Next-generation sequencing (NGS) has revolutionized clinical microbiology, particularly in diagnosing respiratory infectious diseases and conducting epidemiological investigations. This narrative review summarizes conventional methods for routine respiratory infection diagnosis, including culture, smear microscopy, immunological assays, image techniques as well as polymerase chain reaction(PCR). In contrast to conventional methods, there is a new detection technology, sequencing technology, and here we mainly focus on the next-generation sequencing NGS, especially metagenomic NGS(mNGS). NGS offers significant advantages over traditional methods. Firstly, mNGS eliminates assumptions about pathogens, leading to faster and more accurate results, thus reducing diagnostic time. Secondly, it allows unbiased identification of known and novel pathogens, offering broad-spectrum coverage. Thirdly, mNGS not only identifies pathogens but also characterizes microbiomes, analyzes human host responses, and detects resistance genes and virulence factors. It can complement targeted sequencing for bacterial and fungal classification. Unlike traditional methods affected by antibiotics, mNGS is less influenced due to the extended survival of pathogen DNA in plasma, broadening its applicability. However, barriers to full integration into clinical practice persist, primarily due to cost constraints and limitations in sensitivity and turnaround time. Despite these challenges, ongoing advancements aim to improve cost-effectiveness and efficiency, making NGS a cornerstone technology for global respiratory infection diagnosis.

We reported a rare β-thalassemia patient, a 41-year-old Chinese male with small cell hypopigmentation anemia, jaundice and splenomegaly as the main clinical symptoms. By using Next-Generation Sequencing (NGS), we identified a novel de novo HBB mutation(c.358_365dup, p.Phe123Alafs*39) which resulted in an abnormally prolonged β-globin chain comprising 159 amino acid residues. The secondary and three-dimensional structures of the β-globin predicted that the novel prolonged β-globin chain has a considerable risk of instability in the hemoglobin, and leads to clinical phenotype. This study contributes to the enrichment of the genetic pathogenic mutation database for thalassemia and underscores the significance of NGS in the screening of mutations for thalassemia families.

BACKGROUND: Herpes zoster is an infectious skin disease caused by the reactivation of the varicella zoster virus (VZV), which has been latent in the posterior root ganglia of the spinal cord or cranial ganglia for an extended period. Neurological complications caused by herpes zoster include aseptic meningitis, white matter disease, peripheral motor neuropathy, and Guillain-Barré syndrome. However, reduced unilateral sweating caused by the VZV is very rare.
METHODS: This article reports the case of a 34-year-old woman who was admitted to our hospital with sore throat, dizziness, and reduced sweating on the left side of her body. Physical examination found herpes lesions on the left upper lip and left external ear canal (scabbed) and reduced sweating on the left side of the body. Head magnetic resonance imaging (MRI) with contrast showed no abnormalities. After a lumbar puncture, the patient was diagnosed with viral meningitis by VZV infection. The electromyographic skin sympathetic reflex indicated damage to the left sympathetic nerve.
CONCLUSIONS: Secondary unilateral sweating reduction is a rare neurological complication of herpes zoster, caused by damage to the autonomic nervous system. Literature review and comprehensive examination indicated that the reduced unilateral sweating was due to the activation of latent herpes zoster virus in the autonomic ganglia which has damaged the autonomic nervous system. For patients who exhibit acute hemibody sweat reduction, doctors should consider the possibility of secondary autonomic nervous system damage caused by herpes zoster.

