Non-exposed endoscopic wall-inversion surgery with one-step nucleic acid amplification for early gastrointestinal tumors:Personal experience and literature review

BACKGROUND Laparoscopic and endoscopic cooperative surgery is a safe,organ-sparing surgery that achieves full-thickness resection with adequate margins.Recent studies have demonstrated the safety and efficacy of these procedures.However,these techniques are limited by the exposure of the tumor and mucosa to the peritoneal cavity,which could lead to viable cancer cell seeding and the spillage of gastric juice or enteric liquids into the peritoneal cavity.Non-exposed endoscopic wallinversion surgery(NEWS)is highly accurate in determining the resection margins to prevent intraperitoneal contamination because the tumor is inverted into the visceral lumen instead of the peritoneal cavity.Accurate intraoperative assessment of the nodal status could allow stratification of the extent of resection.One-step nucleic acid amplification(OSNA)can provide a rapid method of evaluating nodal tissue,whilst nearinfrared laparoscopy together with indocyanine green can identify relevant nodal tissue intraoperatively.AIM To determine the safety and feasibility of NEWS in early gastric and colon cancers and of adding rapid intraoperative lymph node(LN)assessment with OSNA.METHODS The patient-based experiential portion of our investigations was conducted at the General and Oncological Surgery Unit of the St.Giuseppe Moscati Hospital(Avellino,Italy).Patients with early-stage gastric or colon cancer(diagnosed via endoscopy,endoscopic ultrasound,and computed tomography)were included.All lesions were treated by NEWS procedure with intraoperative OSNA assay between January 2022 and October 2022.LNs were examined intraoperatively with OSNA and postoperatively with conventional histology.We analyzed patient demographics,lesion features,histopathological diagnoses,R0 resection(negative margins)status,adverse events,and follow-up results.Data were collected prospectively and analyzed retrospectively.RESULTS A total of 10 patients(5 males and 5 females)with an average age of 70.4±4.5 years(range:62-78 years)were enrolled in this study.Five patients were diagnosed with gastric cancer.The remaining 5 patients were diagnosed with early-stage colon cancer.The mean tumor diameter was 23.8±11.6 mm(range:15-36 mm).The NEWS procedure was successful in all cases.The mean procedure time was 111.5±10.7 min(range:80-145 min).The OSNA assay revealed no LN metastases in any patients.Histologically complete resection(R0)was achieved in 9 patients(90.0%).There was no recurrence during the follow-up period.CONCLUSION NEWS combined with sentinel LN biopsy and OSNA assay is an effective and safe technique for the removal of selected early gastric and colon cancers in which it is not possible to adopt conventional endoscopic resection techniques.This procedure allows clinicians to acquire additional information on the LN status intraoperatively.

Role of one-step nucleic acid amplification in colorectal cancer lymph node metastases detection

Current histopathological staging procedures in colorectal cancer(CRC)depend on midline division of the lymph nodes(LNs)with one section of hematoxylin and eosin staining.Cancer cells outside this transection line may be missed,which could lead to understaging of Union for International Cancer Control Stage II high-risk patients.The one-step nucleic acid amplification(OSNA)assay has emerged as a rapid molecular diagnostic tool for LN metastases detection.It is a molecular technique that can analyze the entire LN tissue using a reversetranscriptase loop-mediated isothermal amplification reaction to detect tumorspecific cytokeratin 19 mRNA.Our findings suggest that the OSNA assay has a high diagnostic accuracy in detecting metastatic LNs in CRC and a high negative predictive value.OSNA is a standardized,observer-independent technique,which may lead to more accurate staging.It has been suggested that in stage II CRC,the upstaging can reach 25%and these patients can access postoperative adjuvant chemotherapy.Moreover,intraoperative OSNA sentinel node evaluation may allow early CRC to be treated with organ-preserving surgery,while in more advanced-stage disease,a tailored lymphadenectomy can be performed considering the presence of aberrant lymphatic drainage and skip metastases.

Research progress and prospects of nucleic acid isothermal amplification technology

Nucleic acid(DNA and RNA)detection and quantification methods play vital roles in molecular biology.With the development of molecular biology,isothermal amplification of DNA/RNA,as a new molecular biology technology,can be amplified under isothermal condition,it has the advantages of high sensitivity,high specificity,and high efficiency,and has been applied in various fields of biotechnology,including disease diagnosis,pathogen detection,food hygiene and safety detection and so on.This paper introduces the progress of isothermal amplification technology,including rolling circle amplification(RCA),nucleic acid sequence-dependent amplification(NASBA),strand displacement amplification(SDA),loop-mediated isothermal amplification(LAMP),helicase-dependent amplification(HDA),recombinase polymerase amplification(RPA),cross-priming amplification(CPA),and its principle,advantages and disadvantages,and application development are briefly summarized.

