A novel method for simultaneous detection of hematological tumors and infectious pathogens by metagenomic next generation sequencing of plasma.

BACKGROUND: Metagenomic next-generation sequencing (mNGS) is valuable for pathogen identification; however, distinguishing between infectious diseases and conditions with potentially similar clinical manifestations, including malignant tumors, is challenging. Therefore, we developed a method for simultaneous detection of infectious pathogens and cancer in blood samples. METHODS: Plasma samples (n = 244) were collected from 150 and 94 patients with infections and hematological malignancies, respectively, and analyzed by mNGS for pathogen detection, alongside human tumor chromosomal copy number variation (CNV) analysis (≥5Mbp or 10Mbp CNV region). Further, an evaluation set, comprising 87 plasma samples, was analyzed by mNGS and human CNV analysis, to validate the feasibility of the method. RESULTS: Among 94 patients with hematological malignancy, sensitivity values of CNV detection for tumor diagnosis were 69.15 % and 32.98 % for CNV region 5Mbp and 10Mbp, respectively, with corresponding specificities of 92.62 % and 100 % in the infection group. Area under the ROC curve (AUC) values for 5Mbp and 10Mbp region were 0.825 and 0.665, respectively, which was a significant difference of 0.160 (95 % CI: 0.110-0.210; p < 0.001), highlighting the superiority of 5Mbp output region data. Six patients with high-risk CNV results were identified in the validation study: three with history of tumor treatment, two eventually newly-diagnosed with hematological malignancies, and one with indeterminate final diagnosis. CONCLUSIONS: Concurrent CNV analysis alongside mNGS for infection diagnosis is promising for detecting malignant tumors. We recommend adopting a CNV region of 10Mbp over 5Mbp for our model, because of the lower false-positive rate (FPR).

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.

Comprehensive evaluation of plasma microbial cell-free DNA sequencing for predicting bloodstream and local infections in clinical practice: a multicenter retrospective study.

Background: Metagenomic next-generation sequencing (mNGS) of plasma cell-free DNA (cfDNA) shows promising application for complicated infections that cannot be resolved by conventional microbiological tests (CMTs). The criteria for cfDNA sequencing are currently in need of agreement and standardization. Methods: We performed a retrospective cohort observation of 653 patients who underwent plasma cfDNA mNGS, including 431 with suspected bloodstream infections (BSI) and 222 with other suspected systemic infections. Plasma mNGS and CMTs were performed simultaneously in clinical practice. The diagnostic efficacy of plasma mNGS and CMTs in the diagnosis of blood-borne and other systemic infections was evaluated using receiver operating characteristic (ROC) curves. The sensitivity and specificity of the two methods were analyzed based on the final clinical outcome as the gold standard. Results: The mNGS test showed an overall positive rate of 72.3% (472/653) for detecting microorganisms in plasma cfDNA, with a range of 2 to 6 different microorganisms detected in 171 patient specimens. Patients with positive mNGS results were more immunocompromised and had a higher incidence of severe disease (P<0·05). The sensitivity of mNGS was higher for BSI (93·5%) and other systemic infections (83·6%) compared to CMTs (37·7% and 14·3%, respectively). The mNGS detected DNA from a total of 735 microorganisms, with the number of microbial DNA reads ranging from 3 to 57,969, and a higher number of reads being associated with clinical infections (P<0·05). Of the 472 patients with positive mNGS results, clinical management was positively affected in 203 (43%) cases. Negative mNGS results led to a modified clinical management regimen in 92 patients (14.1%). The study also developed a bacterial and fungal library for plasma mNGS and obtained comparisons of turnaround times and detailed processing procedures for rare pathogens. Conclusion: Our study evaluates the clinical use and analytic approaches of mNGS in predicting bloodstream and local infections in clinical practice. Our results suggest that mNGS has higher positive predictive values (PPVs) for BSI and systemic infections compared to CMTs, and can positively affect clinical management in a significant number of patients. The standardized whole-process management procedure for plasma mNGS developed in this study will ensure improved pre-screening probabilities and yield clinically valuable data.

