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Ceftriaxone-resistant Neisseria gonorrhoeae FC428-like strains have disseminated across the Asia-Pacific region, with a continuous rise in prevalence during 2015-2022. To mitigate the effect of these strains, we advocate for enhanced molecular diagnostics, expanded surveillance networks, and a regionally coordinated effort to combat the global spread of FC428-like strains.

Abstract Background Antimicrobial resistance (AMR) of Neisseria gonorrhoeae has spread worldwide. Rapid and comprehensive methods are needed to describe N. gonorrhoeae AMR profiles accurately. A method based on multiplex amplicon sequencing was developed to simultaneously sequence 13 genes related to AMR in N. gonorrhoeae directly from clinical samples. Methods Nine N. gonorrhoeae strains were used for the establishment and validation of the method. Eleven urethral swabs and their corresponding cultured isolates were matched as pairs to determine the accuracy of the method. Mock samples with different dilutions were prepared to determine the sensitivity of the method. Five nongonococcal Neisseria strains and 24 N. gonorrhoeae negative clinical samples were used to evaluate the cross-reactivity. Finally, the method was applied to 64 clinical samples to assess its performance. Results Using Sanger sequencing as a reference method, sequences recovered from amplicon sequencing had a base accuracy of over 99.5% and the AMR sites were correctly identified. The limit of detection (LOD) was lower than 31 copies/reaction. No significant cross-reactivity was observed. Furthermore, target genes were successfully recovered from 64 clinical samples including 9 urines, demonstrating this method could be used in different types of samples. For clinical samples, the results can be obtained within a time frame of 7 h 40 min to 10 h 40 min, while for isolates, the turnaround time was approximately 2 h shorter. Conclusions This method can serve as a versatile and convenient culture-free diagnostic method with the advantages of high sensitivity and accuracy.

Infectious diseases contribute significantly to the global disease burden. Sensitive and accurate screening methods are some of the most effective means of identifying sources of infection and controlling infectivity. Conventional detecting strategies such as quantitative polymerase chain reaction (qPCR), DNA sequencing, and mass spectrometry typically require bulky equipment and well-trained personnel. Therefore, mass screening of a large population using conventional strategies during pandemic periods often requires additional manpower, resources, and time, which cannot be guaranteed in resource-limited settings. Recently, emerging microfluidic technologies have shown the potential to replace conventional methods in performing point-of-care detection because they are automated, miniaturized, and integrated. By exploiting the spatial separation of detection sites, microfluidic platforms can enable the multiplex detection of infectious diseases to reduce the possibility of misdiagnosis and incomplete diagnosis of infectious diseases with similar symptoms. This review presents the recent advances in microfluidic platforms used for multiplex detection of infectious diseases, including microfluidic immunosensors and microfluidic nucleic acid sensors. As representative microfluidic platforms, lateral flow immunoassay (LFIA) platforms, polymer-based chips, paper-based devices, and droplet-based devices will be discussed in detail. In addition, the current challenges, commercialization, and prospects are proposed to promote the application of microfluidic platforms in infectious disease detection.

Mitigating the spread of global infectious diseases requires rapid and accurate diagnostic tools. Conventional diagnostic techniques for infectious diseases typically require sophisticated equipment and are time consuming. Emerging clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated proteins (Cas) detection systems have shown remarkable potential as next‐generation diagnostic tools to achieve rapid, sensitive, specific, and field‐deployable diagnoses of infectious diseases, based on state‐of‐the‐art microfluidic platforms. Therefore, a review of recent advances in CRISPR‐based microfluidic systems for infectious diseases diagnosis is urgently required. This review highlights the mechanisms of CRISPR/Cas biosensing and cutting‐edge microfluidic devices including paper, digital, and integrated wearable platforms. Strategies to simplify sample pretreatment, improve diagnostic performance, and achieve integrated detection are discussed. Current challenges and future perspectives contributing to the development of more effective CRISPR‐based microfluidic diagnostic systems are also proposed. The CRISPR/Cas detection systems that are armed with state‐of‐the‐art microfluidic platforms have shown remarkable potential as next‐generation diagnostic tools. In this review, CRISPR‐based microfluidic systems for infectious diseases diagnosis, including the mechanisms of CRISPR/Cas biosensing and the cutting‐edge microfluidic devices are systematically summarized. Additionally, current challenges and future perspectives are discussed to develop more effective CRISPR‐based microfluidic diagnostic systems.

The cutting‐edge CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas (CRISPR‐associated proteins) system, as an emerging molecular diagnostic technique, is driving revolutionary developments in the detection field due to its high specificity and efficiency. However, the CRISPR‐based assays typically require the combination with an additional pre‐amplification step based on isothermal nucleic acid amplification to meet the requirements of clinical diagnosis, which brings issues including complicated operation and the risk of aerosol contamination. To address these challenges, one‐pot CRISPR platforms are emerging as an attractive solution to streamline workflows, enabling rapid, cost‐effective, and high‐sensitivity diagnostics. This review outlines the current status, challenges, and three key strategies to realize highly efficient one‐pot CRISPR‐based detection. In addition, further perspectives are outlined that will inspire new exploration and promote one‐pot CRISPR/Cas detection as the next generation of diagnostic tools. This review combines the current status of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)‐based molecular diagnostic technology to expound the necessity and challenges faced in promoting the development of one‐pot detection. Besides, the focus is on a comprehensive summary and incisive analysis of emerging strategies for one‐pot CRISPR‐based detection and prospects for future research directions to provide guidance for relevant researchers.

