Application of MALDI-TOF mass spectrometry for identification of Nocardia species.

BACKGROUND: Nocardiosis, despite its rarity and underreporting, is significant due to its severe impact, characterized by high morbidity and mortality rates. The development of a precise, reliable, rapid, and straightforward technique for identifying the pathogenic agent in clinical specimens is crucial to reduce fatality rates and facilitate timely antimicrobial treatment. In this study, we aimed to identify Nocardia spp. in clinical isolates, using MALDI-TOF MS as the primary method, with molecular methods as the gold standard. Clinical Nocardia isolates were identified using 16S rRNA/hsp65/gyrB/secA1/rpoB gene sequencing. Identification performance of the Bruker MALDI Biotyper 3.1 (V09.0.0.0_8468) and MBT Compass 4.1 (V11.0.0.0_10833) for Nocardia identification was evaluated. RESULTS: Seventy-six Nocardia isolates were classified into 12 species through gene sequencing. The MALDI Biotyper 3.1 (V09.0.0.0_8468) achieved 100% genus-level accuracy and 84.2% species accuracy (64/76). The MBT Compass 4.1 with the BDAL Database (V11.0.0.0_10833) improved species identification to 98.7% (75/76). The updated database enhanced species level identification with scores > 1.7, increasing from 77.6% (59/76) to 94.7% (72/76), a significant improvement (P = 0.001). The new and simplified extraction increased the proportion of strains identified to the species level with scores > 1.7 from 62.0% (18/29) to 86.2% (25/29) (P = 0.016). An in-house library construction ensured accurate species identification for all isolates. CONCLUSIONS: The Bruker mass spectrometer can accurately identify Nocardia species, albeit with some variations observed between different database versions. The MALDI Biotyper 3.1 (V09.0.0.0_8468) has limitations in identifying Nocardia brasiliensis, with some strains only identifiable to the genus level. MBT Compass 4.1 (V11.0.0.0_10833) effectively addresses this shortfall, improving species identification accuracy to 98.7%, and offering quick and reliable identification of Nocardia. Both database versions incorrectly identified the clinically less common Nocardia sputorum as Nocardia araoensis. For laboratories that have not upgraded their databases and are unable to achieve satisfactory identification results for Nocardia, employing the new and simplified extraction method can provide a degree of improvement in identification outcomes.

Relationship between virulence and carbapenem resistance phenotype of Klebsiella pneumoniae from blood infection: identification of a carbapenem-resistant and hypervirulent strain. / è¡€æ¶²æ„ŸæŸ“æ¥æºè‚ºç‚Žå ‹é›·ä¼¯èŒæ¯’åŠ›ä¸Žç¢³é’éœ‰çƒ¯ç±»è€è¯è¡¨åž‹çš„å ³ç³»åŠç¢³é’éœ‰çƒ¯ç±»è€è¯é«˜æ¯’åŠ›èŒæ ªçš„é‰´å®š.

OBJECTIVES: To investigate the relationship between the virulence and the carbapenem resistance phenotype of Klebsiella pneumoniae from blood infection, and to identify carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-HVKP)strains. METHODS: A total of 192 Klebsiella pneumoniae strains were isolated from blood culture of patients with bloodstream infections from 2016 to 2019, of which 96 isolates were carbapenem-resistant Klebsiella pneumoniae (CRKP) and 96 were carbapenem-sensitive Klebsiella pneumoniae (CSKP). The drug susceptibility was detected by VITEK-2 automatic microbial analyzer; carbapenemase genes, virulence genes and capsule typing were detected by polymerase chain reaction; the high viscosity phenotype of strains was detected by string test, and the genome characteristics of CR-HVKP were detected by whole genome sequencing. Serum killing and biofilm formation test were used to further verify the virulence of CR-HVKP. RESULTS: There were significant differences in drug resistance to common antibiotics, except for minocycline between CSKP and CRKP isolates (all P<0.05). 92 out of 96 CRKP isolates carried carbapenemase genes, mainly blaKPC-2. The string tests were positive in 4 isolates of CRKP and 36 isolates of CSKP (P<0.05). The detection rates of virulence genes Kfu, aerobictin, iutA, ybtS, rmpA, magA, allS, and capsule antigen K1 and K2 in CSKP group were significantly higher than those in CRKP group (all P<0.05). One HVKP strain was detected in the CRKP group (CR-HVKP) and 36 HVKP was detected in the CSKP group (P<0.05). The CR-HVKP strain belonged to the MLST412, serotype K57, expressed iutA, entB, mrkD, fimH, and rmpA virulence genes, and showed strong biofilm formation and significantly increased serum resistance. Whole genome sequencing results showed that this CR-HVKP isolate carried blaSHV-145, blaTEM-1, blaCTX-M-3, fosA6, oqxA5, oqxB26, and aac(3)-IId resistance genes, accompanied by abnormalities in outer membrane protein K (OmpK) 35 and OmpK36. CONCLUSIONS: The drug resistance of CRKP is significantly higher than that of CSKP, while CRKP carrying fewer virulence genes in both number and types compared to CSKP. A new MLST type of carbapenem-resistant and hypervirulent Klebsiella pneumoniae strain has been detected, which requires clinical awareness and epidemiological monitoring.