UNASSIGNED: Current surveillance modalities of osteosarcoma relapse exhibit limited sensitivity and specificity. Although circulating tumor DNA (ctDNA) has been established as a biomarker of minimal residual disease (MRD) in many solid tumors, a sensitive ctDNA detection technique has not been thoroughly explored for longitudinal MRD detection in osteosarcoma.
UNASSIGNED: From August 2019 to June 2023, 59 patients diagnosed with osteosarcoma at the First Affiliated Hospital of Sun Yat-sen University were evaluated in this study. Tumor-informed MRD panels were developed through whole exome sequencing (WES) of tumor tissues. Longitudinal blood samples were collected during treatment and subjected to multiplex PCR-based next-generation sequencing (NGS). Kaplan-Meier curves and Log-rank tests were used to compare outcomes, and Cox regression analysis was performed to identify prognostic factors.
UNASSIGNED: WES analysis of 83 patients revealed substantial mutational heterogeneity, with non-recurrent mutated genes accounting for 58.1%. Tumor-informed MRD panels were successfully obtained for 85.5% of patients (71/83). Among 59 patients with successful MRD panel customization and available blood samples, 13 patients exhibited positive ctDNA detection after surgery. Patients with negative post-operative ctDNA had better event-free survival (EFS) compared to those with positive ctDNA, at 1-6 months after surgery, after adjuvant chemotherapy, and more than 6 months after surgery (p < 0.05). In both univariate and multivariate Cox regression analysis, ctDNA results emerged as a significant predictor of EFS (p < 0.05). ctDNA detection preceded positive imaging in 5 patients, with an average lead time of 92.6 days. Thirty-nine patients remained disease-free, with ctDNA results consistently negative or turning negative during follow-up.
UNASSIGNED: Our study underscores the applicability of tumor-informed deep sequencing of ctDNA in osteosarcoma MRD surveillance and, to our knowledge, represents the largest cohort to date. ctDNA detection is a significant prognostic factor, enabling the early identification of tumor relapse and progression compared to standard imaging, thus offering valuable insights in guiding osteosarcoma patient management.
UNASSIGNED: The Grants of National Natural Science Foundation of China (No. 82072964, 82072965, 82203798, 82203026), the Natural Science Foundation of Guangdong (No. 2023A1515012659, 2023A1515010302), and the Regional Combination Project of Basic and Applied Basic Research Foundation of Guangdong (No. 2020A1515110010).

UNASSIGNED: Driver genes are essential predictors of targeted therapeutic efficacy. Detecting driver gene mutations in lung adenocarcinoma (LUAD) patients can help to screen for targeted drugs and improve patient survival benefits. This study aims to investigate the mutation characterization of driver genes and their correlation with clinicopathological features in LUAD.
UNASSIGNED: A total of 440 LUAD patients were selected from Sir Run Run Shaw Hospital between July 2019 and September 2022. Postoperative tissue specimens were analyzed for gene mutations using next-generation sequencing technology, focusing, including epidermal growth factor receptor EGFR, ALK, ROS1, RET, KRAS, MET, BRAF, HER2, PIK3CA and NRAS. At the same time, clinicopathological data were collected and organized for multidimensional correlation analysis.
UNASSIGNED: Of 440 LUAD patients, driver gene mutations were not detected in 48 patients. The proportion of patients with driver gene mutations was as high as 89.09%. The top three driver genetic mutations were EGFR, KRAS, and MET. Sixty-nine types of EGFR mutations were detected and distributed in the protein tyrosine kinase catalytic domain (56, 81.16%), Furin-like cysteine-rich region (9, 13.04%), receptor binding domain (3, 4.35%), and EGFR transmembrane domain (1, 1.45%). Single gene locus mutation occurred in 343 LUAD patients, but the mutation gene types covered all tested genes. Our findings showed that EGFR mutations were more commonly observed in non-smoking and female patients (P<0.01), KRAS mutations were more prevalent in male patients and smokers (P<0.01), ROS1 mutations had larger tumor diameters (P<0.01) and RET mutations were more prevalent in smokers (P<0.05).
UNASSIGNED: LUAD patients exhibit diverse genetic mutations, which may co-occur simultaneously. Integrated analysis of multiple mutations is essential for accurate diagnosis and effective treatment of the disease. The use of NGS can significantly expand our understanding of gene mutations and facilitate integrated analysis of multiple gene mutations, providing critical evidence for targeted treatment methods.

