It is difficult to differentiate between coronavirus disease 2019 (COVID-19) and influenza based on the symptoms. In the present study, a newly developed antigen rapid diagnostic test (Ag-RDT) called Panbio™ COVID-19/Flu A&B that can simultaneously detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A/B virus was evaluated. Its accuracy was evaluated using 235 pairs of nasopharyngeal samples collected from patients with respiratory symptoms and fever (>37.5°C). Reverse transcription polymerase chain reaction was used as a reference method to evaluate the accuracy of the SARS-CoV-2 detection. We confirmed the accuracy of the developed Ag-RDT against the Omicron variant where the sensitivity and specificity were 94.8% and 100%, respectively. In addition, to identify the influenza A virus, a noninferiority test was conducted using a commercial Ag-RDT, which has a sensitivity and specificity in comparison with viral culture of 94.8% and 98.4%, respectively. The positive and negative predictive values for influenza A virus were 98.5% and 98.1%, respectively, for the Panbio COVID-19/Flu A&B test. The evaluation of this newly developed Ag-RDT using clinical samples suggests that it has a high efficacy in clinical settings.

UNASSIGNED: Monkeypox virus (MPXV) is spreading globally and nearly half of the infected people were human immunodeficiency virus (HIV) positive. Therefore, an in-depth understanding of the effects of HIV infection on the outcomes of MPXV infection is urgently needed. This study aimed to explore the clinical features, viral dynamics, and antibody response to MPXV infections in men who had sex with men (MSM) with and without HIV co-infection.
UNASSIGNED: MPXV-infected patients diagnosed by PCR were recruited in this study and were divided into MPXV and MPXV + HIV groups based on whether they were co-infected with HIV. Clinical data and samples were collected during of the hospital stay and follow up interviews. The symptoms and signs, laboratory examinations, viral shedding in various body fluids or swabs, antibody dynamics were tracked and compared between the two groups.
UNASSIGNED: A total of 41 MPXV patients were recruited through June 2023 to September 2023 in Guangzhou. The MPXV group and MPXV + HIV group comprised 20 and 21 MSM, respectively. Patients in the two groups exhibited similar clinical characteristics except for pruritus and eschar, both were significantly fewer in MPXV + HIV group than in MPXV only group. Among the 355 clinical samples collected, MPXV DNA was detected in 100% of scabs, 97.4% of skin swabs, and 92.3% of exudate swabs from lesions, while the positive rate was 87.5% from oropharyngeal swabs, 59% from saliva, 51.3% from anal swabs, 50% from feces, 30.6% from urine samples, 37.5% of semen, and 28.2% from sera. Dynamics analysis revealed that viral DNA was undetectable in most patients 20 days after symptom onset. IgM and IgG antibodies to MPXV were detected in all patients with 3-5 days earlier in the MPXV group than in the MPXV + HIV group.
UNASSIGNED: This cohort analysis based on a large outbreak among MSM in Guangzhou indicated no obvious differences in clinical symptoms, viral DNA data, but antibody responses were 3-5 days later in mpox patients with HIV infection.