Determination of the predictive factors for diagnostic positivity of nucleic acid amplification tests for diagnosing pulmonary tuberculosis

Background:Tuberculosis(TB)remains a major threat to human health,and TB diagnostic methods remain unsatisfactory.Nucleic acid amplification tests(NAATs)show higher sensitivity compared with culture for the diagnosis of pulmonary TB(PTB).However,NAATs are expensive and cannot be easily implemented outside major medical centers.To improve the sensitivity of NAATs for PTB diagnosis,we investigated the predictive factors that might optimize NAAT utilization.Methods:A total of 1263 patients with suspected PTB were enrolled for evaluation.The sensitivity,specificity,and accuracy of methods including smear-microbiology,culture of Mtb and NAAT for Mycobacterium tuberculosis(Mtb)detection in sputum and bronchoalveolar lavage fluid samples were compared.Odds ratios and 95%confidence intervals were used to assess variables that might be associated with positive NAAT results for sputum and bronchoalveolar lavage fluid from patients with suspected PTB.Results:NAAT showed higher sensitivity for Mtb detection(61.1%)when compared with smear(9.0%)and Mtb culture(47.8%).We found that an elevated erythrocyte sedimentation rate,the presence of cavities,and positive interferon-𝛾release assay(IGRA)results were indicative of positive Mtb detection by NAAT.Moreover,individuals who had all three of these characteristics showed an 86%diagnostic positivity for PTB from Mtb detection by NAAT.Conclusions:Our study suggests that an elevated erythrocyte sedimentation rate,a positive IGRA result,and the presence of pulmonary cavities are helpful factors for predicting positive Mtb detection by NAAT.Patients with the three positive clinical markers should undergo NAAT for Mtb detection because they are the most likely individuals to be bacteriologically confirmed as having TB.

Development of Nucleic Acid Sequence-Based Amplification Assay for Detection of Macrobrachium rosenbergii Nodavirus

A nucleic acid sequence-based amplification（NASBA）assay was established for the detection of Macrobrachium rosenbergii Nodavirus（MrNV）.The specific primers were designed according to the high conserved region of RNA2 sequence of MrNV.The 224 bp specific amplification product was obtained in positive sample determined with 3%agarose gel electrophoresis,while no product was generated from shrimp infected with other viruses including DNA viruses（IHHNV,WSSV）and RNA viruses（TSV,IMNV,YHV）.The detecting limit of the assay was 8pg nucleic acid,which is more sensitive than that of PCR method.

A microfluidic surface-enhanced Raman scattering(SERS) sensor for microRNA in extracellular vesicles with nucleic acid-tyramine cascade amplification

Exosomal micro RNA(mi RNA) is an ideal candidate of noninvasive biomarker for the early diagnosis of cancer. Sensitive and accurate sensing of abnormal exosomal mi RNA plays essential role for clinical promotion due to its close correlation with tumor proliferation and progression. Herein, a microfluidic surface-enhanced Raman scattering(SERS) sensor was proposed for an on-line detection of exosomal mi RNA based on rolling circle amplification(RCA) and tyramine signal amplification(TSA) strategy. The microfluidic chip consists of a magnetic enrichment chamber, a serpentine fluidic mixer and a plasmonic SERS substrate functionalized with capture probes. The released mi RNA activates the capture probe, triggers RCA reaction, and generates a large number of single-stranded DNA products to drive the catalysis of nanotags deposition via TSA, producing numerous “hot spots” to enhance the SERS signals. In merit of the microfluidics chip and nucleic acid-tyramine cascade amplification, the developed SERS sensor significantly improves the sensitivity for the exosomal mi RNA assay, resulting in a limit of detection(LOD) as low as 1 pmol/L and can be successfully applied in the analysis of exosomes secreted from breast tumor cells, which demonstrates the potential utility in practical applications.