Hepatitis A virus (HAV), a member of the genus Hepatovirus (Picornaviridae HepV), remains a significant viral pathogen, frequently causing enterically transmitted hepatitis worldwide. In this study, we conducted an epidemiological survey of HepVs carried by small terrestrial mammals in the wild in Yunnan Province, China. Utilizing HepV-specific broad-spectrum RT-PCR, next-generation sequencing (NGS), and QNome nanopore sequencing (QNS) techniques, we identified and characterized two novel HepVs provisionally named EpMa-HAV and EpLe-HAV, discovered in the long-tailed mountain shrew (Episoriculus macrurus) and long-tailed brown-toothed shrew (Episoriculus leucops), respectively. Our sequence and phylogenetic analyses of EpMa-HAV and EpLe-HAV indicated that they belong to the species Hepatovirus I (HepV-I) clade II, also known as the Chinese shrew HepV clade. Notably, the codon usage bias pattern of novel shrew HepVs is consistent with that of previously identified Chinese shrew HepV. Furthermore, our structural analysis demonstrated that shrew HepVs differ from other mammalian HepVs in RNA secondary structure and exhibit variances in key protein sites. Overall, the discovery of two novel HepVs in shrews expands the host range of HepV and underscores the existence of genetically diverse animal homologs of human HAV within the genus HepV.

UNASSIGNED: The significant progress has been made in targeted therapy for lung adenocarcinoma (LUAD) in the past decade. Only few targeted therapeutics have yet been approved for the treatment of lung squamous cell carcinoma (LUSC). Several higher frequency of gene alterations are identified as potentially actionable in LUSC. Our work aimed to explore the complex interplay of multiple genetic alterations and pathways contributing to the pathogenesis of LUSC, with a very low frequency of a single driver molecular alterations to develop more effective therapeutic strategies in the future.
UNASSIGNED: We retrospectively analyzed the targeted next-generation sequencing (NGS) data (approximately 600 genes) of 335 patients initially diagnosed with non-small cell lung cancer (NSCLC) at our institution between January 2019 and March 2023 and explored the somatic genome alteration difference between LUSC and LUAD.
UNASSIGNED: We analyzed that the presence of loss-of-function (LoF) mutations (nonsense, frameshift, and splice-site variants) in histone-lysine N-methyltransferase 2D (KMT2D) was much more prevalent in LUSC (11/53, 20.8%) than in LUAD (6/282, 2.1%). Moreover, our data indicated TP53 co-mutated with KMT2D LoF in 90.9% (10/11) LUSC and 33.3% (2/6) LUAD. Notably, the mutation allele fraction (MAF) of KMT2D was very similar to that of TP53 in the co-mutated cases. Genomic profiling of driver gene mutations of NSCLC showed that 81.8% (9/11) of the patients with LUSC with KMT2D LoF mutations had PIK3CA amplification and/or FGFR1 amplification.
UNASSIGNED: Our results prompted that somatic LoF mutations of KMT2D occur frequently in LUSC, but are less frequent in LUAD and therefore may potentially contribute to the pathogenesis of LUSC. Concurrent TP53 mutations, FGFR1 amplification, and PIK3CA amplification are very common in LUSC cases with KMT2D LoF mutations. It needs more deeper investigation on the interplay of the genes and pathways and uses larger cohorts in the future.

Nocardia disease is an opportunistic infection, the occurrence is rare and mostly occurs in patients with immune deficiency. Even if the patient is immunocompetent, it can still be life-threatening. This case report describes a previously healthy 78-year-old male farmer with lung lesions discovered on a computerized tomography scan. Combined with the patient\'s history of fever and the results of elevated laboratory markers associated with inflammation, the patient was diagnosed with a lung infection. After escalating empirical broad-spectrum antibiotics, antiviral and antifungal therapy, the patient continued to deteriorate to septic shock. In the meanwhile, the patient\'s sputum was cultured repeatedly, and no obvious positive pathogenic bacteria were found. Considering the patient was elderly and that these lesions were solid with burr signs, as well as the progression after antimicrobial therapy cancer was considered in the differential diagnosis. Artificial intelligence (YITU, Hangzhou Yitu Medical Technology Limited Company) was also applied, and it also calculated that these lesions were cancerous. The patient received a puncture biopsy of the largest lung lesion. During the puncture pus was withdrawn from largest lung lesion. Culture and metagenome next-generation sequencing (mNGS) detection performed on pus indicated Nocardia otitidiscaviarum. The test report of the mNGS is also attached with a susceptibility report of commonly used clinical antibiotics to this Nocardia spp. Using this result, the patient\'s disease was quickly controlled after selecting the targeted drug compound sulfamethoxazole and intravenous meropenem for treatment. In view of the high misdiagnosis rate and poor sensitivity of culture for Nocardia spp., this case emphasized mNGS playing a key role in the diagnosis and selection of effective antibiotics for the treatment of Nocardia spp. lung infections.

