Impact of next-generation sequencing on antimicrobial treatment in immunocompromised adults with suspected infections

BACKGROUND:Prompt pathogen identification can have a substantial impact on the optimization of antimicrobial treatment.The objective of the study was to assess the diagnostic value of next-generation sequencing(NGS)for identifying pathogen and its clinical impact on antimicrobial intervention in immunocompromised patients with suspected infections.METHODS:This was a retrospective study.Between January and August 2020,47 adult immunocompromised patients underwent NGS testing under the following clinical conditions:1)prolonged fever and negative conventional cultures;2)new-onset fever despite empiric antimicrobial treatment;and 3)afebrile with suspected infections on imaging.Clinical data,including conventional microbial test results and antimicrobial treatment before and after NGS,were collected.Data were analyzed according to documented changes in antimicrobial treatment(escalated,no change,or deescalated)after the NGS results.RESULTS:The median time from hospitalization to NGS sampling was 19 d.Clinically relevant pathogens were detected via NGS in 61.7% of patients(29/47),more than half of whom suffered from fungemia(n=17),resulting in an antimicrobial escalation in 53.2% of patients(25/47)and antimicrobial de-escalation in 0.2% of patients(1/47).Antimicrobial changes were mostly due to the identification of fastidious organisms such as Legionella,Pneumocystis jirovecii,and Candida.In the remaining three cases,NGS detected clinically relevant pathogens also detected by conventional cultures a few days later.The antimicrobial treatment was subsequently adjusted according to the susceptibility test results.Overall,NGS changed antimicrobial management in 55.3%(26/47)of patients,and conventional culture detected clinically relevant pathogens in 14.9% of the patients(7/47).CONCLUSION:With its rapid identification and high sensitivity,NGS could be a promising tool for identifying relevant pathogens and enabling rapid appropriate treatment in immunocompromised patients with suspected infections.

Brain abscess from oral microbiota approached by metagenomic next-generation sequencing: A case report and review of literature

BACKGROUND Brain abscess is a serious and potentially fatal disease caused primarily by microbial infection.Although progress has been made in the diagnosis and treatment of brain abscesses,the diagnostic timeliness of pathogens needs to be improved.CASE SUMMARY We report the case of a 54-year-old male with a brain abscess caused by oral bacteria.The patient recovered well after receiving a combination of metagenomic next-generation sequencing(mNGS)-assisted guided medication and surgery.CONCLUSION Therefore,mNGS may be widely applied to identify the pathogenic microor-ganisms of brain abscesses and guide precision medicine.

Bronchoalveolar lavage fluid metagenomic next-generation sequencing assay for identifying pathogens in lung cancer patients

Background:For patients with lung cancer,timely identification of new lung lesions as infectious or non-infectious,and accurate identification of pathogens is very important in improving OS of patients.As a new auxiliary examination,metagenomic next-generation sequencing(mNGS)is believed to be more accurate in diagnosing infectious diseases in patients without underlying diseases,compared with conventional microbial tests(CMTs).We designed this study tofind out whether mNGS has better performance in distinguishing infectious and non-infectious diseases in lung cancer patients using bronchoalveolar lavagefluid(BALF).Materials and Methods:This study was a real-world retrospective review based on electronic medical records of lung cancer patients with bronchoalveolar lavage(BAL)and BALF commercial mNGS testing as part of clinical care from 1 April 2019 through 30 April 2022 at The First Affiliated Hospital of Sun Yat-sen University.164 patients were included in this study.Patients were categorized into the pulmonary non-infectious disease(PNID)group(n=64)and the pulmonary infectious disease(PID)group(n=100)groups based onfinal diagnoses.Results:BALF mNGS increased the sensitivity rate by 60%compared to CMTs(81%vs.21%,p<0.05),whereas there was no significant difference in specificity(75%vs.98.4%,p>0.1).Among the patients with PID,bacteria were the most common cause of infection.Fungal infections occurred in 32%of patients,and Pneumocystis Yersini was most common.Patients with Tyrosine kinase inhibitors(TKIs)therapy possess longer overall survival(OS)than other anti-cancer agents,the difference between TKIs and immuno-checkpoint inhibitors(ICIs)was insignificant(median OS TKIs vs.ICIs vs.Anti-angiogenic vs.Chemo vs.Radiotherapy=76 vs.84 vs.61 vs.58 vs.60).Conclusions:our study indicates that BALF mNGS can add value by improving overall sensitivity in lung cancer patients with potential pulmonary infection,and was outstanding in identifying Pneumocystis infection.It could be able to help physicians adjust the follow-up treatment to avoid the abuse of antibiotics.