Progress of Microfluidics for Biology and Medicine

Microfluidics has been considered as a potential technology to miniaturize the conventional equipments and technologies. It offers advantages in terms of small volume, low cost, short reaction time and highthroughput. The applications in biology and medicine research and related areas are almost the most extensive and profound. With the appropriate scale that matches the scales of cells, microfluidics is well positioned to contribute significantly to cell biology. Cell culture, fusion and apoptosis were successfully performed in microfluidics. Microfluidics provides unique opportunities for rare circulating tumor cells isolation and detection from the blood of patients, which furthers the discovery of cancer stem cell biomarkers and expands the understanding of the biology of metastasis. Nucleic acid amplification in microfluidics has extended to single-molecule, high-throughput and integration treatment in one chip. DNA computer which is based on the computational model of DNA biochemical reaction will come into practice from concept in the future. In addition, microfluidics offers a versatile platform for protein-protein interactions, protein crystallization and high-throughput screening. Although microfluidics is still in its infancy, its great potential has already been demonstrated and will provide novel solutions to the high-throughput applications.

An outlook on coronavirus disease 2019 detection methods

Diagnostic testing plays a fundamental role in the mitigation and containment of coronavirus disease 2019(COVID-19),as it enables immediate quarantine of those who are infected and contagious and is essential for the epidemiological characterization of the virus and estimating the number of infected cases worldwide.Confirmation of viral infections,such as COVID-19,can be achieved through two general approaches:nucleic acid amplification tests(NAATs)or molecular tests,and serological or antibody-based tests.The genetic material of the pathogen is detected in NAAT,and in serological tests,host antibodies produced in response to the pathogen are identified.Other methods of diagnosing COVID-19 include radiological imaging of the lungs and in vitro detection of viral antigens.This review covers different approaches available to diagnosing COVID-19 by outlining their advantages and shortcomings,as well as appropriate indications for more accurate testing.

Turnaround times for molecular testing of pediatric viral cerebrospinal fluid samples in United Kingdom laboratories

BACKGROUND Rapid molecular testing has revolutionized the management of suspected viral meningitis and encephalitis by providing an etiological diagnosis in<90 min with potential to improve outcomes and shorten inpatient stays.However,use of molecular assays can vary widely.AIM To evaluate current practice for molecular testing of pediatric cerebrospinal fluid(CSF)samples across the United Kingdom using a structured questionnaire.METHODS A structured telephone questionnaire survey was conducted between July and August 2020.Data was collected on the availability of viral CSF nucleic acid amplification testing(NAAT),criteria used for testing and turnaround times including the impact of the coronavirus disease 2019 pandemic.RESULTS Of 196/212(92%)microbiology laboratories responded;63/196(32%)were excluded from final analysis as they had no on-site microbiology laboratory and outsourced their samples.Of 133 Laboratories included in the study,47/133(35%)had onsite facilities for viral CSF NAAT.Hospitals currently undertaking onsite NAAT(n=47)had much faster turnaround times with 39 centers(83%)providing results in≤24 h as compared to those referring samples to neighboring laboratories(5/86;6%).CONCLUSION Onsite/near-patient rapid NAAT(including polymerase chain reaction)is recommended wherever possible to optimize patient management in the acute setting.

Clinical diagnostic performance of the simultaneous amplification and testing methods for detection of the Mycobacterium tuberculosis complex for smear-negative or sputum-scarce pulmonary tuberculosis in China

Background Early detection of pulmonary tuberculosis （PTB） is a big challenge in smear negative and sputum scarce patients in China.Simultaneous amplification and testing methods for detection of the Mycobactedum tuberculosis （MTB） complex （SAT-TB assay） is a novel molecular technique established in our hospital.This method has a high sensitivity and specificity in the lab.In this study,the clinical diagnostic performance of this method in smear-negative or sputum-scarce PTB suspects was investigated and evaluated.Methods Two hundred smear negative and 80 sputum-scarce patients were recruited in this study.Samples that included sputum or bronchial washing fluid were collected and sent for both bacteria culture and SAT-TB assay.Diagnosis for these patients was based on the comprehensive evaluation of chestX-ray/CT study,histology examination,lab results,and treatment response.Sensitivity,specificity,positive predictive value （PPV） and negative predictive value （NPV） for each diagnostic test were investigated and calculated using confirmed tuberculosis （TB） and non-TB cases.The time required for detection of MTB was also measured for each method.Results Ninety-two patients （33％） were diagnosed as definitive TB,112 patients （40％） were probable PTB,and 76 （27％） were non-TB.The sensitivity,specificity,PPV,and NPV of SAT-TB in smear-negative PTB suspects were 93％ （95％ CI,84％-98％）,98％ （95％ CI,90％-100％）,98％ （95％ Cl,91％-100％）,and 93％ （95％ CI,83％-98％）.In sputum scarce PTB suspects,the sensitivity,specificity,PPV,and NPV of the SAT-TB assay on bronchial washing fiuids were 90％ （95％Cl,74％-98％）,100％ （95％ Cl,85％-100％）,100％ （95％ Cl,88％-100％）,and 88％ （95％ CI,69％-97％）.The accuracy of the SAT-TB assay is consistent with the bacteria culture assay.The median time required for detecting MTB in the SAT-TB assay was 0.5 day,which was much faster than bacteria culture （28 days）.Conclusions The SAT-TB assay is a fast and accurate method for the detection of MTB.It can be widely applied in the clinic and be an asset in early detection and management of PTB suspects,especially in those patients who are smear negative or sputum scarce.