Esophageal cancer is a common malignancy worldwide with a poor prognosis without radical resection. Neoadjuvant concurrent chemoradiotherapy (NACRT) followed by esophagectomy is widely used for treating locally advanced esophageal cancer in the thorax. The study aimed to assess mutation profiles and their correlation with therapeutic outcomes in patients diagnosed with locally advanced thoracic esophageal squamous cell carcinoma (ESCC). A retrospective analysis was conducted on 62 patients with ESCC who underwent NACRT. All patients received concurrent chemoradiotherapy (CCRT) utilizing intensity-modulated radiation therapy alongside concurrent chemotherapy with a cisplatin-based regimen. A 35-gene next-generation sequencing (NGS) panel detecting 402 genetic variants was used, which has been proven predictive in ESCC patients who received definitive chemoradiation. The 35-gene mutation profiles were analyzed in pre-treatment biopsies. The results reveled there were variants correlated with pathological complete remission or partial response, overall survival, and progression-free survival. A combination of p.Pro1319Ser and p.Arg2159Gly mutations in the MUC17 gene demonstrated an adverse impact on pathological response (OR [95% CI] = 7.00 (3.07-15.94), P < 0.001). Additionally, the variants located in the MUC17, MUC4, and MYH4 genes exhibited notably effects on tumor recurrence or mortality. Patients harboring either the MUC17 p.Thr2702Val or MUC4 p.Thr3355Ser mutation displayed a more than four-fold increased risk for disease recurrence or mortality. We concluded that specific mutations correlated to the pathological complete response in ESCC receiving neoadjuvant chemoradiation can be identified through the utilization of 35-gene expression profiles. Further investigation into the pathophysiological roles of MUC17 and MUC4 mutations in ESCC is warranted.

UNASSIGNED: Circulating tumor DNA (ctDNA) is a potential biomarker not only capable of monitoring the treatment response during neoadjuvant therapy (NAT) or rescue therapy, but also identifying minimal residual disease (MRD) and detecting early relapses after primary treatment. However, it remains uncertain whether the detection of ctDNA at diagnosis, before any treatment, can predict the prognosis for patients with early breast cancer. The objective of our study was to evaluate the predictive value of baseline ctDNA for prognosis in patients with early breast cancer.
UNASSIGNED: A total of 90 patients with early breast cancer and 24 healthy women were recruited between August 2016 and October 2016. Peripheral blood samples were collected from patients at diagnosis, before any treatment. Blood samples were processed and subjected to targeted deep sequencing with a next-generation sequencing (NGS) panel of 1,021 cancer-related genes. The recurrence-free survival (RFS) and invasive disease-free survival (iDFS) were reported.
UNASSIGNED: The 90 patients with breast cancer included 6 patients with ductal carcinoma in situ (DCIS) and 84 patients with invasive breast cancer. Within the cohort of patients with invasive breast cancer, ctDNA were detected in 57 patients, with a ctDNA detection rate of 67.9%. Meanwhile, no ctDNA was detected in DCIS patients. Among 84 patients with invasive breast cancer, patients with high-level ctDNA had a significantly lower RFS compared to patients with low-level ctDNA (log-rank P=0.0036).
UNASSIGNED: Our study suggested that ctDNA at diagnosis, before any treatment, could potentially serve as a biomarker to predict the prognosis for patients with early breast cancer. However, further follow-up and more studies with large sample sizes are required to confirm these findings.