Clinical application and evaluation of metagenomic next-generation sequencing in suspected adult central nervous system infection.

BACKGROUND: Accurate etiology diagnosis is crucial for central nervous system infections (CNS infections). The diagnostic value of metagenomic next-generation sequencing (mNGS), an emerging powerful platform, remains to be studied in CNS infections. METHODS: We conducted a single-center prospective cohort study to compare mNGS with conventional methods including culture, smear and etc. 248 suspected CNS infectious patients were enrolled and clinical data were recorded. RESULTS: mNGS reported a 90.00% (9/10) sensitivity in culture-positive patients without empirical treatment and 66.67% (6/9) in empirically-treated patients. Detected an extra of 48 bacteria and fungi in culture-negative patients, mNGS provided a higher detection rate compared to culture in patients with (34.45% vs. 7.56%, McNemar test, p < 0.0083) or without empirical therapy (50.00% vs. 25.00%, McNemar test, p > 0.0083). Compared to conventional methods, positive percent agreement and negative percent agreement was 75.00% and 69.11% separately. mNGS detection rate was significantly higher in patients with cerebrospinal fluid (CSF) WBC > 300 * 106/L, CSF protein > 500 mg/L or glucose ratio ≤ 0.3. mNGS sequencing read is correlated with CSF WBC, glucose ratio levels and clinical disease progression. CONCLUSION: mNGS showed a satisfying diagnostic performance in CNS infections and had an overall superior detection rate to culture. mNGS may held diagnostic advantages especially in empirically treated patients. CSF laboratory results were statistically relevant to mNGS detection rate, and mNGS could dynamically monitor disease progression.

Universal screening for Lynch syndrome in a large consecutive cohort of Chinese colorectal cancer patients: High prevalence and unique molecular features.

The prevalence of Lynch syndrome (LS) varies significantly in different populations, suggesting that ethnic features might play an important role. We enrolled 3330 consecutive Chinese patients who had surgical resection for newly diagnosed colorectal cancer. Universal screening for LS was implemented, including immunohistochemistry for mismatch repair (MMR) proteins, BRAFV600E mutation test and germline sequencing. Among the 3250 eligible patients, MMR protein deficiency (dMMR) was detected in 330 (10.2%) patients. Ninety-three patients (2.9%) were diagnosed with LS. Nine (9.7%) patients with LS fulfilled Amsterdam criteria II and 76 (81.7%) met the revised Bethesda guidelines. Only 15 (9.7%) patients with absence of MLH1 on IHC had BRAFV600E mutation. One third (33/99) of the MMR gene mutations have not been reported previously. The age of onset indicates risk of LS in patients with dMMR tumors. For patients older than 65 years, only 2 patients (5.7%) fulfilling revised Bethesda guidelines were diagnosed with LS. Selective sequencing of all cases with dMMR diagnosed at or below age 65 years and only of those dMMR cases older than 65 years who fulfill revised Bethesda guidelines results in 8.2% fewer cases requiring germline testing without missing any LS diagnoses. While the prevalence of LS in Chinese patients is similar to that of Western populations, the spectrum of constitutional mutations and frequency of BRAFV600E mutation is different. Patients older than 65 years who do not meet the revised Bethesda guidelines have a low risk of LS, suggesting germline sequencing might not be necessary in this population.

Identification of vaccine candidates from differentially expressed outer membrane proteins of Vibrio alginolyticus in response to NaCl and iron limitation.

Vibrio alginolyticus is the etiological agent that causes great losses in aquacultures and clinical emanating cases in humans. Identification of highly efficient vaccine candidates to control V. alginolyticus infection has been highly concerned since vaccines offer a powerful approach to provide efficient protection from bacterial infections. In the present study, we firstly investigated the altered outer membrane proteins (OM proteins) of V. alginolyticus in response to NaCl concentrations and iron limitation using Western blotting, and then identified the protective activity of these altered OM proteins by bacterial challenge post immunization. Ten OM proteins were differentially expressed in response to the osmolarity changing or/and iron limitation, in which VA2212, OmpV, VPA1186, OmpU, VPA1644, VA1061, VA1631 and VPA0860 were markedly altered in response to osmolarity, and VPA1186, OmpU, OmpV, VA0449, VPA0860, VPA1435 and VA1631 were determined to be iron-limited responsive proteins. Out of the ten OM proteins, VA1061, OmpU, VPA1435 and VPA0860 could be effective vaccine candidates against infection by V. alginolyticus in vivo. Further results indicated that VA1061 and VPA0860 were dominant antigens and could stimulate hosts to produce stronger antibody response than other two in live or inactivated whole-cell vaccines. These results not only expand knowledge on osmolarity-, iron-responsive proteins, but also provide a valuable strategy for identify protective proteins suitable for use in vaccine development.