Human norovirus (HuNoV) is the leading cause of nonbacterial acute gastroenteritis, which is highly infectious, rapidly evolving, and easily transmitted through feces. The accurate and early detection of HuNoV subtypes is essential for effective treatment, early surveillance, risk assessment, and disease prevention. In this study, a portable multiplex HuNoV detection platform that combines integrated microfluidics and cascade isothermal amplification, using a streamlined protocol for clinical fecal‐based diagnosis is presented. To overcome the problems of carryover contamination and the incompatibility between recombinase polymerase amplification (RPA) and loop‐mediated isothermal amplification (LAMP), a Dynamic confined‐space‐implemented One‐pot RPA‐LAMP colorimetric detection system (DORLA) is developed by creating a hydrogen bond network. The DORLA system exhibits excellent sensitivity, with detection limits of 10 copies µL−1 and 1 copy µL−1 for HuNoV GI and GII, respectively. In addition, a portable diagnostic platform consisting of a thermostatic control module and an integrated 3D‐printed microfluidic chip for specific HuNoV capture, nucleic acid pretreatment, and DORLA detection, which enables simultaneous diagnosis of HuNoV GI and GII is developed. A DORLA‐based microfluidic platform exhibits satisfactory performance with high sensitivity and portability, and has high potential for the rapid point‐of‐care detection of HuNoV in clinical fecal samples, particularly in resource‐limited settings. Herein, a dynamic confined space implemented one‐pot recombinase polymerase amplification and Loop‐mediated isothermal amplification colorimetric detection system (DORLA) is established by creating a hydrogen bond network, which can achieve a simultaneous diagnosis of Human norovirus GI and GII on the portable diagnostic platform.

Sexually transmitted infections (STIs), representing a major global health problem, are caused by different microbes, including bacteria, viruses, and protozoa. Unfortunately, infections of different sexually transmitted pathogens often present similar clinical symptoms, so it is almost impossible to distinguish them clinically. Therefore, the aim of the current study was to develop a sensitive, multitarget, and high-throughput method that can detect various agents responsible for STIs. We developed and tested a 23-plex PCR coupled with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) assay (sexually transmitted infection-mass spectrometry, STI-MS) that simultaneously targets 11 different agents, including 8 most common clinical pathogens related to STIs (HSV-1, HSV-2, , and ) and 3 controversial microorganisms as pathogens ( , and ). The results showed that the STI-MS approach can accurately detect the expected agents, without cross-reaction with other organisms. The limit of detection of each STI-MS assay was ranged from 1.739 to 10.009 copies/reaction, using probit analyses. The verification rate for each target organism of the STI-MS ranged from a minimum of 89.3% to a maximum of 100%, using conventional assays and ultrasensitive digital PCR to confirm the STI-MS-positive results. To further evaluate the clinical performance of this assay, 241 clinical specimens (124 urethral/cervical swabs and 117 urine) were tested in parallel using the STI-MS assay and monoplex real-time PCR for each agent. The overall validation parameters of STI-MS were extremely high including sensitivity (from 85.7% to 100%), specificity (from 92.3% to 100%), PPV (from 50% to 100%), and NPV (from 99.1% to 100%) for each target. STI-MS is a useful high-throughput screening tool for detecting mixed infections of STIs and has great potential for application in large-scale epidemiological programs for specific microorganisms of STI.

Background Antimicrobial resistance in gonorrhea has become a growing global public health burden. Neisseria gonorrhoeae isolates with resistance to ceftriaxone, the last remaining first-line option, represent an emerging threat of untreatable gonorrhea. Methods A total of ten ceftriaxone-resistant N. gonorrhoeae FC428 isolates and two isolates harboring a novel mosaic penA -232.001 allele from 160 gonococcal isolates in Chengdu in 2019–2020 was described in the present study. Multilocus sequence typing (MLST) and N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) were performed to characterize the isolates. Whole genome sequencing and maximum-likelihood method were performed to infer how the genetic phylogenetic tree of these isolates looks like. Recombination analysis was performed using the RDP4 software. This study was registered in the Chinese Clinical Trial Registry (ChiCTR2100048771, registration date: 20210716). Results The genetic phylogeny showed that the ten FC428 isolates sporadically clustered into different phylogenetic clades, suggesting different introductions and local transmission of FC428. Two isolates showed close genetic relatedness to ceftriaxone-resistant clone A8806, which was only reported from Australia in 2013. Homologous recombination events were detected in penA between Neisseria gonorrhoeae and commensal Neisseria species ( N. perflava and N. polysaccharea ), providing evidence of commensal Neisseria species might serve as reservoirs of ceftriaxone resistance-mediating penA sequences in clinical gonococcal strains. Conclusions Our results demonstrate further dissemination of FC428 in China and resurgence risks of sporadic ceftriaxone-resistant A8806 to become the next clone to spread.

Chlamydia trachomatis is the most common sexually transmitted pathogen globally, causing serious health problems and representing a burden on public health. A new variant of C. trachomatis (nvCT) that carries mutations (C1514T, C1515T and G1523A) in the 23S rRNA gene has eluded detection in Aptima Combo 2 assays. This has led to false negatives in diagnostics tests and poses a challenge for C. trachomatis diagnostics on a global level. In this study, we developed a simple and cost‐effective assay to identify C. trachomatis , with a potential application to screen for nvCT. We developed a screening assay based on high‐resolution melting (HRM), targeting the 23S rRNA gene and cryptic plasmid. To evaluate the performance of the assay, 404 archived C. trachomatis DNA specimens and 570 extracted clinical specimens were analysed. Our HRM assay not only identified C. trachomatis in clinical specimens, but also correctly differentiated nvCT carrying C1514T, C1515T and G1523A mutations from the wild‐type. We observed no cross‐reactions with other clinically related agents, and the limit of detection was 11.26 (95% CI; 7.61–31.82) copies per reaction. Implementation of this screening assay could reduce detection times and costs for C. trachomatis diagnoses, and facilitate increased research on the presence and monitoring of nvCT.