Construction of SNP genetic maps based on targeted next-generation sequencing and QTL mapping of vital agronomic traits in faba bean(Vicia faba L.)

Owing to the limitation of a large genome size(~13 Gb),the genetic and gene mapping studies on faba bean(Vicia faba L.)are lagging far behind those for other legumes.In this study,we selected three purified faba bean lines(Yundou 8137,H0003712,and H000572)as parents and constructed two F2 populations.These two F2 populations,namely 167 F2 plants in Pop1(Yundou 8137×H0003712)and 204 F2 plants in Pop2(H000572×Yundou 8137),were genotyped using a targeted next-generation sequencing(TNGS)genotyping platform,and two high-density single nucleotide polymorphisms(SNP)genetic linkage maps of faba bean were constructed.The map constructed from Pop1 contained 5103 SNPs with a length of 1333.31 cM and an average marker density of 0.26 cM.The map constructed from Pop2 contained 1904 SNPs with a greater length of 1610.61 cM.In these two F2 populations,QTL mapping identified 98 QTLs for 14 agronomic traits related to the flowers,pods,plant types and grains.The two maps were then merged into an integrated genetic linkage map containing 6895 SNPs,with a length of 3324.48 cM.These results not only lay the foundation for fine mapping and map-based cloning of related genes,but can also accelerate the molecular marker-assisted breeding of faba bean.

Next-generation Sequencing of MHC Class Ⅰ Genes Reveals Trans-species Polymorphism in Eutropis multifasciata and Other Species of Scincidae

The genes of the major histocompatibility complex(MHC) encode cell surface proteins that are essential for adaptive immunity. MHC genes show the most prominent genetic diversity in vertebrates,reflecting the adaptation of populations to their evolving environment, population survival and reproduction. In the present study, we used nextgeneration sequencing(NGS) to study the loci polymorphism of exon 3 of the MHC class Ⅰ genes in an ovoviviparous skink, the many-lined sun skink,Eutropis multifasciata and five other species of Scincidae, to quantify genetic variation. In addition,we genotyped the same MHC class Ⅰ genes of E.multifasciata using clone sequencing, to directly compare the effectiveness of both analytical techniques for MHC genotyping. NGS detected 20MHC class Ⅰ alleles in E. multifasciata, and 2 to 15 alleles in the other five Scincidae species. However,clone sequencing detected only 15 of those MHC class Ⅰ alleles in E. multifasciata. In addition, transspecies polymorphism of MHC class Ⅰ genes was studied by constructing a phylogenetic tree using the gene sequences obtained by NGS. Phylogenetic analysis revealed that MHC class I alleles were shared among different species of Scincidae with trans-species polymorphism, and did not exhibit specific genealogical inheritance. These results have important implications for understanding polymorphism interspecies diversity in the MHC genes of Scincidae, and the evolution of the MHC more broadly.

Metagenomic next-generation sequencing for pleural effusions induced by viral pleurisy:A case report

BACKGROUND Viral pleurisy is a viral infected disease with exudative pleural effusions.It is one of the causes for pleural effusions.Because of the difficult etiology diagnosis,clinically pleural effusions tend to be misdiagnosed as tuberculous pleurisy or idiopathic pleural effusion.Here,we report a case of pleural effusion secondary to viral pleurisy which is driven by infection with epstein-barr virus.Viral infection was identified by metagenomic next-generation sequencing(mNGS).CASE SUMMARY A 40-year-old male with a history of dermatomyositis,rheumatoid arthritis,and secondary interstitial pneumonia was administered with long-term oral prednisone.He presented with fever and chest pain after exposure to cold,accompanied by generalized sore and weakness,night sweat,occasional cough,and few sputums.The computed tomography scan showed bilateral pleural effusions and atelectasis of the partial right lower lobe was revealed.The pleural fluids were found to be yellow and slightly turbid after pleural catheterization.Thoracoscopy showed fibrous adhesion and auto-pleurodesis.Combining the results in pleural fluid analysis and mNGS,the patient was diagnosed as viral pleuritis.After receiving Aciclovir,the symptoms and signs of the patient were relieved.CONCLUSION Viral infection should be considered in cases of idiopathic pleural effusion unexplained by routine examination.mNGS is helpful for diagnosis.