Rapid characterization of the causative agent(s) during a disease outbreak can aid in the implementation of effective control measures. However, isolation of the agent(s) from crude clinical samples can be challenging and time-consuming, hindering the establishment of countermeasures. In the present study, we used saliva specimens collected for the diagnosis of SARS-CoV-2-a good example of a practical target-and attempted to characterize the virus within the specimens without virus isolation. Thirty-four saliva samples from coronavirus disease 2019 patients were used to extract RNA and synthesize DNA amplicons by PCR. New primer sets were designed to generate DNA amplicons of the full-length spike (S) gene for subsequent use in a circular polymerase extension reaction (CPER), a simple method for deriving recombinant viral genomes. According to the S sequence, four clinical specimens were classified as BA. 1, BA.2, BA.5, and XBB.1 and were used for the de novo generation of recombinant viruses carrying the entire S gene. Additionally, chimeric viruses carrying the gene encoding GFP were generated to evaluate viral propagation using a plate reader. We successfully used the RNA purified directly from clinical saliva samples to generate chimeric viruses carrying the entire S gene by our updated CPER method. The chimeric viruses exhibited robust replication in cell cultures with similar properties. Using the recombinant GFP viruses, we also successfully characterized the efficacy of the licensed antiviral AZD7442. Our proof-of-concept demonstrates the novel utility of CPER to allow rapid characterization of viruses from clinical specimens.
OBJECTIVE: Characterization of the causative agent(s) for infectious diseases helps in implementing effective control measurements, especially in outbreaks. However, the isolation of the agent(s) from clinical specimens is often challenging and time-consuming. In this study, saliva samples from coronavirus disease 2019 patients were directly subjected to purifying viral RNA, synthesizing DNA amplicons for sequencing, and generating recombinant viruses. Utilizing an updated circular polymerase extension reaction method, we successfully generated chimeric SARS-CoV-2 viruses with sufficient in vitro replication capacity and antigenicity. Thus, the recombinant viruses generated in this study were applicable for evaluating the antivirals. Collectively, our developed method facilitates rapid characterization of specimens circulating in hosts, aiding in the establishment of control measurements. Additionally, this approach offers an advanced strategy for controlling other (re-)emerging viral infectious diseases.

BACKGROUND: Antimicrobial resistance is one of the common global public health problems. The emergence of antimicrobial resistance is multifactorial, and tackling its development is challenging. Consequently, infections caused by resistant bacteria are unresponsive to conventional drugs, resulting in prolonged and severe illnesses, higher mortality rates, and considerable healthcare costs. Therefore, understanding the antimicrobial resistance profiles of bacterial pathogens is essential to optimize treatments and reduce the risks associated with infections. This study aimed to determine the antimicrobial resistance patterns of bacterial isolates from different clinical specimens at the Ethiopian Public Health Institute (EPHI).
METHODS: The retrospective cross-sectional study was conducted on the bacterial culture and antibiotic susceptibility reports of different clinical specimens referred to the Bacteriology Laboratory of EPHI from September 2015 to August 2019. Standard bacteriological techniques were used for the isolation and identification of the bacteria. Data were extracted from 840 patients\' records, which included the type of clinical sample cultured, the name of the bacteria, the representations of the antibiotics used for susceptibility testing, and the susceptibility results. Descriptive statistics were used to describe the bacterial isolates and the antimicrobial resistance profiles.
RESULTS: Eight types of clinical specimens were analyzed for bacterial isolates and urine specimens were the most analyzed. Ten different genera of bacteria were identified by culture. Almost all the isolates were gram-negative bacteria, while only one species of gram-positive (Staphylococcus aureus) was reported. Antibiotic sensitivity patterns were tested on 840 culture isolates. Escherichia coli strains revealed more than 57% resistance to seventeen antibiotics. Klebsiella pneumoniae showed nearly 70% or greater resistance rates for 17 of the antibiotics used. The overall detected multidrug resistance (MDR) was 64.29%. The highest MDR was reported in Acinetobacter strains (84%) followed by K. pneumoniae (80%).
CONCLUSIONS: The multidrug resistance rates found in this study were alarming. Strengthening antimicrobial resistance surveillance at the national level is mandatory, and antimicrobial sensitivity testing should be accessible at local diagnostic centers.