Clinical manifestations, antimicrobial resistance and genomic feature analysis of multidrug-resistant Elizabethkingia strains.

BACKGROUND: Elizabethkingia is emerging as an opportunistic pathogen in humans. The aim of this study was to investigate the clinical epidemiology, antimicrobial susceptibility, virulence factors, and genome features of Elizabethkingia spp. METHODS: Clinical data from 71 patients who were diagnosed with Elizabethkingia-induced pneumonia and bacteremia between August 2019 and September 2021 were analyzed. Whole-genome sequencing was performed on seven isolates, and the results were compared with a dataset of 83 available Elizabethkingia genomes. Genomic features, Kyoto Encyclopedia of Genes and Genomes (KEGG) results and clusters of orthologous groups (COGs) were analyzed. RESULTS: The mean age of the patients was 56.9 ± 20.7 years, and the in-hospital mortality rate was 29.6% (21/71). Elizabethkingia strains were obtained mainly from intensive care units (36.6%, 26/71) and emergency departments (32.4%, 23/71). The majority of the strains were isolated from respiratory tract specimens (85.9%, 61/71). All patients had a history of broad-spectrum antimicrobial exposure. Hospitalization for invasive mechanical ventilation or catheter insertion was found to be a risk factor for infection. The isolates displayed a high rate of resistance to cephalosporins and carbapenems, but all were susceptible to minocycline and colistin. Genomic analysis identified five ß-lactamase genes (blaGOB, blaBlaB, blaCME, blaOXA, and blaTEM) responsible for ß-lactam resistance and virulence genes involved in stress adaptation (ureB/G, katA/B, and clpP), adherence (groEL, tufA, and htpB) and immune modulation (gmd, tviB, cps4J, wbtIL, cap8E/D/G, and rfbC). Functional analysis of the COGs revealed that "metabolism" constituted the largest category within the core genome, while "information storage and processing" was predominant in both the accessory and unique genomes. The unique genes in our 7 strains were mostly enriched in KEGG pathways related to microRNAs in cancer, drug resistance (ß-lactam and vancomycin), ABC transporters, biological metabolism and biosynthesis, and nucleotide excision repair mechanisms. CONCLUSION: The Elizabethkingia genus exhibits multidrug resistance and carries carbapenemase genes. This study presents a comparative genomic analysis of Elizabethkingia, providing knowledge that facilitates a better understanding of this microorganism.

[Carbapenemase Genes, Virulence Genes, and Molecular Epidemiology of Carbapenem-Resistant Klebsiella pneumoniae Derived From Bloodstream Infections]. / è¡€æµæ„ŸæŸ“ç¢³é’éœ‰çƒ¯è€è¯è‚ºç‚Žå ‹é›·ä¼¯èŒçš„è€è¯åŸºå› å’Œæ¯’åŠ›åŸºå› åŠåˆ†å­æµè¡Œç— å­¦ç ”ç©¶.

Objective: To investigate the clinical characteristics and molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolated from patients with bloodstream infections in a large tertiary-care general hospital in Southwest China. Methods: A total of 131 strains of non-repeating CRKP were collected from the blood cultures of patients who had bloodstream infections in 2015-2019. The strains were identified by VITEK-2, a fully automated microbial analyzer, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The minimum inhibitory concentration (MIC) was determined by microbroth dilution method. The common carbapenemase resistant genes and virulence factors were identified by PCR. Homology analysis was performed by multilocus sequencing typing. Whole genome sequencing was performed to analyze the genomic characteristics of CRKP without carbapenemase. Results: The 131 strains of CRKP showed resistance to common antibiotics, except for polymyxin B (1.6% resistance rate) and tigacycline (8.0% resistance rate). A total of 105 (80.2%) CRKP strains carried the Klebsiella pneumoniae carbapenemase (KPC) resistance gene, 15 (11.4%) strains carried the New Delhi Metallo-ß-lactamase (NDM) gene, and 4 (3.1%) isolates carried both KPC and NDM genes. Sequence typing (ST) 11 (74.0%) was the dominant sequence type. High detection rates for mrkD (96.2%), fimH (98.5%), entB (100%), and other virulence genes were reported. One hypervirulent CRKP strain was detected. The seven strains of CRKP that did not produce carbapenemase were shown to carry ESBL or AmpC genes and had anomalies in membrane porins OMPK35 and OMPK36, according to whole genome sequencing. Conclusion: In a large-scale tertiary-care general hospital, CRKP mainly carries the KPC gene, has a high drug resistance rate to a variety of antibiotics, and possesses multiple virulence genes. Attention should be paid to CRKP strains with high virulence.