Differentially expressed outer membrane proteins of Vibrio alginolyticus in response to six types of antibiotics.

Vibrio alginolyticus is an opportunistic pathogen that occasionally causes life-threatening infections in individuals and results in great losses in marine aquacultures of crustaceans and fish. Recently, antibiotic-resistant strains of the bacterium from clinical and environmental sources have been reported with increasing frequency. However, few reports were involved in the antibiotic resistance of this bacterium at molecular levels. In the present study, Western blotting was utilized to investigate altered OM proteins of V. alginolyticus in response to six types of antibiotics: erythromycin, kanamycin, tetracycline, streptomycin, nalidixic acid, and chloromycetin. Seventeen OM proteins have been reported here for the first time to be related to antibiotic resistance. They were porins OmpU, OmpN, putative OmpU and LamB; transport proteins VA0802, VA2212 (FadL) and VPA0860; TolC family TolC and VA1631; lipoprotein VA0449; OmpA family VPA1186 and VA0764; iron-regulated proteins OmpV, VPA1435, and VA2602; and receptor protein OmpK; hypothetical protein VA1475. Importantly, VA2212 was up-regulated in response to the five antibiotics except nalidixic acid, and VPA1186 was down-regulated in response to the six antibiotics in antibiotic-stressed bacteria. They might be potentially universal targets for designing the new drugs that inhibit multi-resistant bacteria. These findings suggested that parallel investigations into a bacterium responding to several types of antibiotics would be helpful not only for the further understanding of antibiotic-resistant mechanisms but also for the screening of valuable targets of new drugs controlling antibiotic-resistant bacteria.

Immunoproteomic identification of polyvalent vaccine candidates from Vibrio parahaemolyticus outer membrane proteins.

Bacterium is still a major cause of many infectious diseases and a global threat to human health, aquaculture, and animal feeding. Prevention by vaccination is the most efficient and economical way of fighting bacterial diseases, but one of the persistent challenges to prevent bacterial infections and disease transmissions is the existence of multiple bacterial species, families, and genera and the lack of efficient polyvalent vaccines against them. The information on candidate immunogens for polyvalent vaccine development is elusive, as well. For the development of broad cross-protective vaccines, we have employed heterogeneous antiserum-based immunoproteomics approaches to identify antigenically similar outer membrane (OM) proteins that could be used as potential polyvalent vaccine candidates against Vibrio parahaemolyticus , V. alginolyticus , V. fluvialis , Aeromonas hydrophila , and A. sobria infections. VPA1435, VP0764, VPA1186, VP1061, and VP2850 could be recognized by at least three antisera and demonstrated significantly passive and active immune protection against V. parahaemolyticus infection in a crucian carp model. VP1061 and VP2850 induced higher immune and protective abilities than the other three OM proteins. Furthermore, the abilities of VP1061 and VP2850 in the generation of broad cross-protective immune reaction against the infections of V. alginolyticus , A. hydrophila , and Pseudomonas fluorescens were also investigated in fish and mouse models. Our results suggested that VP1061 and VP2850 could potentially be used as polyvalent vaccine candidates for the development of novel polyvalent vaccines against V. parahaemolyticus and other Gram-negative pathogens. On the basis of these results, characteristics of OM proteins as polyvalent vaccine candidates have been addressed.

Identification of broad cross-protective immunogens using heterogeneous antiserum-based immunoproteomic approach.

Bacterium is still one of the major causes of life-threatening microbial infections. The most effective way to control bacterial infections is probably vaccine prevention. However, development of bacterial vaccine, especially polyvalent vaccines that could be used to fight against a variety of bacterial serotypes and species, is challenging due to the difficulty in identifying broad cross-protective antigens for different serotypes and species of pathogenic bacteria. In the present study, we developed a new approach to identify polyvalent vaccine candidates from outer membrane (OM) proteins of Vibrio alginolyticus with the ability to fight against infections caused by different genera and families of Gram-negative bacteria. This approach combined heterogeneous antiserum-based immunoproteomics with bacterial immunization challenging method. Four of the 35 protein spots resolved in a 2-DE gel of V. alginolyticus sarcosine-insoluble fraction could be recognized not only by homogeneous antiserum, but also by heterogeneous antisera obtained from other bacterial species, genera and families. The genes encoding the four OM proteins were then cloned and expressed in E. coli BL21. The expressed recombinant proteins were used as broadly cross-protective immunogens to immunize carps for investigation of their cross-protective spectra, activities and protective abilities in carps. The carps immunized with OmpA (VA0764) and Pal (VA1061) have abilities to fight against infections not only caused by V. alginolyticus, but also by Aeromonas hydrophila and Pseudomonas fluorescens. This study provides a novel approach for the identification of broadly cross-protective antigens, and possibly polyvalent vaccines against a variety of microbial infections.