Application of metagenomic next-generation sequencing in the diagnosis of pulmonary invasive fungal disease.

Background: Metagenomic next-generation sequencing (mNGS) is increasingly being used to detect pathogens directly from clinical specimens. However, the optimal application of mNGS and subsequent result interpretation can be challenging. In addition, studies reporting the use of mNGS for the diagnosis of invasive fungal infections (IFIs) are rare. Objective: We critically evaluated the performance of mNGS in the diagnosis of pulmonary IFIs, by conducting a multicenter retrospective analysis. The methodological strengths of mNGS were recognized, and diagnostic cutoffs were determined. Methods: A total of 310 patients with suspected pulmonary IFIs were included in this study. Conventional microbiological tests (CMTs) and mNGS were performed in parallel on the same set of samples. Receiver operating characteristic (ROC) curves were used to evaluate the performance of the logarithm of reads per kilobase per million mapped reads [lg(RPKM)], and read counts were used to predict true-positive pathogens. Result: The majority of the selected patients (86.5%) were immunocompromised. Twenty species of fungi were detected by mNGS, which was more than was achieved with standard culture methods. Peripheral blood lymphocyte and monocyte counts, as well as serum albumin levels, were significantly negatively correlated with fungal infection. In contrast, C-reactive protein and procalcitonin levels showed a significant positive correlation with fungal infection. ROC curves showed that mNGS [and especially lg(RPKM)] was superior to CMTs in its diagnostic performance. The area under the ROC curve value obtained for lg(RPKM) in the bronchoalveolar lavage fluid of patients with suspected pulmonary IFIs, used to predict true-positive pathogens, was 0.967, and the cutoff value calculated from the Youden index was -5.44. Conclusions: In this study, we have evaluated the performance of mNGS-specific indicators that can identify pathogens in patients with IFIs more accurately and rapidly than CMTs, which will have important clinical implications.

Diagnosis of Ureaplasma parvum meningitis by mNGS in an extremely low birth weight infant with multi-system lesions.

Ureaplasma parvum encephalitis is a rare disease with high mortality in the neonates. While the manifestations are atypical and identification of U. parvum is difficult, diagnosis would always be delayed. Metagenomic next-generation sequencing (mNGS) is a pre-hypothesis free technique which could theoretically detect all the microbes in a sample. Herein we report a case of U. parvum meningitis identified by mNGS in an extremely low birth weight neonate complicated with multi-system lesions. The patient was treated with erythromycin and ciprofloxacin, symptoms were relieved in the following days and the patient was transferred to treat complications after three weeks' therapy.

Detection of aerobe-anaerobe mixed infection by metagenomic next-generation sequencing in an adult suffering from descending necrotizing mediastinitis.

BACKGROUND: Descending necrotizing mediastinitis (DNM) is one of the most virulent forms of mediastinitis. The main causes of high mortality in DNM are believed to stem from difficulty and delay in the diagnosis. Fast and accurate identification of pathogens is important for the treatment of these patients. Metagenomics next-generation sequencing (mNGS) is a powerful tool to identify all kinds of pathogens, especially for rare and complex infections. CASE PRESENTATION: A 64-year-old male patient was admitted to the intensive care unit (ICU) with unconsciousness, dyspnea, and swelling in the mandible and neck. Computed tomography (CT) scan results combined with clinical laboratory examination indicated DNM. Vancomycin and imipenem were used, and vacuum sealing drainage was applied for debridement and drainage of the infected area. The positive mNGS results of drainage fluid confirmed the presence of mixed infection caused by Streptococcus anginosus, Prevotella oris, and several other anaerobes. The antibiotics were adjusted to piperacillin/tazobactam and tinidazole according to the mNGS results and antimicrobial susceptibility testing of cultured pathogens. After 11 days of antibiotic therapy, the infection symptoms of the neck and mediastinum improved, and the patient was transferred out of the ICU on the 26th day after negative result of drainage fluid culture. CONCLUSION: This case suggested that mNGS is a promising technology for precise and fast pathogens identification with high sensitivity, which may guide the diagnosis of infectious diseases in the future trend.

