Rapid,high-throughput,timely,multiplex diagnosis of respiratory-tract infections still relies on laboratory infrastructure,sequential assays,and trained personnel,thereby delaying targeted therapy and outbreak contain...Rapid,high-throughput,timely,multiplex diagnosis of respiratory-tract infections still relies on laboratory infrastructure,sequential assays,and trained personnel,thereby delaying targeted therapy and outbreak containment.In this study,a Fully Automated rotary microfluidic platform(FA-RMP)for high-throughput multiplex respiratory tract pathogens detection was presented.FA-RMP enables a true“sample-in,result-out”workflow through the integration of swab lysis,reagent partitioning,lyophilized reverse transcription loop-mediated isothermal amplification(RT-LAMP),and movingprobe fluorescence read-out,all encapsulated with a disposable microfluidic cartridge and paired with a 9 kg,fourchannel benchtop reader.The FA-RMP enables parallel processing of 16 independent reactions within 30 min,supporting simultaneous detection of up to 4 distinct clinical samples.Analytical validation using serially diluted Mycoplasma pneumoniae(MP)DNA established a limit of detection(LoD)of 50 copies μL^(-1) and a log-linear correlation between threshold time and template load(R^(2)=0.9528).Testing with eight non-target respiratory pathogens yielded no amplification,confirming high analytical specificity.FA-RMP successfully detected the clinical samples with influenza A,influenza B,and MP,further demonstrating its robust multiplex detection capability.By integrating automated sample preparation,multiplex isothermal amplification and quantitative detection into a portable,high-throughput system,the platform delivers laboratory-grade performance at the point of care,serving as a scalable tool for routine respiratory pathogens screening and rapid epidemic response.展开更多
BACKGROUND Klebsiella pneumoniae(K.pneumoniae)is an infective microorganism of worldwide concern because of its varied manifestations and life-threatening potential.Genetic analyses have revealed that subspecies of K....BACKGROUND Klebsiella pneumoniae(K.pneumoniae)is an infective microorganism of worldwide concern because of its varied manifestations and life-threatening potential.Genetic analyses have revealed that subspecies of K.pneumoniae exhibit higher virulence and mortality.However,infections with Klebsiella subspecies are often misdiagnosed and underestimated in the clinic because of difficulties in distinguishing K.pneumoniae from its subspecies using routine tests.This case study reports the rapid and fatal effects of K.pneumoniae subspecies.CASE SUMMARY A 52-year-old male patient was febrile and admitted to hospital.Examinations excluded viral and fungal causes along with mycoplasma/chlamydia and parasitic infections.Bacterial cultures revealed blood-borne K.pneumoniae sensitive to carbapenem antibiotics,although corresponding treatment failed to improve the patient’s symptoms.His condition worsened and death occurred within 72 h of symptom onset from sepsis shock.Application of the PMseq-DNA Pro high throughput gene detection assay was implemented with results obtained after death showing a mixed infection of K.pneumoniae and Klebsiella variicola(K.variicola).Clinical evidence suggested that K.variicola rather than K.pneumoniae contributed to the patient’s poor prognosis.CONCLUSION This is the first case report to show patient death from Klebsiella subspecies infection within a short period of time.This case provides a timely reminder of the clinical hazards posed by Klebsiella subspecies and highlights the limitations of classical laboratory methods in guiding anti-infective therapies for complex cases.Moreover,this report serves as reference for physicians diagnosing similar diseases and provides a recommendation to employ early genetic detection to aid patient diagnosis and management.展开更多
文摘Rapid,high-throughput,timely,multiplex diagnosis of respiratory-tract infections still relies on laboratory infrastructure,sequential assays,and trained personnel,thereby delaying targeted therapy and outbreak containment.In this study,a Fully Automated rotary microfluidic platform(FA-RMP)for high-throughput multiplex respiratory tract pathogens detection was presented.FA-RMP enables a true“sample-in,result-out”workflow through the integration of swab lysis,reagent partitioning,lyophilized reverse transcription loop-mediated isothermal amplification(RT-LAMP),and movingprobe fluorescence read-out,all encapsulated with a disposable microfluidic cartridge and paired with a 9 kg,fourchannel benchtop reader.The FA-RMP enables parallel processing of 16 independent reactions within 30 min,supporting simultaneous detection of up to 4 distinct clinical samples.Analytical validation using serially diluted Mycoplasma pneumoniae(MP)DNA established a limit of detection(LoD)of 50 copies μL^(-1) and a log-linear correlation between threshold time and template load(R^(2)=0.9528).Testing with eight non-target respiratory pathogens yielded no amplification,confirming high analytical specificity.FA-RMP successfully detected the clinical samples with influenza A,influenza B,and MP,further demonstrating its robust multiplex detection capability.By integrating automated sample preparation,multiplex isothermal amplification and quantitative detection into a portable,high-throughput system,the platform delivers laboratory-grade performance at the point of care,serving as a scalable tool for routine respiratory pathogens screening and rapid epidemic response.
基金Supported by Science and Technology Fund of Guizhou Provincial Health CommissionNo. gzwjkj2019-1-067+1 种基金Doctor Foundation of Guizhou Provincial People’s HospitalNo. GZSYBS[2019]04
文摘BACKGROUND Klebsiella pneumoniae(K.pneumoniae)is an infective microorganism of worldwide concern because of its varied manifestations and life-threatening potential.Genetic analyses have revealed that subspecies of K.pneumoniae exhibit higher virulence and mortality.However,infections with Klebsiella subspecies are often misdiagnosed and underestimated in the clinic because of difficulties in distinguishing K.pneumoniae from its subspecies using routine tests.This case study reports the rapid and fatal effects of K.pneumoniae subspecies.CASE SUMMARY A 52-year-old male patient was febrile and admitted to hospital.Examinations excluded viral and fungal causes along with mycoplasma/chlamydia and parasitic infections.Bacterial cultures revealed blood-borne K.pneumoniae sensitive to carbapenem antibiotics,although corresponding treatment failed to improve the patient’s symptoms.His condition worsened and death occurred within 72 h of symptom onset from sepsis shock.Application of the PMseq-DNA Pro high throughput gene detection assay was implemented with results obtained after death showing a mixed infection of K.pneumoniae and Klebsiella variicola(K.variicola).Clinical evidence suggested that K.variicola rather than K.pneumoniae contributed to the patient’s poor prognosis.CONCLUSION This is the first case report to show patient death from Klebsiella subspecies infection within a short period of time.This case provides a timely reminder of the clinical hazards posed by Klebsiella subspecies and highlights the limitations of classical laboratory methods in guiding anti-infective therapies for complex cases.Moreover,this report serves as reference for physicians diagnosing similar diseases and provides a recommendation to employ early genetic detection to aid patient diagnosis and management.