[Epidemiological Analysis of Carbapenem-Resistant Enterobacteriaceae Strains in the Clinical Specimens of a Hospital].

Objective: To analyze the detection rate, in vitro susceptibility to antibiotics, and carbapenemase types of carbapenem-resistant Enterobacteriaceae (CRE) strains in the clinical samples of a hospital and to provide support for the prevention, control and treatment of CRE-related infections. Methods: Clinical specimens were examined according to the operating procedures of bacteriological tests. Species identification and in vitro drug susceptibility testing were performed on the isolated strains. Carbapenemase inhibitor enhancement testing, which combined the use of 3-aminobenzeneboronic acid and ethylenediaminetetraacetic acid, was conducted to identify the types of carbapenemase in the CRE strains. Results: In 2021, 2215 CRE strains were isolated from 157196 clinical samples collected in this hospital, presenting a detection rate of 1.4% (2215/157196). A total of 1134 non-repetitive strains of CRE were isolated from 903 patients. The main sources of samples were respiratory tract (494/1134, 43.6%), secretion (191/1134, 16.8%) and blood (173/1134, 15.3%) samples. The cases with the same CRE strain isolated from the samples of two, three and four sites accounted for 12.5%, 4.9%, and 1.1%, respectively. The most common species was Klebsiella pneumoniae (883/1134, 77.9%), followed by Enterobacter cloacae complex (107/1134, 9.4%) and Escherichia coli (96/1134, 8.5%). The rates of resistance to polymyxin B and tigecycline of different species of CRE strains were not significantly different ( P<0.05). Serine carbapenemase-producing strains, metallo-ß-lactamase-producing strains, and those producing both enzymes accounted for 82.6% (809/979), 17.2% (168/979), and 0.2% (2/979), respectively. Conclusion: CRE strains are frequently isolated from samples collected from the respiratory tract, secretion, and blood. The most common strain is serine carbapenemase-producing K. pneumoniae, which has a high resistance rate to various antimicrobial drugs, and risk factors of its associated infections deserve more attention.

[Application of Rapid Detection Method Based on NG-Test® CARBA 5 in Bloodstream Infections Associated With Carbapenem-Resistant Enterobacterales].

Objective: To compare the consistency and accuracy of a rapid test method and a traditional test method for pathogen identification, antimicrobial susceptibility and carbapenemase type identification of positive blood culture samples. Methods: A total of 51 positive blood culture samples of bloodstream infection (BSI) were collected between March 2022 and May 2022. All samples were found to be "positive for Gram-negative bacilli" according to the blood smear results. The rapid method was adopted to perform rapid antimicrobial susceptibility test (RAST) and analysis of the positive blood culture samples. According to the RAST result interpretation standards, NG-Test® CARBA 5 was used for rapid carbapenemase detection of the imipenem-resistant strains and the results were confirmed by PCR. In addition, mass spectrometry, VITEK 2 Compact drug sensitivity analysis, and carbapenemase type identification were performed with the colonies cultured with positive samples according to the traditional method. Results: In the identification of bacteria, the rapid method and the traditional method had 100% consistency rate in the identification results of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. In the antimicrobial susceptibility test, the consistency rate between the results of the two methods was high and the consistency rate for results for susceptibility to imipenem was 100%. In the identification of carbapenemase type, 18 serinase-producing strains and 3 metal-ß-lactamase-producing strains of Enterobacterales were detected by the traditional method. With the rapid method, 18 Klebsiella pneumoniae carbapenemase (KPC)-producing strains, 2 New Delhi metallo-betalactamase (NDM)-producing strains, and 1 imipenem enzyme (IMP)-producing strain were identified in the blood culture samples by using a testing kit. Compared with the PCR results, the sensitivity and specificity of the rapid test for determining carbapenemase types were 100%. In this study, we investigated a rapid method for bacteria and carbapenemase type identification of positive blood culture specimens and found that the turnaround time (TAT) of the rapid method was reduced by 1.94 days on average in comparison with the TAT of the traditional method. Conclusion: The rapid method established in the study can effectively shorten the TAT for pathogenic microorganism identification and antimicrobial susceptibility test of blood culture samples, and the joint report of colloidal gold carbapenemase type identification results can provide a reference for clinicians to use antibiotics appropriately and accurately manage multi-drug resistant bacterial infections.