Soft tissue tuberculosis detected by next-generation sequencing:A case report and review of literature

BACKGROUND Soft tissue tuberculosis is rare and insidious,with most patients presenting with a localized enlarged mass or swelling,which may be factors associated with delayed diagnosis and treatment.In recent years,next-generation sequencing has rapidly evolved and has been successfully applied to numerous areas of basic and clinical research.A literature search revealed that the use of next-generation sequencing in the diagnosis of soft tissue tuberculosis has been rarely reported.CASE SUMMARY A 44-year-old man presented with recurrent swelling and ulcers on the left thigh.Magnetic resonance imaging suggested a soft tissue abscess.The lesion was surgically removed and tissue biopsy and culture were performed;however,no organism growth was detected.Finally,Mycobacterium tuberculosis was confirmed as the pathogen responsible for infection through next-generation sequencing analysis of the surgical specimen.The patient received a standardized anti-tuberculosis treatment and showed clinical improvement.We also performed a literature review on soft tissue tuberculosis using studies published in the past 10 years.CONCLUSION This case highlights the importance of next-generation sequencing for the early diagnosis of soft tissue tuberculosis,which can provide guidance for clinical treatment and improve prognosis.

Pneumocystis jirovecii diagnosed by next-generation sequencing of bronchoscopic alveolar lavage fluid: A case report and review of literature

BACKGROUND The advent of molecular targeted agents and immune checkpoint inhibitors has greatly improved the treatment of advanced renal cell carcinoma(RCC), thus significantly improving patient survival. The incidence of rare drug-related adverse events has gained increased attention.CASE SUMMARY We report a patient with advanced RCC treated with multiple lines of molecular targeted agents and immune checkpoint inhibitors, who developed a pulmonary infection after treatment with everolimus in combination with lenvatinib. Determining the pathogenic organism was difficult, but it was eventually identified as Pneumocystis jirovecii by next-generation sequencing(NGS) of bronchoscopic alveolar lavage fluid(BALF) and successfully treated with trimethoprim-sulfamethoxazole.CONCLUSION Rare pulmonary infections caused by molecular targeted agents are not uncommon in clinical practice, but their diagnosis is difficult. Evaluating BALF with NGS is a good method for rapid diagnosis of such infections.

Pulmonary nocardiosis with bloodstream infection diagnosed by metagenomic next-generation sequencing in a kidney transplant recipient:A case report

BACKGROUND Pulmonary nocardiosis is difficult to diagnose by culture and other conventional testing,and is often associated with lethal disseminated infections.This difficulty poses a great challenge to the timeliness and accuracy of clinical detection,especially in susceptible immunosuppressed individuals.Metagenomic nextgeneration sequencing(mNGS)has transformed the conventional diagnosis pattern by providing a rapid and precise method to assess all microorganisms in a sample.CASE SUMMARY A 45-year-old male was hospitalized for cough,chest tightness and fatigue for 3 consecutive days.He had received a kidney transplant 42 d prior to admission.No pathogens were detected at admission.Chest computed tomography showed nodules,streak shadows and fiber lesions in both lung lobes as well as right pleural effusion.Pulmonary tuberculosis with pleural effusion was highly suspected based on the symptoms,imaging and residence in a high tuberculosisburden area.However,anti-tuberculosis treatment was ineffective,showing no improvement in computed tomography imaging.Pleural effusion and blood samples were subsequently sent for mNGS.The results indicated Nocardia farcinica as the major pathogen.After switching to sulphamethoxazole combined with minocycline for antinocardiosis treatment,the patient gradually improved and was finally discharged.CONCLUSION A case of pulmonary nocardiosis with an accompanying bloodstream infection was diagnosed and promptly treated before the dissemination of the infection.This report emphasizes the value of mNGS in the diagnosis of nocardiosis.mNGS may be an effective method for facilitating early diagnosis and prompt treatment in infectious diseases,which overcomes the shortcomings of conventional testing.