Metagenomic Analysis Identifying a Rare Leishmania Infection in an Adult With AIDS.

Leishmania belongs to a genus of the protozoan parasites that causes leishmaniasis, and includes cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). In this case, Leishmania amastigotes were found on cytomorphology examination of the bone marrow specimen, followed by 1,076 Leishmania donovani reads using metagenomic next generation sequencing (mNGS). Since being definitely diagnosed with VL/HIV coinfection, the patient was treated with liposomal amphotericin B as the parasite-resistant therapy and was discharged after clinical cure. But nearly a year later, on the mNGS follow-up, L. donovani was detected in the patient's blood plasma specimen with 941 reads, suggesting that a relapse of leishmaniasis had occurred. These results indicate that leishmaniasis still exists in China and may represent a public health concern. This case could be helpful in the differential diagnosis of leishmaniasis, and for determining disease progression, prevention, and control of vectors and reservoir hosts.

Identification of a Panel of MiRNAs as Positive Regulators of Insulin Release in Pancreatic Β-Cells.

BACKGROUND/AIMS: MicroRNAs (miRNAs) are a novel class of small RNAs that participate in a variety of biological processes. Although miRNAs have been linked to insulin synthesis and glucose homeostasis, their role in the targeting of mitochondrial uncoupling protein 2 (UCP2), a negative modulator of insulin secretion, remains unclear. METHODS: miRNA levels were determined by real-time quantitative PCR analysis using TaqMan probes, and insulin secretion from isolated islets was quantified by ELISA. Effects of miRNAs on UCP2 expression were checked with a luciferase assay and western blotting analysis. RESULTS: An overall change in a set of miRNAs was discovered, with miR-15a, miR-424, miR-497, and miR-185 coinciding with insulin levels in islets maintained under high-glucose conditions. Moreover, experiments in MIN6 cells illustrated that miR-15a, miR-424, miR-497, and miR-185 positively regulated insulin biosynthesis by co-inhibiting UCP2 expression. Furthermore, the four miRNAs were found to post-transcriptionally repress UCP2 expression by directly targeting the 3'UTR of UCP2 mRNA. CONCLUSIONS: Thus, our results shed further light on the regulatory network in ß-cells consisting of miRNAs, UCP2, and insulin and provide novel therapeutic targets for diabetes.

UCP3 Ablation Exacerbates High-Salt Induced Cardiac Hypertrophy and Cardiac Dysfunction.

BACKGROUND/AIMS: Excessive salt intake and left ventricular hypertrophy (LVH) are both critical for the development of hypertension and heart failure. The uncoupling protein 3 (UCP3) plays a cardio-protective role in early heart failure development. However, the potential role for UCP3 in salt intake and LVH is unclear. METHODS: UCP3-/- and C57BL/6 mice were placed on either a normal-salt (NS, 0.5%) or a high-salt (HS, 8%) diet for 24 weeks. The cardiac function, endurance capacity, energy expenditure, and mitochondrial functional capacity were measured in each group. RESULTS: Elevated blood pressure was only observed in HS-fed UCP3-/- mice. High salt induced cardiac hypertrophy and dysfunction were observed in both C57BL/6 and UCP3-/- mice. However, the cardiac lesions were more profound in HS-fed UCP3-/- mice. Furthermore, HS-fed UCP3-/-mice experienced more severe mitochondrial respiratory dysfunction compared with HS-fed C57BL/6 mice, represented by the decreased volume of oxygen consumption and heat production at the whole-body level. CONCLUSION: UCP3 protein was involved in the incidence of high-salt induced hypertension and the progression of cardiac dysfunction in the early stages of heart failure. UCP3 ablation exacerbated high-salt-induced cardiac hypertrophy and cardiac dysfunction.