Molecular epidemiology characteristics and detecting transmission of carbapenemase-producing enterobacterales in southwestern China.

BACKGROUND: To investigate the genotype and clinical characteristics of carbapenem-resistant Enterobacteriaceae (CRE) strains in southwest China and provide information on the treatment stopping the spread of the infection. METHODS: The clinical information of CRE isolates was collected from 19 hospitals in 12 cities across Sichuan Province, China, between June 2018 and April 2019. The isolates were detected by DNA sequencing of genes encoding carbapenem enzymes and multilocus sequence types (MLSTs). RESULTS: A total of 166 nonrepetitive CRE isolates were isolated during the study period from sputum, blood, urine, and other samples. Klebsiella pneumoniae carbapenemase (KPC) was dominant in Klebsiella pneumoniae (53.9%), followed by New Delhi metallo-ß-lactamase (NDM) (42.1%). A total of 43 STs were detected. The most common ST of K. pneumoniae was ST11, and that of Escherichia coli was ST410. Pairwise single nucleotide polymorphism (SNP) distances and the likelihood of local transmission by epidemiology were plotted for each species. About 65% of these pairs had ≤ 20 pairwise SNPs. CONCLUSION: A large number of CRE strains carried carbapenemase. Although NDM-ST12 K. pneumoniae should not be disregarded, KPC-ST11is the predominant strain. Thus, the possibility of transmission between E. coli and K. pneumoniae could not be ignored.

Epidemiology, Clinical Characteristics, Risk Factors, and Outcomes of Candidemia in a Large Tertiary Teaching Hospital in Western China: A Retrospective 5-Year Study from 2016 to 2020.

The aim of this study was to investigate the current status of candidemia and evaluate the clinical characteristics, risk factors and outcomes among different species. We conducted a retrospective study by univariate and multivariate analysis between Candida albicans and non-albicans Candida (NAC) species in a Chinese national medical center from 2016 to 2020. Among the 259 episodes, C. albicans (38.6%) was the leading species, followed by C. tropicalis (24.3%), C. parapsilosis (20.5%), and C. glabrata (12.4%). Most C. albicans and C. parapsilosis were susceptible to nine tested antifungal agents, whereas C. tropicalis showed 30.2~65.9% resistance/non-wild-type to four azoles with great cross-resistance, indicating that fluconazole should not be used for empirical antifungal treatment. In multivariable models, the factor related to an increased risk of NAC was glucocorticoid exposure, whereas gastrointestinal hemorrhage and thoracoabdominal drainage catheters were associated with an increased risk in C. albicans. Subgroup analysis revealed leukemia and lymphoma, as well as glucocorticoid exposure, to be factors independently associated with C. tropicalis in comparison with C. albicans candidemia. No significant differences in 7-day mortality or 30-day mortality were observed between C. albicans and NAC. This study may provide useful information with respect to choosing empirical antifungal agents and exploring differences in molecular mechanisms.

Detection and Characterization of Carbapenemases in Enterobacterales With a New Rapid and Simplified Carbapenemase Detection Method Called rsCDM.