We developed a convenient method for amplifying the complete SARS-CoV-2 sequence using in-house RT-PCR without virus culture. Forty-one stored throat swabs and blood specimens were collected from eight SARS-CoV-2 infections at multiple time points. Total RNA was extracted using the QIAamp viral RNA mini kit and pooled for higher RNA levels. Only those positive specimens by commercial real-time RT-PCR (RT-qPCR) were selected and amplified by in-house RT-PCR for complete sequences, followed by sequencing. Phylogenetic trees and exploratory analyses were performed using MEGA 11 and Simplot 3.5.1 software. Swab samples had significantly higher total RNA concentrations than plasma (p < 0.01). Positive results were found mainly in swabs, but one was found in plasma. Successful gene amplification depended on Ct values (Ct < 38). A non-synonymous substitution was found in ORF1ab/Nsp3 (at NC045512.2 position 6312, C to A) and most spike protein mutations occurred in the S1 subunit (residues 14-685). The proposed method is time-saving and reliable for rapid genomic analysis. Increasing sample volume and pooling them for RNA extraction increases RNA concentration without culture. Combining nucleotide sequences from specific variable regions of the genome is more efficient than conventional methods.

Papillary thyroid cancer (PTC) is a common malignancy of the endocrine system, and its morbidity and mortality are increasing year by year. Traditional two-dimensional culture of cell lines lacks tissue structure and is difficult to reflect the heterogeneity of tumors. The construction of mouse models is inefficient and time-consuming, which is difficult to be applied to individualized treatment on a large scale. Clinically relevant models that recapitulate the biology of their corresponding parental tumors are urgently needed. Based on clinical specimens of PTC, we have successfully established patient-derived organoids by exploring and optimizing the organoid culture system. These organoids have been cultured stably for more than 5 passages and successfully cryopreserved and retried. Histopathological and genome analysis revealed a high consistency of the histological architectures as well as mutational landscapes between the matched tumors and organoids. Here, we present a fully detailed method to derive PTC organoids from clinical specimens. Using this approach, we have developed PTC organoid lines from thyroid cancer samples with a success rate of 77.6% (38/49) until now.

In the 2022 mpox outbreak, several studies have explored longitudinal DNA shedding of mpox virus (MPXV) using PCR. However, there are fewer studies assessing infectivity in cell culture, and, by inference, MPXV transmissibility. Such information could help inform infection control and public health guidelines.
The aim of this study was to correlate cell culture infectivity of clinical samples with viral loads in clinical samples. Between May to October 2022, clinical samples from different body sites sent to the Victorian Infectious Diseases Reference Laboratory in Melbourne, Australia for MPXV PCR detection were cultured in Vero cells as a surrogate for infectivity.
In the study period, 144 samples from 70 patients were tested by MPXV PCR. Viral loads in skin lesions were significantly higher than those in throat or nasopharyngeal samples (median Ct 22.0 vs 29.0, p = 0.0013 and median Ct 22.0 vs 36.5, p = 0.0001, respectively). Similarly, viral loads were significantly higher in anal samples compared to throat or nasopharyngeal samples (median Ct 20.0 vs. 29.0, p=<0.0001 and median Ct 20.0 vs. 36.5, p=<0.0001, respectively). Viral culture was successfully performed in 80/94 samples. Using logistic regression analysis, 50% of the samples were positive in viral culture at Ct 34.1 (95% confidence intervals 32.1-37.4).
Our data further validate recent findings showing that samples with a higher MPXV viral load are more likely to demonstrate infectivity in cell culture. Although the presence of infectious virus in cell culture may not directly translate with clinical transmission risk, our data may be used as an adjunct help inform guidelines on testing and isolation policies in individuals with mpox.