[Therapeutic Effect of Autologous Platelet-rich Gel in the Treatment of Chronic Skin Ulcer with Tophus].

OBJECTIVE: To evaluate the clinical effectiveness and safety of autologous platelet-rich gel (APG) in the treatment of chronic skin ulcer with tophus. METHODS: Four patients of chronic skin ulcer with tophus received routine debridement to remove necrotic tissue and erasion tophus as far as possible,and then received the treatment of APG. RESULTS: All of the patients had their wounds healed after the treatment of APG (one wound was treated twice). The wounds were healed between 8 to 22 d, average (13. 7±6. 8) d, while there were no adverse effects observed. CONCLUSION: Topical therapy with APG may be considered as an effective and safe adjuvant method for the treamtment of chronic skin ulcer with tophus.

Characterization of miR-218/322-Stxbp1 pathway in the process of insulin secretion.

MicroRNAs (miRNAs) have been implicated in a variety of physiological processes, however, the function of miRNAs in insulin secretion and type 2 diabetes is still unclear. Stxbp1 plays an essential role in exocytosis, and is crucial for insulin secretion. In this study, we focused on the molecular mechanism of Stxbp1 in insulin secretion by identifying its upstream regulators: miR-218 and miR-322. The expression of Stxbp1 was significantly increased in isolated mouse islets exposed to high levels of glucose within 1 h; while two of its predicted upstream miRNAs were found to be downregulated. Further study found that miR-218 and miR-322 directly interact with Stxbp1 by targeting the 3'UTR of its mRNA. MIN6 cells overexpressing the two miRNAs showed a sharp decline in insulin secretion and a decreased sensitivity to glucose; while the inhibition of the two miRNAs promoted insulin secretion. However, islets treated with prolonged high levels of glucose, which is known as glucolipotoxicity, displayed relatively high expression of miR-218 and miR-322, and a reduced level of expression of Stxbp1 accompanied by the blocking of insulin secretion. In summary, this study identified a pathway consisting of miR-218/322 and Stxbp1 in insulin secretion, contributing to a network of ß-cell function involving miRNA.

Prauserella coralliicola sp. nov., isolated from the coral Galaxea fascicularis.

A novel Gram-stain-positive actinobacterium, designated strain SCSIO 11529(T), was isolated from tissues of the stony coral Galaxea fascicularis, and characterized by using a polyphasic approach. The temperature range for growth was 22-50 °C (optimum 28-45 °C), the pH range for growth was 6.0-8.0 (optimum pH 7.0), and the NaCl concentration range for growth was 0-7% (w/v) NaCl. The polar lipid profile contained diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine and an unknown polar lipid. The predominant menaquinone was MK-9(H4). The major fatty acids (>10%) were iso-C16:0, iso-C17:1ω6c, iso-C16:1 H and C16:1ω7c/iso-C15:0 2-OH. The DNA G+C content of strain SCSIO 11529(T) was 70.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain SCSIO 11529(T) belongs to the genus Prauserella, with the closest neighbours being Prauserella marina MS498(T) (97.0% 16S rRNA gene sequence similarity), Prauserella rugosa DSM 43194(T) (96.4%) and Prauserella flava YIM 90630(T) (95.9%). Based on the evidence of the present study, strain SCSIO 11529(T) is considered to represent a novel species of the genus Prauserella, for which the name Prauserella coralliicola sp. nov. is proposed. The type strain is SCSIO 11529(T) ( = DSM 45821(T) = NBRC 109418(T)).

[Joint diagnostic value of four temperature sensation tests in elderly patients with type 2 diabetic peripheral neuropathy].