Objective: This study aimed to develop a new rapid and simplified carbapenemase detection method (rsCDM) for detection and characterization of carbapenemase with 3-aminophenylboronic acid (APBA), ethylenediaminetetraacetic acid (EDTA), and cloxacillin (CLO) ß-lactamase inhibitors. Methods: A panel of 182 carbapenem-resistant Enterobacterales (CRE) strains with blaKPC (88), blaNDM (60), blaIMP (10), blaVIM (3), blaOXA-181 (5), blaKPC, and blaNDM (7), porin changes in combination with an extended-spectrum ß-lactamase (ESBL) (3) or AmpC hyper-production (6) and 43 carbapenem-susceptible Enterobacterales isolates were used to evaluate the performance of rsCDM and EDTA-carbapenem inactivation method (eCIM). Carbapenemase class was determined with specific inhibitors at 4, 6, and 18 h by rsCDM, and the difference between imipenem (IMI) and meropenem (MEM) disks was simultaneously compared. Results: The sensitivity of rsCDM using IMI was 97.1% at the three time points, with a specificity of 100%, independent of the culture duration. Similar to IPM, MEM disk also showed high sensitivity (97.1%) and specificity (100%) at 6 h. And the sensitivity of eCIM was 95.4% and the specificity was 100%. Based on a decision algorithm, the characterization number of IMI and MEM in KPC-producing isolates was 88 vs. 87, metallo-ß-lactamases (MBLs) was 73 vs. 72, KPC and NDM carbapenemase was 7 vs. 7 at 4 h, respectively. After 6 h, the category number changed insignificantly except for isolates with combined AmpC overproduction and porin changes, showing an increase in IMI (6) and MEM (2), and there was no difference in the results between 6 and 18 h for the two tablets. OXA-181-producing strains can't be distinguished by rsCDM. For eCIM, the characterization number in KPC-, OXA- 181-, and MBLs-producing strains was 88, 5, and 72, but it failed to detect multi-enzyme-producing isolates (KPC and NDM). Conclusion: rsCDM accurately discriminated carbapenemase within 4 h and could differentiate multi-enzyme (KPC and NDM) and AmpC in conjunction with porin changes strains. Hence, rsCDM represents a rapid, simple, easy readout, and accurate tool that can be used without any specialized equipment.

Evaluation of a Cryptococcus capsular polysaccharide detection FungiXpert LFA (lateral flow assay) for the rapid diagnosis of Cryptococcosis.

Cryptococcus is an opportunistic pathogenic fungus and is the major cause of fungal meningitis. The cryptococcal antigen (CrAg) lateral flow assay (LFA) is an immunochromatographic test system that has simplified diagnosis as a point-of-care test. In this study, we evaluated the diagnostic performance of Cryptococcal capsular polysaccharide detection FungiXpert (Genobio Pharmaceutical, Tianjin, China) using serum and cerebrospinal fluid (CSF) samples for the diagnosis of cryptococcosis and investigated the cross-reaction of the assays to pathogenic fungi and bacterium by comparing it to the U.S. Food and Drug Administration (US FDA)-approved IMMY CrAg LFA. Eighty CSF and 119 serum/plasma samples from 158 patients were retrospectively collected to test for qualitative or semi-quantitative detection of CrAg. Cross-reaction of the assays was tested using 28 fungi and 1 bacterium. Compared to IMMY CrAg LFA, the FungiXpert LFA demonstrated 99.1% sensitivity and 98.9% specificity in the qualitative test. In the 96 semi-quantitative CrAg assay results, 39 (40.6%) test titers of FungiXpert LFA were 1-2 dilutions higher than those of IMMY CrAg LFA. The Intraclass Correlation Coefficient of the Semi-quantitative results of CrAg titer tests via the two assays was 0.976. Similar to IMMY CrAg LFA, FungiXpert LFA showed cross-reactivity with Trichosporon asahii. Compared with the IMMY CrAg LFA, the FungiXpert LFA showed an equal, yet, excellent performance. However, it is important to note that these two assays have potential cross-reactivity to T. asahii when diagnosing patients. FungiXpert LFA is a rapid screening method for the effective and practical diagnosis and treatment of cryptococcosis. LAY SUMMARY: The FungiXpert LFA was developed to diagnose fungal meningitis caused by Cryptococcus yeasts, by using serum or cerebrospinal fluid. It was compared to an existing lateral flow assay (LFA). The FungiXpert LFA performed well in qualitative and semi-quantitative tests.

Emergence of ceftazidime-avibactam resistance due to a novel blaKPC-2 mutation during treatment of carbapenem-resistant Klebsiella pneumoniae infections.

OBJECTIVE: Klebsiella pneumoniae carbapenemase (KPC)-producing K.pneumoniae has represented a serious health problem in worldwide. The resistance to ceftazidime-avibactam (CAZ-AVI) began to emerge since its approval in 2015. We aim to explore the resistance mechanism of CAZ-AVI. METHODS: Phenotypic test and whole-genome sequencing (WGS) analysis were performed in KP-HX0917 and KP-HX1016 Klebsiella pneumoniae isolates, collected from the same patient following treatment with CAZ-AVI. RESULTS: We report a case of emergence of CAZ-AVI resistance in ST 11 KPC-2-producing K. pneumoniae (KP-HX1016) during 14 days of exposure with CZA-AVI. Molecular analysis highlighted the A533C mutation in the blaKPC-2 gene, resulting a D179A substitution in protein sequence, which restored the hydrolysis ability of imipenem and meropenem, but not for ertapenem, and the result of phenotypic test was negative. However, KP-HX0917 produced serine-carbapenemase by phenotypic detection and lost its capacity of hydrolyzing carbapenems. CONCLUSION: The emergence of CAZ-AVI resistance should arouse our attention, the susceptibility testing should be followed by a combination of phenotypic and molecular methods, to make sure that no potential carbapenemase-producing bacteria are missed.