16S rRNA gene sequencing is increasingly used in clinical practice for bacterial identification of clinical specimens. However, studies on its applicability to direct clinical specimens are limited. Here, we studied the diagnostic yield and impact of 16S rRNA gene sequencing from direct clinical specimens on antimicrobial management. Adult inpatients whose attending physician requested 16S rRNA gene sequencing and corresponding bacterial culture from a direct clinical specimen between January and December 2021 in a university hospital were prospectively included in this study. A total of 434 specimens from 374 patients were requested. Of these, 253 (58.3%) specimens were collected from patients whose final diagnosis indicated a bacterial infection, whereas 181 (41.7%) specimens were from nonbacterial infections. Using the final diagnosis as a \"gold standard,\" the sensitivity and specificity of 16S rRNA gene sequencing were 38.3% and 93.9%, respectively. Among the bacterial infection cases, the proportion of 16S rRNA gene sequencing-positive and culture-positive cases was 32.4%, and the proportion of sequencing-positive and culture-negative cases was 5.9%. The impact on antimicrobial management was evident in 10 (2.3%) specimens, which all resulted in the continuation of antibiotics. The impact on antimicrobial management was highest in skin and soft tissue infections, followed by bone and joint infections. In this study, the long turnaround time of 16S rRNA gene sequencing of clinical specimens was a limiting factor. In conclusion, the overall diagnostic yield of 16S rRNA gene sequencing in bacterial infection cases was fair, being useful in selected cases. Restrictions on test requests may improve test utilization in this setting. IMPORTANCE 16S rRNA gene sequencing has been increasingly used in clinical practice. Using the final diagnosis as a gold standard, the sensitivity of 16S rRNA gene sequencing was fair. In the setting with no 16S rRNA gene sequencing test ordering restrictions, only small percentages of the test results had an impact on antimicrobial management. Restrictions on test requests should be developed to maximize the benefit of the test.

The ongoing monkeypox virus outbreak includes at least 7553 confirmed cases in previously non-endemic countries worldwide as of July 2022. Clinical presentation has been reported as highly variable, sometimes lacking classically described systemic symptoms, and only small numbers of cutaneous lesions in most patients. The aim of this study was to compare clinical data with longitudinal qPCR results from lesion swabs, oropharyngeal swabs and blood in a well characterized patient cohort.
16 male patients (5 hospitalized, 11 outpatients) were included in the study cohort and serial testing for monkeypox virus-DNA carried out in various materials throughout the course of disease. Laboratory analysis included quantitative PCR, next-generation sequencing, immunofluorescence tests and virus isolation in cell culture.
All patients were male, between age 20 and 60, and self-identified as men having sex with men. Two had a known HIV infection, coinciding with an increased number of lesions and viral DNA detectable in blood. In initial- and serial testing, lesion swabs yielded viral DNA-loads at, or above 106 cp/ml and only declined during the third week. Oropharyngeal swabs featured lower viral loads and returned repeatedly negative in some cases. Viral culture was successful only from lesion swabs but not from oropharyngeal swabs or plasma.
The data presented underscore the reliability of lesion swabs for monkeypox virus-detection, even in later stages of the disease. Oropharyngeal swabs and blood samples alone carry the risk of false negative results, but may hold value in pre-/asymptomatic cases or viral load monitoring, respectively.

Diagnosis of botulism caused by multiple serotypes of botulinum neurotoxin (BoNT) is still a challenge due to the lack of a reliable detection method. The present study develops a feasible laboratorial method based on an isotope dilution Immuno-Endopep-MS to detect BoNTs and determine their serotypes and activities in clinical samples. Eleven positive foodborne botulism cases out of a total of 17 suspected cases in China, 2019-2022, were determined by the established method. Blood, urine, vomitus, gastric mucosa samples, and food samples were employed and evidenced to be suitable for the detection. Results showed that, although single type A-intoxication was still the first cause among these foodborne botulism cases, other causes involving type E, type B, and their mixed types were also determined, providing a glimpse to the serotype profile of botulism happened in recent years in China. Furthermore, in order to provide insights into in vivo profiles of toxin serotypes, a comprehensive analysis of clinical specimens collected from one family of four patients was performed during a clinically and therapeutically relevant time frame. Serotypes and concentrations of BoNT in specimens revealed a good correlation with symptoms and progresses of disease. Additionally, serum was proved to be more suitable for detection of BoNT/A with a detection window up to 12 days. A urine sample, although rarely reported for foodborne botulism diagnosis, was validated to be suitable for testing BoNTs, with a longer detection window up to 25 days. To the best of our knowledge, this is the first comprehensive analytical research on in vivo profiles of serotypes A, B, and E in different types of specimens from mixed botulism cases. Our method and findings facilitate the toxin detection and identification by clinical diagnostic laboratories.