OBJECTIVE: To explore the joint diagnostic value of four temperature sensation tests in elderly patients with type 2 diabetic peripheral neuropathy. METHODS: Thermal sensory analyzer-II were applied to measure cool sensation (CS), warm sensation (WS), cold pain sensation (CP)and heat pain sensation (HP) of 308 elderly patients with type 2 diabetes. Logistic regression model was adopted to create the new variable Temp4 from four temperature sensation tests to diagnose type 2 diabetic peripheral neuropathy. The ROC curve analysis was used to determine the best cut-off points of the four temperature sensation and Temp4, and the diagnostic value of it was evaluated. RESULTS: The means of temperature sensation tests of the diabetic peripheral neuropathy (DPN) group were significantly different from those of the non-DPN group (P < 0.05). According to the current reference intervals of the four temperature sensation tests to diagnose the DPN, the sensitivity of WS test was the highest, and the value was 0.710; but the specificity, positive predictive value, negative predictive value, Youden index, diagnostic accuracy and Kappa value of cold sensation test were the highest, and the values were 0.842, 0.746, 0.799, 0.528, 77.92% and 0.535, respectively; the Kappa values of the other three temperature sensation tests were all greater than 0.4 (P < 0.05). The area under the ROC curve of the new variable Temp4 was 0.93 (95% CI 0.91-0.96), and was larger than the four temperature sensation tests (P < 0.05). The sensitivity, specificity, Youden index and diagnostic accuracy of Temp4 were 0.823, 0.897, 0.719 and 86.69%, respectively. The new best cut-off points of the CS test, WS test, CP test, HP test and Temp4 was 27.5 degrees C, 34.7 degrees C, 20.5 degrees C, 43.5 degrees C and 0.416, respectively. CONCLUSION: The results of the four temperature sensation quantitative tests were in good agreementand could be applied to diagnose DPN; the new variable Temp4 could be used for diagnosis of DPN with a higher diagnostic accuracy.

Bacillus oceani sp. nov., a new slightly halophilic bacterium, isolated from a deep sea sediment environment.

A strictly aerobic, Gram-stain positive, slightly halophilic strain, designated SCSIO 04524(T), was isolated from a deep sea sediment sample collected from the northern South China Sea at a depth of 3415 m. The isolate slightly embedded into the medium after 72 h incubation at 30 °C. Growth was found to occur on media with 0-10 % NaCl but extremely weak growth occurred without supplying NaCl. The predominant menaquinone was determined to be MK-7. The major cellular fatty acid identified was iso-C15:0. The diagnostic polar lipids were determined to be diphosphatidylglycerol, phosphatidyl methylethanolamine, phosphatidylethanolamine and phosphatidylglycerol. The genomic DNA G+C content was determined to be 38 mol%. 16S rRNA gene sequences analysis showed that this strain had the highest similarities with Bacillus carboniphilus JCM 9731(T) (94.7 %) and Bacillus endophyticus 2DT(T) (94.3 %). Phylogenetic analysis revealed that strain SCSIO 04524(T) formed a distinct lineage with Bacillus chungangensis CAU 348(T) and B. carboniphilus JCM 9731(T). Physiological characteristics including utilization of sole nitrogen and carbon sources, and chemotaxonomic properties of cellular fatty acids and polar lipids could readily distinguish strain SCSIO 04524(T) from its most closely related species. Based on this polyphasic taxonomic data, a new species, Bacillus oceani sp. nov., is proposed, with the type strain SCSIO 04524(T) (=DSM 26213(T) = KCTC 33077(T)).

Melghirimyces profundicolus sp. nov., isolated from a deep-sea sediment.