Total Laboratory Automation and Three Shifts Reduce Turnaround Time of Cerebrospinal Fluid Culture Results in the Chinese Clinical Microbiology Laboratory.

Background: Total laboratory automation (TLA) has the potential to reduce specimen processing time, optimize workflow, and decrease turnaround time (TAT). The purpose of this research is to investigate whether the TAT of our laboratory has changed since the adoption of TLA, as well as to optimize laboratory workflow, improve laboratory testing efficiency, and provide better services of clinical diagnosis and treatment. Materials and Methods: Laboratory data was extracted from our laboratory information system in two 6-month periods: pre-TLA (July to December 2019) and post-TLA (July to December 2020), respectively. Results: The median TAT for positive cultures decreased significantly from pre-TLA to post-TLA (65.93 vs 63.53, P<0.001). For different types of cultures, The TAT of CSF changed the most (86.76 vs 64.30, P=0.007), followed by sputum (64.38 vs 61.41, P<0.001), urine (52.10 vs 49,57, P<0.001), blood (68.49 vs 66.60, P<0.001). For Ascites and Pleural fluid, there was no significant difference (P>0.05). Further analysis found that the incidence of broth growth only for pre-TLA was 12.4% (14/133), while for post-TLA, it was 3.4% (4/119). The difference was statistically significant (P=0.01). The common isolates from CSF samples were Cryptococcus neoformans, coagulase-negative Staphylococcus, Acinetobacter baumannii, and Klebsiella pneumonia. Conclusion: Using TLA and setting up three shifts shortened the TAT of our clinical microbiology laboratory, especially for CSF samples.

Comparing three different phenotypic methods for accurate detection of carbapenemase-producing Enterobacterales.

BACKGROUND: Early identification of carbapenemase-producing Enterobacterales (CPE) is highly essential to prevent their dissemination within health care settings. OBJECTIVE: This study aimed to compare 3 reported phenotypic assays for detecting carbapenemase-producing Enterobacterales (CPE). METHODS: 151 Enterobacterales isolates were collected, the sensitivity and specificity of each test was determined, with molecular genotype serving as the gold standard. The phenotypic evaluations were performed using EDTA-synergistic carbapenem inactivation method (esCIM), EDTA-carbapenem inactivation method (eCIM), and enzyme inhibitor enhancement experiment (EIE). RESULTS: The concordance rate was 98% for the EIE for the detection of KPC producer, and 100% for the esCIM and eCIM. Sensitivity differed among the 3 methods, and all assays had excellent sensitivity exceeding 90% for detecting metallo-ß-lactamases (MBLs). The specificity of the eCIM, esCIM and EIE was 100%, 100% and 95%. Both eCIM and esCIM were unsatisfactory in detecting multi-enzyme strains (MBL and class A serine carbapenemase) (0/6). However, EIE increased the positive number to six (6/6). CONCLUSIONS: The eCIM, esCIM and EIE can be used to accurately detect and distinguish carbapenemase and is suitable for routine use in most clinical microbiology laboratories.

PTX3 gene polymorphism associated with cryptococcosis in HIV-uninfected Chinese patients.

BACKGROUND: For Chinese Han populations, cryptococcosis are more likely to occur in HIV-uninfected patients instead of HIV-infected patients compared with other countries and regions, implying that there may be genetic predisposing factors for cryptococcosis in the Chinese Han populations. However, the retail mechanism has not been clarified. OBJECTIVES: We aimed to conduct an association analysis between the single nucleotide polymorphisms (SNPs) of pattern recognition receptors (PRR) genes and the susceptibility to cryptococcosis in HIV-uninfected Chinese patients, which may provide new genetic predisposing factors for early-risk prediction of disease, individualised treatment and prognosis monitoring. PATIENTS/METHODS: Using the SNaPshot SNP typing technique, eight SNPs of PRR genes (Dectin-2, Dectin-1, PTX3, CXCL8, IL12B, IFIH1, TLR1 and CD209) were typed on 97 HIV-uninfected cryptococcosis patients and 120 healthy controls who admitted to West China Hospital, Sichuan University, China, from 1 March 2018 to 30 December 2018. The results were analysed by the SHEsis software and SPSS 20.0 software. RESULTS: It was found that that PTX3 rs2305619 polymorphism was associated with cryptococcosis in HIV-uninfected patients. Compared with the GG genotype, AA genotype increased the risk of cryptococcosis in HIV-uninfected patients (p = .015, OR, 2.579; 95% CI, 1.202-5.535). In the immunocompetent patients, the AA genotype had a higher risk (p = .002, OR, 4.399; 95% CI, 1.745-11.088). Further verification found that the plasma PTX3 level of the AA genotype was significantly higher than the GA or GG genotype (60.28 ± 16.12 vs 7.32 ± 0.79, p < .001). CONCLUSIONS: PTX3 rs2305619 polymorphism was associated with cryptococcosis in HIV-uninfected Chinese patients. The AA genotype increased the risk of cryptococcosis, and its plasma PTX3 level was significantly higher than that of GA or GG genotype.