Enhancement of a recombinase-aided amplification assay using betaine and pullulan

Background:Nucleic acid amplification enhancers suitable for use in a recombinase-aided amplification(RAA)assay were studied for the first time,and amplification of a long-fragment(509 bp)was initially explored.Methods:Using recombinant plasmids and clinical samples,RAA fluorescence and basic methods were used to evaluate the efficacy.The fluorescence method was evaluated by threshold time and fluorescence value,and the basic method was characterized by 2%agarose gel electrophoresis.Results:Taking a previously established RAA assay for HPV18 as an example,we demonstrated that the addition of 0.2 M,0.4 M,and 0.6 M betaine and 10%pullulan could enhance the RAA.The new RAA assays with betaine and pullulan were named B-RAA and P-RAA,respectively.Using the B-RAA and P-RAA fluorescence methods,the threshold time values could be shortened by 1.72-2.32 minutes and 2.60 minutes,respectively,and the fluorescence values could be enhanced by 8847.25-9094.37 mv and 5250 mv,respectively.Using the basic method,the sensitivity could be increased 10-fold.We successfully amplified a long-fragment of 509 bp using a P-RAA assay with a sensitivity of 102 copies/μL(compared with 103 copies/μL in the RAA assay).Conclusions:Thus,we concluded that betaine and pullulan are effective additives to enhance the sensitivity of RAA assays.

Laboratory diagnosis of nonpolio enteroviruses:A review of the current literature

Infections by nonpolio enteroviruses(EVs)are highly prevalent,particularly among children and neonates,where they may cause substantial morbidity and mortality.Laboratory diagnosis of these viral infections is important in patient prognosis and guidance of clinical management.Although the laboratory diagnosis of non-polio EVs is mainly based on molecular techniques,classical virus-isolation techniques are still used in refer-ence laboratories.Other techniques,such as antigen detection and serology,are becoming obsolete and rarely used in diagnosis.An important part of diagnosis and surveillance of EV infections is viral typing by VP1 gene sequencing using conventional Sanger technique and more recently,full-genome next-generation sequencing.The latter allows the typing of all EVs,better investigation of EV outbreaks,detection of coinfec-tion,and identification of severity markers in the EV genome.

Internet of things (IoT) imbedded point-of-care SARS-CoV-2 testing in the pandemic and post-pandemic era

The outbreaks of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)Omicron variant in China have revealed a high rate of asymptomatic cases,making isolation and quarantine measures exceedingly difficult.Public health surveillance and intervention measures will require rapid and accurate testing preferably on-site using point-of-care tests(POCTs)technology for SARS-CoV-2 variants.However,delayed and/or inaccurate surveillance data is a major obstacle blocking the large-scale implementation of POCTs in curbing spread of infectious pathogens and reducing mortality during an outbreak.To determine levels of community transmission and timely strategies accordingly,highly sensitive and specific POCT embedded with the internet of things(IoT)technology could enable on-site screening and real-time data collection.A new Rapid Amplification with Sensitivity And Portability point-of-care test(RASAP-POCT)system based on thermal convection PCR is the first IoT-based isothermal nucleic acid amplification POCT,which can provide test results within 20-30 min using saliva and/or nasopharyngeal swab samples without nucleic acid extraction.With the IoT-imbedded feature,the RASAP-POCT system can be integrated easily and smoothly with China’s existing mobile-phone-based contact tracing system,which has previously proved to be highly effective in maintaining the dynamic zero-COVID policy.Current regulatory guidelines and rules should be modified to accelerate the adoption of new technologies under an emergency use authorization(EUA).