Application of next-generation sequencing in thalassemia screening:A systematic review and meta-analysis

Objective:To evaluate the value of next-generation sequencing(NGS)in the prevention and management of thalassemia.Methods:A systematic search was performed in eight databases including China Biomedical Literature Database,Chinese National Knowledge Infrastructure,Chinese Scientific Journals Database,Wanfang database,PubMed,EMBASE,Web of Science,and Cochrane Library from the inception to 1 June 2022.Stata 17.0 and Review Manager 5.4 were used for the meta-analysis.Results:Nine studies containing 14794 participants were included in the meta-analysis.Compared with the routine genetic testing(including Gap-PCR and reverse dot blot),NGS had higher detection rates in screening thalassemia(RR 1.22,95%CI 1.13-1.31,P<0.01),particularly for theα-thalassaemia mutation carriers(RR 1.24,95%CI 1.07-1.44,P<0.01).However,no significant difference was found in the screening ofβ-thalassemia(RR 1.10,95%CI 0.99-1.23,P>0.05).Conclusions:Compared with routine genetic testing,NGS had a higher detection rate in general,particularly in the detection ofα-thalassemia.

Comprehensive next-generation sequencing reveals double primary colorectal carcinoma missed by diagnostic imaging: A case report

BACKGROUND Multiple primary colorectal carcinoma(MPCC)is a rare clinical disease,which is challenging to differentiate from metastatic disease using histopathological methods.Next-generation sequencing(NGS)has been employed to identify multiple primary cancers.CASE SUMMARY This study a rare case of a 63-year-old male patient diagnosed with MPCC by targeted NGS,which was initially missed by radiological evaluation.The patient was found to have two tumors located on the surface of the colorectum which had distinct genomic alterations.Based on wild-type KRAS detected in the unresected tumor,the patient benefited from the epidermal growth factor receptor(EGFR)inhibitor cetuximab treatment,but developed novel mutations including KIF5B-RET fusion,which provides a possible resistance mechanism to anti-EGFR therapy.CONCLUSION Our case highlights the necessity of using genetic testing for primary tumor diagnosis and the application of serial plasma circulating tumor DNA profiling for dynamic disease monitoring.

Molecular methods for colorectal cancer screening:Progress with next-generation sequencing evolution

Currently,colorectal cancer(CRC)represents the third most common malignancy and the second most deadly cancer worldwide,with a higher incidence in developed countries.Like other solid tumors,CRC is a heterogeneous genomic disease in which various alterations,such as point mutations,genomic rearrangements,gene fusions or chromosomal copy number alterations,can contribute to the disease development.However,because of its orderly natural history,easily accessible onset location and high lifetime incidence,CRC is ideally suited for preventive intervention,but the many screening efforts of the last decades have been compromised by performance limitations and low penetrance of the standard screening tools.The advent of next-generation sequencing(NGS)has both facilitated the identification of previously unrecognized CRC features such as its relationship with gut microbial pathogens and revolutionized the speed and throughput of cataloguing CRC-related genomic alterations.Hence,in this review,we summarized the several diagnostic tools used for CRC screening in the past and the present,focusing on recent NGS approaches and their revolutionary role in the identification of novel genomic CRC characteristics,the advancement of understanding the CRC carcinogenesis and the screening of clinically actionable targets for personalized medicine.

Next-generation sequencing technology for the diagnosis of Pneumocystis pneumonia in an immunocompetent female:A case report

