAO eyes while were not visualised in severe CRAO. This sign gradually faded out over time. Other OCT findings in higher grades of CRAO included inner retinal fluid, neurosensory detachment, internal limiting membrane detachment, hyperreflective foci, and posterior vitreous opacities. Regardless of the CRAO grade, the final end-point seen was inner retinal layer thinning over time.
CONCLUSIONS: OCT in CRAO is a useful for determining the severity of retinal ischemia, disease stage, tissue damage mechanism, and final visual outcome. More prospective studies analysing a larger number of cases at fixed time points will be required in the future.
BACKGROUND: Trial Registration Number: Not applicable.

Low body weight is associated with an increased risk of fractures. However, the effect of temporal changes in the low body weight status on the risk of fracture remains unknown. This study aimed to evaluate the relationships between temporal changes in low body weight status and the risk of fractures in adults over the age of 40 years.
This study included data on adults over 40 years old who underwent two biannual consecutive general health examinations between January 1, 2007 and December 31, 2009 extracted from the National Health Insurance Database, a large nationwide population database. Fracture cases in this cohort were monitored from the time of the last health examination to the end of the designated follow-up period (from January 1, 2010 to December 31, 2018) or the participant\'s death. Fractures were defined as any fracture resulting in hospitalization or outpatient treatment claim after the date of general health screening. The study population was then separated into four groups based on the temporal changes in low body weight status as follows: low body weight to low body weight (L-to-L), low body weight to non-low body weight (L-to-N), non-low body weight to low body weight (N-to-L), and non-low body weight to non-low body weight (N-to-N). The hazard ratios (HRs) for new fractures, depending on weight changes over time, were calculated using Cox proportional hazard analysis.
Adults in the L-to-L, N-to-L, and L-to-N groups had a substantially increased risk of fractures after multivariate adjustment (HR, 1.165; 95% confidence interval [CI], 1.113-1.218; HR, 1.193; 95% CI, 1.131-1.259; and HR, 1.114; 95% CI, 1.050-1.183, respectively). Although the adjusted HR was greater in participants who changed into having a low body weight, followed by those with consistently low body weight, those with low body weight remained to have an elevated risk of fracture independent of weight fluctuation. Elderly men (aged over 65 years), high blood pressure, and chronic kidney disease were significantly associated with an increase in fractures (p < 0.05).
Individuals aged over 40 years with low body weight, even after regaining normal weight, had an increased risk of fracture. Moreover, having a low body weight after having a normal body weight increased the risk of fractures the most, followed by those with consistently low body weight.

UNASSIGNED: To examine the use of proton pump inhibitors (PPIs) in Scandinavian children with focus on the geographical variation, temporal changes and possible contributing factors to observed changes.
UNASSIGNED: An observational population-based study of children and adolescents (1-17 years) in Norway, Sweden, and Denmark during the period 2007-2020. Information concerning dispensed PPIs was obtained from the national prescription databases of each country and presented as means per 1,000 children for each country and calendar year in four age categories (1-4, 5-9, 10-13 and 14-17 years).
UNASSIGNED: In 2007, the PPI use in children was similar across Scandinavian countries. An increased PPI use was observed in all countries during the study period, with gradually increasing differences between the countries. In general, Norway showed both the largest total increase and the largest increase in each age category compared to Sweden and Denmark. In 2020 Norwegian children showed, on average, a 59% higher PPI use compared to Swedish children and a more than double the overall dispensation rate than Denmark. In Denmark there was a 19% reduction in dispensed PPIs from 2015 to 2020.
UNASSIGNED: Despite being countries with similar health care systems and without indications of increased incidence of gastroesophageal reflux disease (GERD), we observed considerable geographical variation and temporal changes of PPI use in children. Although this study did not contain data on the indication for PPI use, these large differences across countries and time may indicate a current overtreatment.

Monitoring early diagnosis is a priority of cancer policy in England. Information on stage has not always been available for a large proportion of patients, however, which may bias temporal comparisons. We previously estimated that early-stage diagnosis of colorectal cancer rose from 32% to 44% during 2008-2013, using multiple imputation. Here we examine the underlying assumptions of multiple imputation for missing stage using the same dataset.
Individually-linked cancer registration, Hospital Episode Statistics (HES), and audit data were examined. Six imputation models including different interaction terms, post-diagnosis treatment, and survival information were assessed, and comparisons drawn with the a priori optimal model. Models were further tested by setting stage values to missing for some patients under one plausible mechanism, then comparing actual and imputed stage distributions for these patients. Finally, a pattern-mixture sensitivity analysis was conducted.
Data from 196,511 colorectal patients were analysed, with 39.2% missing stage. Inclusion of survival time increased the accuracy of imputation: the odds ratio for change in early-stage diagnosis during 2008-2013 was 1.7 (95% CI: 1.6, 1.7) with survival to 1 year included, compared to 1.9 (95% CI 1.9-2.0) with no survival information. Imputation estimates of stage were accurate in one plausible simulation. Pattern-mixture analyses indicated our previous analysis conclusions would only change materially if stage were misclassified for 20% of the patients who had it categorised as late.
Multiple imputation models can substantially reduce bias from missing stage, but data on patient\'s one-year survival should be included for highest accuracy.

UNASSIGNED: Cardiac amyloidosis (CA) has been associated with poor outcomes. Screening studies suggest that CA is overlooked-especially in the elderly. Recent advances in treatment have brought attention to the disease, but data on temporal changes in CA epidemiology are sparse.
UNASSIGNED: The aim of this work was to describe all patients with CA in Denmark, examining changes in patient characteristics from 1998 to 2017.
UNASSIGNED: All patients with any form of amyloidosis diagnosed from 1998 to 2017, as well as their comorbidities and pharmacotherapy, were identified in Danish nationwide registries. CA was defined as any diagnosis code for amyloidosis combined with a diagnosis code for heart failure, cardiomyopathy, or atrial fibrillation or a procedural code for pacemaker implantation, regardless of the order. The index date was defined as the date of meeting those criteria. Patients were divided into 5-year periods by index date. For comparison, we also included control subjects (1:4 ratio) from the general population.
UNASSIGNED: CA criteria were met by 619 patients. Comparing 1998-2002 vs 2013-2017, the median age at baseline increased from 67.4 years (interquartile range [IQR]: 53.9-75.2 years) to 72.3 years (IQR: 66.0-79.3 years). The frequency of male patients increased from 62.1% to 66.2%. The incidence of CA rose from 0.88 to 3.56 per 100,000 person-years in the Danish population aged ≥65 years, and the 2-year mortality decreased from 82.6% (IQR: 69.9%-90.5%) to 50.2% (IQR: 43.1%-56.9%). Compared with control subjects, the mortality among CA patients was significantly higher (log-rank test: P < 0.0001).
UNASSIGNED: CA, as defined in this study, was increasingly diagnosed on a national scale. The increasing frequency of male patients and median age suggest that wild-type transthyretin amyloidosis is driving this increase. Greater recognition of earlier, less advanced cases might explain decreasing mortality.