Bacterial and Fungal Infections After Liver Transplantation: Microbial Epidemiology, Risk Factors for Infection and Death with Infection.

BACKGROUND Infections, especially bacterial and fungal infections, are the leading cause of high mortality after liver transplantation (LT). This research investigated the pathogenic spectrum, antimicrobial susceptibility results, and risk factors of infection and death with infection to better control such infections. MATERIAL AND METHODS A retrospective cohort study was performed, and 433 liver transplant recipients between January 2010 and December 2016 were analyzed. RESULTS We found 290 isolates of bacteria and fungi in 170 infected liver transplant patients. Significant independent risk factors for bacterial and fungal infections were prolonged hospital stay (OR 1.034, 95% CI 1.013~1.056, p=0.002), mechanical ventilation (OR 3.806, 95% CI 1.567~9.248, p=0.003), and liver failure (OR 2.659, 95% CI 1.019~6.940, p=0.046). Furthermore, postoperative MELD scores (OR 1.120, 95% CI 1.020~1.230, p=0.017) and septic shock (OR 12.000, 95% CI 1.124~128.066, p=0.003) were independent risk factors for death with infection. CRAB infection is the main pathogenic bacteria of septic shock in LT patients. CONCLUSIONS We found that 39.3% of recipients had at least 1 bacterial or fungal infection after LT. Shortening the length of hospital stay and early withdrawal of mechanical ventilation will reduce the risk of infection after LT. Patients with liver failure should be more vigilant against postoperative infection. Once an infection occurs, immediate assessment of the postoperative MELD score, early diagnosis of septic shock, and active search for pathogenic evidence for precise treatment will help improve patient prognosis. Routine screening for CRAB colonization before surgery will facilitate empirical use of effective antibiotics.

Rapid Nanopore Assay for Carbapenem-Resistant Klebsiella pneumoniae.

The prevalence of carbapenem-resistant Klebsiella pneumoniae (CRKP) is rapidly increasing worldwide in recent decades and poses a challenge for today's clinical practice. Rapid detection of CRKP can avoid inappropriate antimicrobial therapy and save lives. Traditional detection methods for CRKP are extremely time-consuming; PCR and other sequencing methods are too expensive and technologically demanding, making it hard to meet the clinical demands. Nanopore assay has been used for screening biomarkers of diseases recently because of its high sensitivity, real-time detection, and low cost. In this study, we distinguished CRKP from carbapenem-sensitive K. pneumoniae (CSKP) by the detection of increasing amount of extracted 16S ribosomal RNA (16S rRNA) from bacterial culture with antibiotics imipenem, indicating the uninhibited growth of CRKP by the imipenem. Specific signals from single channel recording of 16S rRNA bound with probes by MspA nanopore allowed the ultra-sensitive and fast quantitative detection of 16S rRNA. We proved that only 4 h of CRKP culture time was needed for nanopore assay to distinguish the CRKP and CSKP. The time-cost of the assay is only about 5% of disk diffusion method while reaching the similar accuracy. This new method has the potential application in the fast screening of drug resistance in clinical microorganism samples.

Development and evaluation of the method for detecting metallo-carbapenemases among carbapenemase-producing Enterobacteriaceae.