BACKGROUND Pneumocystis pneumonia(PCP)is a serious fungal infection usually seen in patients with human immunodeficiency virus,and it is more frequently found and has a high fatality rate in immunocompromised people.Surprisingly,it rarely occurs in immunocompetent patients.However,the clinical diagnosis of this pathogen is made more difficult by the difficulty of obtaining accurate microbiological evidence with routine tests.This case reports a PCP patient with normal immune function who was diagnosed through next-generation sequencing(NGS).CASE SUMMARY A 23-year-old female who had no special disease in the past was admitted to the hospital with a persistent fever and cough.Based on the initial examination results,the patient was diagnosed with bipulmonary pneumonia,and empirical broad-spectrum antibiotic therapy was administered.However,due to the undetermined etiology,the patient's condition continued to worsen.She was transferred to the intensive care unit because of acute respiratory failure.After the diagnosis of Pneumocystis jirovecii infection through NGS in bronchoalveolar lavage fluid and treatment with trimethoprim/sulfamethoxazole and caspofungin,the patient gradually recovered and had a good prognosis.CONCLUSION This case emphasizes that,for patients with normal immune function the possibility of PCP infection,although rare,cannot be ignored.NGS plays an important role in the diagnosis of refractory interstitial pneumonia and acute respiratory failure.

Metagenomic next-generation sequencing in the diagnosis of neurocysticercosis:A case report

BACKGROUND The clinical symptoms and imaging manifestations of neurocysticercosis(NCC)are very different,and the difficulty and delay of clinical diagnoses may lead to an increase in mortality and disability.Rapid and accurate pathogen identification is important for the treatment of these patients.Metagenomic next-generation sequencing(mNGS)is a powerful tool to identify pathogens,especially in infections that are difficult to identify by conventional methods.CASE SUMMARY A 43-year-old male patient was admitted due to a recurrent headache for a few months.Imaging examinations showed hydrocephalus and cystic lesions,which were considered to be a central nervous system infection,but no etiology was found by routine examination.mNGS of the cerebrospinal fluid revealed high Taenia solium reads,and the positive results of a cysticercosis antibody test confirmed the infection.Combined with the patient’s clinical manifestations,the etiological evidence,and the imaging manifestation,the patient was finally diagnosed with NCC and he was prescribed dexamethasone,albendazole,neurotrophic drugs,and intracranial pressure reduction therapy.The headaches disappeared after anti-parasite treatment,and no associated symptoms recurred prior to the three-and six-month follow-up.CONCLUSION As an accurate and sensitivity detection method,mNGS can be a reliable approach for the diagnosis of NCC.

Identification of 8 Rare Deleterious Variants in ADAMTS13 by Next-generation Sequencing in a Chinese Population with Thrombotic Thrombocytopenic Purpura

Objective Thrombotic thrombocytopenic purpura(TTP)is a rare and fatal disease caused by a severe deficiency in the metalloprotease ADAMTS13 and is characterized by thrombotic microangiopathy.The present study aimed to investigate the genes and variants associated with TTP in a Chinese population.Methods Target sequencing was performed on 220 genes related to complements,coagulation factors,platelets,fibrinolytic,endothelial,inflammatory,and anticoagulation systems in 207 TTP patients and 574 controls.Subsequently,logistic regression analysis was carried out to identify the TTP-associated genes based on the counts of rare deleterious variants in the region of a certain gene.Moreover,the associations between common variants and TTP were also investigated.Results ADAMTS13 was the only TTP-associated gene(OR=3.77;95%CI:1.82–7.81;P=3.6×10^(-4))containing rare deleterious variants in TTP patients.Among these 8 variants,5 novel rare variants that might contribute to TTP were identified,including rs200594025,rs782492477,c.T1928G(p.I643S),c.3336_3361del(p.Q1114Afs*20),and c.3469_3470del(p.A1158Sfs*17).No common variants associated with TTP were identified under the stringent criteria of correction for multiple testing.Conclusion ADAMTS13 is the primary gene related to TTP.The genetic variants associated with the occurrence of TTP were slightly different between the Chinese and European populations.