A novel filamentous bacterium, strain SCSIO 11153(T), was isolated from a sediment sample collected from the Indian Ocean (80° 03.099' E 01° 03.300' N) at a depth of 4593 m. Good growth was observed at 50-55 °C and pH 7.0 with 3 % NaCl. It formed ivory-white colonies with radial wrinkles. Aerial mycelium was absent on the media tested. Phenotypic characteristics and 16S rRNA gene sequence analysis indicated that strain SCSIO 11153(T) belonged to the family Thermoactinomycetaceae. It exhibited 96.4% and 96.2% 16S rRNA gene sequence similarities to Melghirimyces algeriensis NariEX(T) and Melghirimyces thermohalophilus Nari11A(T), respectively, while lower sequence similarity values (<95.4%) were observed between strain SCSIO 11153(T) and other species of genera in the family Thermoactinomycetaceae. The menaquinone type was MK-7. Major cellular fatty acids were iso-C15:0, anteiso-C15:0 and iso-C17:0. The polar lipids were diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content of strain SCSIO 11153(T) was 52.6 mol%. On the basis of the genotypic and phenotypic characteristics, it is proposed that strain SCSIO 11153(T) represents a novel species of the genus Melghirimyces with the name Melghirimyces profundicolus sp. nov. The type strain is SCSIO 11153(T) ( = DSM 45787(T) = CCTCC AA 2012007(T) = NBRC 109068(T)).

Georgenia sediminis sp. nov., a moderately thermophilic actinobacterium isolated from sediment.

A Gram-stain-positive actinobacterium, designated strain SCSIO 15020(T), was isolated from sediment of the South China Sea, and characterized by using a polyphasic approach. The temperature range for growth was 24-60 °C, with optimal growth occurring at 50 °C. The pH range for growth was 6-10 (optimum pH 8-9). The NaCl concentration range for growth was 0-5 % (w/v). The peptidoglycan type was A4α. Polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and an unknown polar lipid. The major menaquinone was MK-8(H4); MK-7(H4) was present as a minor component. The major fatty acids (>5 %) were anteiso-C15 : 0, iso-C15 : 0 and iso-C16 : 0. The DNA G+C content of strain SCSIO 15020(T) was 73.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain SCSIO 15020(T) belonged to the genus Georgenia, with the closest neighbours being Georgenia muralis 1A-C(T) (96.3 % similarity), Georgenia thermotolerans TT02-04(T) (95.7 %) and Georgenia ruanii YIM 004(T) (95.6 %). Based on evidence from the present polyphasic study, strain SCSIO 15020(T) is considered to represent a novel species of the genus Georgenia, for which the name Georgenia sediminis sp. nov. is proposed. The type strain is SCSIO 15020(T) ( = DSM 25884(T) = NBRC 108941(T)).

Bacillus abyssalis sp. nov., isolated from a sediment of the South China Sea.

A Gram-positive bacterium, designated SCSIO 15042(T), was isolated from a sediment of the South China Sea and was subjected to a polyphasic taxonomic study. The isolate grew at 20-60 °C, pH 6.0-10.0 and it could grow with up to 10 % (w/v) NaCl. The cell-wall diamino acid was found to be meso-diaminopimelic acid. Polar lipids were found to be diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine and an unknown polar lipid. The only menaquinone was determined to be MK-7. The major fatty acids were identified as C16:1 ω7c/C16:1 ω6c, C16:0, iso-C15:0, anteiso-C15:0, and iso-C16:0. The DNA G+C content of strain SCSIO 15042(T) was determined to be 43.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequences affiliated strain SCSIO 15042(T) to the genus Bacillus. Levels of 16S rRNA gene sequence similarities between strain SCSIO 15042(T) and Bacillus herbersteinensis D-1-5a(T), Bacillus infantis SMC 4352-1(T), Bacillus novalis LMG 21837(T) and Bacillus drentensis LMG 21831(T) were 96.2, 96.2, 96.1 and 96.1 %, respectively. Based on the evidence of the present polyphasic study, strain SCSIO 15042(T) is considered to represent a novel species of the genus Bacillus, for which the name Bacillus abyssalis sp. nov. is proposed. The type strain is SCSIO 15042(T) (=DSM 25875(T) = CCTCC AB 2012074(T) = NBRC 109102(T)).