A simple EDTA synergistic carbapenem inactivation method (esCIM) based on the simplified carbapenem inactivation method (sCIM) and EDTA synergistic carbapenem inactivation test (eCIM) detected the levels of metallo-ß-lactamases (MBLs) carbapenemase. The esCIM method uses EDTA-impregnated antibiotic disk to detect carbapenemase-producing Enterobacteriaceae (CPE) directly instead of inculating the disk in the trypticase soy broth (TSB). To determine the sensitivity and specificity of esCIM, 167 carbapenemase-resistant Enterobacteriaceae (CRE) isolates were collected, of which, 161 were CPE strains confirmed by PCR. The carbapenemase genes included blaKPC (50.9%), blaNDM (36.6%), blaIMP (6.8%), blaVIM (3.1%), and blaOXA-181 (0.6%). Three isolates carried two different types of genes (blaKPC and blaNDM), and the remaining six CRE strains lacked the carbapenemase genes. The phenotypic evaluations were performed using both esCIM and eCIM. The esCIM performs better than eCIM in the detection of blaNDM and blaIMP, and the positive rate of eCIM was 83% and 55% for blaNDM and blaIMP, respectively. However, in the case of esCIM, the rate increased to 97% and 73%, respectively. For all MBLs, the sensitivity of esCIM and eCIM observed was 91% and 76%, respectively, while the specificity of the two methods was 100%. Taken together, esCIM could be performed easily and interpreted quickly.

Development and validation of an advanced fragment analysis-based assay for the detection of 22 pathogens in the cerebrospinal fluid of patients with meningitis and encephalitis.

BACKGROUND: Meningitis and encephalitis (ME) are central nervous system (CNS) infections mainly caused by bacteria, mycobacteria, fungi, viruses, and parasites that result in high morbidity and mortality. The early, accurate diagnosis of pathogens in the cerebrospinal fluid (CSF) and timely medication are associated with better prognosis. Conventional methods, such as culture, microscopic examination, serological detection, CSF routine analysis, and radiological findings, either are time-consuming or lack sensitivity and specificity. METHODS: To address these clinical needs, we developed an advanced fragment analysis (AFA)-based assay for the multiplex detection of 22 common ME pathogens, including eight viruses, 11 bacteria, and three fungi. The detection sensitivity of each target was evaluated with a recombinant plasmid. The limits of detection of the 22 pathogens ranged from 15 to 120 copies/reaction. We performed a retrospective study to analyze the pathogens from the CSF specimens of 170 clinically diagnosed ME patients using an AFA-based assay and compared the results with culture (bacteria and fungi), microscopic examination (fungi), polymerase chain reaction (PCR) (Mycobacterium tuberculosis), and Sanger sequencing (virus) results. RESULTS: The sensitivity of the AFA assay was 100% for 10 analytes. For Cryptococcus neoformans, the sensitivity was 63.6%. The overall specificity was 98.2%. The turnaround time was reduced to 4-6 hours from the 3-7 days required using conventional methods. CONCLUSIONS: In conclusion, the AFA-based assay provides a rapid, sensitive, and accurate method for pathogen detection from CSF samples.

[Etiology, Prognosis and Risk Factors of 181 Adult Community-acquired Acute Bacterial Meningitis].

OBJECTIVE: To understand the etiology, clinical prognosis and risk factors of adult community-acquired acute bacterial meningitis (ABM) and provide the evidence for clinical diagnosis and treatment. METHODS: We performed a retrospective study of 181 clinically diagnosed hospitalized patients with community-acquired adult ABM from Jan.2010 to Jan.2018. The patients were categorized as non-elderly (16≤age<65 years old, n=156 ) and elderly (age≥65 years old, n=25) group. The etiology, clinical features, prognosis and risk factors of the two groups were compared. RESULTS: Sixty-four of 181 patients (35.4%) had pathogens detected. The most common pathogens were Streptococcus pneumoniae (17.9%), Listeria monocytogenes (13.4%) and Klebsiella pneumoniae (10.5%). The mortality of the elderly group was higher than that of the non-elderly group (P<0.05). Univariate analysis showed that there was a significant difference between the elderly group and the non-elderly group in the incidence of hypertension, hypokalemia, pulmonary infection, ear-nose-throat ( ENT) infection, cerebrospinal fluid (CSF) protein concentration, head CT abnormalities and mortality. Logistic regression analysis showed that pulmonary infection and temperature ≥38.5 ℃ were independent risk factors for poor prognosis in the non-elderly group. CSF pressure ≥200 mmH2O was a independent risk factors for poor prognosis in the elderly group. CONCLUSION: The pathogens that cause acute bacterial meningitis in adult community are mainly Streptococcus pneumoniae, Listeria monocytogenes and Klebsiella pneumoniae.Pulmonary infection and temperature ≥38.5 ℃ are independent risk factors of poor prognosis in the non-elderly patients, as CSF pressure ≥200 mmH2O a independent risk factor in the elderly patients.