Endoscopic ultrasound-guided fine-needle aspiration pancreatic adenocarcinoma samples yield adequate DNA for next-generation sequencing:A cohort analysis

BACKGROUND Genetic tests are increasingly performed for the management of unresectable pancreatic cancer.For genotyping aimed samples current guidelines recommend using core specimens,although based on moderate quality evidence.However,in clinical practice among the endoscopic ultrasound(EUS) guided tissue acquisition methods,fine needle aspiration(FNA) is the most widely performed.AIM To assess the adequacy for next generation sequencing(NGS) of the DNA yielded from EUS-FNA pancreatic adenocarcinoma(PDAC) samples.METHODS Between November 2018 and December 2021,105 patients with PDAC confirmed by EUS-FNA were included in the study at our tertiary gastroenterology center.Either 22 gauge(G) or 19G FNA needles were used.One pass was dedicated to DNA extraction.DNA concentration and purity(A260/280,A260/230) were assessed by spectrophotometry.We assessed the differences in DNA parameters according to needle size and tumor characteristics(size,location) and the adequacy of the extracted DNA for NGS(defined as A260/280 ≥ 1.7,and DNA yield:≥ 10 ng for amplicon based NGS,≥ 50 ng for whole exome sequencing [WES],≥ 100 ng for whole genome sequencing [WGS]) by analysis of variance and ttest respectively.Moreover,we compared DNA purity parameters across the different DNA yield categories.RESULTS Our cohort included 49% male patients,aged 67.02 ± 8.38 years.The 22G needle was used in 71%of the cases.The DNA parameters across our samples varied as follows:DNA yield:1289 ng(inter quartile range:534.75-3101),A260/280 = 1.85(1.79-1.86),A260/230 = 2.2(1.72-2.36).DNA yield was > 10 ng in all samples and > 100 ng in 93% of them(one sample < 50 ng).There were no significant differences in the concentration and A260/280 between samples by needle size.Needle size was the only independent predictor of A260/230 which was higher in the 22G samples(P =0.038).NGS adequacy rate was 90% for 19G samples regardless of NGS type,and for 22G samples it reached 89% for WGS adequacy and 91% for WES and amplicon based NGS.Samples with DNA yield > 100 ng had significantly higher A260/280(1.89 ± 0.32 vs 1.34 ± 0.42,P = 0.013).Tumor characteristics were not corelated with the DNA parameters.CONCLUSION EUS-FNA PDAC samples yield DNA adequate for subsequent NGS.DNA amount was similar between 22G and 19G FNA needles.DNA purity parameters may vary indirectly with needle size.

Effects of viral infection and microbial diversity on patients with sepsis:A retrospective study based on metagenomic next-generation sequencing

BACKGROUND: The study aims to investigate the performance of a metagenomic next-generationsequencing (NGS)-based diagnostic technique for the identifi cation of potential bacterial and viral infectionsand eff ects of concomitant viral infection on the survival rate of intensive care unit (ICU) sepsis patients.METHODS: A total of 74 ICU patients with sepsis who were admitted to our institution from February1, 2018 to June 30, 2019 were enrolled. Separate blood samples were collected from patients for bloodcultures and metagenomic NGS when the patients’ body temperature was higher than 38 °C. Patients’demographic data, including gender, age, ICU duration, ICU scores, and laboratory results, were recorded.The correlations between pathogen types and sepsis severity and survival rate were evaluated.RESULTS: NGS produced higher positive results (105 of 118;88.98%) than blood cultures(18 of 118;15.25%) over the whole study period. Concomitant viral infection correlated closelywith sepsis severity and had the negative effect on the survival of patients with sepsis. However,correlation analysis indicated that the bacterial variety did not correlate with the severity of sepsis.CONCLUSIONS: Concurrent viral load correlates closely with the severity of sepsis and thesurvival rate of the ICU sepsis patients. This suggests that prophylactic administration of antiviraldrugs combined with antibiotics may be benefi cial to ICU sepsis patients.

Next-generation sequencing technology for diagnosis and efficacy evaluation of a patient with visceral leishmaniasis: A case report

BACKGROUND Visceral leishmaniasis(VL)is a parasitic disease caused by Leishmania and transmitted by infected sand flies.VL has a low incidence in China,and its clinical presentation is complex and atypical.This disease is easily misdiagnosed and can become life-threatening within a short period of time.Therefore,early,rapid and accurate diagnosis and treatment of the disease are essential.CASE SUMMARY A 25-year-old male patient presented with the clinical manifestations of irregular fever,hepatosplenomegaly,increased polyclonal globulin,and pancytopenia.The first bone marrow puncture biopsy did not provide a clear diagnosis.In order to relieve the pressure and discomfort of the organs caused by the enlarged spleen and to confirm the diagnosis,splenectomy was performed,and hemophagocytic syndrome was diagnosed by pathological examination of the spleen biopsy.Following bone marrow and spleen pathological re-diagnosis and metagenomic next-generation sequencing(mNGS)technology detection,the patient was finally diagnosed with VL.After treatment with liposomal amphotericin B,the body temperature quickly returned to normal and the hemocytes recovered gradually.Post-treatment re-examination of the bone marrow puncture and mNGS data showed that Leishmania was not detected.CONCLUSION As a fast and accurate detection method,mNGS can diagnose and evaluate the efficacy of treatment in suspicious cases of leishmaniasis.

Gene mutations in stool from gastric and colorectal neoplasia patients by next-generation sequencing

AIM To study cancer hotspot mutations by next-generation sequencing(NGS) in stool DNA from patients with different gastrointestinal tract(GIT) neoplasms. METHODS Stool samples were collected from 87 Finnish patients diagnosed with various gastric and colorectal neoplasms, including benign tumors, and from 14 healthy controls. DNA was isolated from stools by usingthe PSP~? Spin Stool DNA Plus Kit. For each sample, 20 ng of DNA was used to construct sequencing libraries using the Ion AmpliS eq Cancer Hotspot Panel v2 or Ion AmpliS eq Colon and Lung Cancer panel v2. Sequencing was performed on Ion PGM. Torrent Suite Software v.5.2.2 was used for variant calling and data analysis.RESULTS NGS was successful in assaying 72 GIT samples and 13 healthy controls, with success rates of the assay being78% for stomach neoplasia and 87% for colorectal tumors. In stool specimens from patients with gastric neoplasia, five hotspot mutations were found in APC,CDKN2 A and EGFR genes, in addition to seven novel mutations. From colorectal patients, 20 mutations were detected in AKT1, APC, ERBB2, FBXW7, KIT, KRAS,NRAS, SMARCB1, SMO, STK11 and TP53. Healthy controls did not exhibit any hotspot mutations, except for two novel ones. APC and TP53 were the most frequently mutated genes in colorectal neoplasms, with five mutations, followed by KRAS with two mutations.APC was the most commonly mutated gene in stools of patients with premalignant/benign GIT lesions.CONCLUSION Our results show that in addition to colorectal neoplasms,mutations can also be assayed from stool specimens of patients with gastric neoplasms.

The Diagnostic Value of Metagenomic Next-Generation Sequencing in <i>Angiostrongylus cantonensis</i>Encephalitis/Meningitis

Objective: Angiostrongylus cantonensis (A. cantonensis) is an important parasite, which causes neurological infection in children, and is generally difficult to diagnose. We analyzed the significance of metagenomic next-generation sequencing in diagnosing A. cantonensis encephalitis/meningitis in children to provide a reference for clinical diagnosis. Methods: The case collection of A. cantonensis encephalitis/meningitis was from July 2018 to August 2020. The patients’ clinical characteristics and pathogen were described, and diagnostic sensitivity methods for A. cantonensis encephalitis/meningitis were compared and analyzed, including parasite antibody detection and metagenomic next-generation sequencing (mNGS) detection in different samples. Results: Eleven cases were diagnosed with A. cantonensis encephalitis/meningitis, including six males (54.5%) and five females (45.5%), and the age ranged from 1 to 13 years with a median of 21 months (IQR: 15.6, 96). All patients were undiagnosed upon admission, of which ten cases had neurological symptoms or signs, six cases (54.5%) had a history of definite or suspicious exposure to parasites, and eight cases (72.7%) had abnormal changes in cranial MRI. The results of A. cantonensis antibodies in the peripheral blood and cerebrospinal fluid (CSF) were compared with CSF’s metagenomic next-generation sequencing (mNGS). The antibody-positive rate of peripheral blood was 54.5%, CSF antibody-positive rate was 27.2%, and mNGS-positive rate of CSF was 81.8%, and the positive diagnostic rate of the latter was significantly higher than the former two (p = 0.035 < 0.05 and p = 0.030 < 0.05, respectively). Conclusion: Metagenomic next-generation sequencing has a higher positive rate for diagnosing infection and plays an important role in clarifying diagnosis and reducing misdiagnosis.