[Pathogen Diagnosis of a Febrile HIV Case by the Metagenomic Next-generation Sequencing].

This study is aimed to explore the value of metagenomic next-generation sequencing (mNGS) in diagnosing pathogen in fever patients. It is often a challenge to identify the pathogen that caused the infection in the HIV patients with fever. How could the mNGS be helpful for pathogen diagnosis is unclear. Here we reported a case of human immunodeficiency virus (HIV) patient with 2-month period of fever. After routine clinical laboratory tests including the conventional smear, culture, serological tests and pathological examinations, the causal pathogen still remained undiagnosed. Then the lymph node biopsy tissue was subjected to broad-range polymerase chain reaction (PCR) and the peripheral blood was subjected to mNGS. At the same time, peripheral blood culture was carried out with an extension of culture time to acquire the pathogen. Results from both broad-range PCR and mNGS revealed the pathogen was Talaromyces marneffei. The isolate recovered from the peripheral blood culture was subjected to the whole-genome sequencing. Whole genome sequencing revealed that the antimicrobial resistance gene FLU1 existed in this pathogen's genome, but mNGS did not detect the FLU1 gene. Phylogenetic analysis based on whole genome sequence revealed that this isolate was far from other clones published in NCBI database. Here we reported a case of Talaromyces marneffei infection diagnosed by mNGS, showing that mNGS is helpful in etiological diagnosis for HIV patients with unexplained fever. However, application of mNGS in antimicrobial resistant genes detection and pathogen tracing need to be well-studied in the future.

[Metagenomic Sequencing for Pathogens Detection in a Critically Ill Patient].

OBJECTIVE: To detect pathogens in a critically ill patient using metagenomic sequencing. METHODS: A critically ill patient with severe acute pancreatitis suffered from abdominal pain and progressed into unconsciousness. Tissue smear, culture, automated biochemical identification and antibiotic susceptibility test, viral load determination by real-time fluorescence quantitative PCR, and immunohistochemical pathological tests were performed to detect pathogens, in addition to metagenomic sequencing based on the BGISEQ-100 high throughput sequencing platform. The sequences exclusive of host sequences were searched in the microbial genome database including viruses, bacteria, fungi and parasites. RESULTS: The patient was infected with methicillin-resistant Staphylococcus aureus, carbapenem-resistant Klebsiella pneumoniae and carbapenem-resistant Acinetobacter baumannii, verified by both the routine methods and the metagenomic sequencing. The metagenomic sequencing also detected cytomegalovirus (CMV) with a turn-around time of 5 days. Real-time fluorescent quantitative PCR confirmed 189 000 copies/mL CMV load. CONCLUSION: In this case, three species of bacteria and one virus were detected by metagenomic sequencing quickly and accurately. Metagenomic sequencing may be helpful for diagnosing infectious diseases in critically ill patients.

[Trends of Antimicrobial Susceptibility of Clostridium difficileIsolates in West China Hospital of Sichuan University from 2015 to 2017].

OBJECTIVE: To summarize the drug sensitivity and its trends of Clostridium difficile diarrhea pathogenic strains in a large tertiary hospital, so as to provide basic reference data for the treatment and control of Clostridium difficile infection. METHODS: There were 73 toxigenic isolates collected from fecal sample of diarrheal patients in West China Hospital of Sichuan University during two periods. One was from August to December in 2015 (44 strains) , and another was from July 2016 to July 2017 (29 strains) . Enhanced nosocomial infection control measures were implemented during the second sample collection period. The toxin gene was amplified by PCR and sequenced for identification. Minimum inhibitory concentration (MIC) of metronidazole, vancomycin, clindamycin, moxifloxacin, rifaximin, fidaxomicin and linezolid were determined using agar double dilution method. We analyzed the drug resistance characteristics of Clostridium difficile and compared the changes of antimicrobial resistance before and after the enhanced control measures implementation. RESULTS: All 73 strains tested were sensitive to metronidazole and vancomycin. Resistance rate to clindamycin, moxifloxacin, tetracycline and rifaximin were 79.5%, 26.0%, 27.4%, and 9.5%, respectively. Fidaxomicin and nitazoxanide were highly susceptible in vitro against these strains with MIC ranges<0.008-0.5 mg/L ( P<0.05). Resistance to clindamycin and moxifloxacin were significantly decreased after enhanced control measures implementation (resistance rates were 99.5% vs. 44.8%, 36.4% vs. 10.3%, P<0.05). Additionally, isolate with decreased susceptibility to tinezolid as MIC 16 mg/L was found. CONCLUSION: Clostridium difficileis highly resistant to clindamycin and quinolones. Since strains remain highly sensitive to metronidazole and vancomycin in our hospital, empirical application is reasonable without routine antimicrobial susceptibility testing.

Antimicrobial resistance-encoding plasmid clusters with heterogeneous MDR regions driven by IS26 in a single Escherichia coli isolate.

BACKGROUND: IS26-flanked transposons played an increasingly important part in the mobilization and development of resistance determinants. Heterogeneous resistance-encoding plasmid clusters with polymorphic MDR regions (MRRs) conferred by IS26 in an individual Escherichia coli isolate have not yet been detected. OBJECTIVES: To characterize the complete sequence of a novel blaCTX-M-65- and fosA3-carrying IncZ-7 plasmid with dynamic MRRs from an E. coli isolate, and to depict the mechanism underlying the spread of resistance determinants and genetic polymorphisms. METHODS: The molecular characterization of a strain carrying blaCTX-M-65 and fosA3 was analysed by antimicrobial susceptibility testing and MLST. The transferability of a plasmid bearing blaCTX-M-65 and fosA3 was determined by conjugation assays, and the complete structure of the plasmid was obtained by Illumina, PacBio and conventional PCR mapping, respectively. The circular forms derived from IS26-flanked transposons were detected by reverse PCR and sequencing. RESULTS: A novel IncZ-7 plasmid pEC013 (∼118kb) harbouring the blaCTX-M-65 and fosA3 genes was recovered from E. coli isolate EC013 belonging to D-ST117. The plasmid was found to have heterogeneous and dynamic MRRs in an individual strain and the IS26-flanked composite transposon-derived circular intermediates were identified and characterized in pEC013. CONCLUSIONS: The heterogeneous MRRs suggested that a single plasmid may actually be a cluster of plasmids with the same backbone but varied MRRs, reflecting the plasmid's heterogeneity and the survival benefits of having a response to antimicrobial-related threatening conditions in an individual strain.

Comparative genomics of rmtB-carrying IncI1 ST136 plasmids in avian escherichia coli isolates from chickens in China.

Eight rmtB-carrying avian Escherichia coli strains from a farm in China were characterised in our previous study, but little is known about the backbones and entire multiresistance regions (MRRs) of these plasmids. Here, three rmtB-carrying IncI1 ST136 plasmids were analysed by whole-plasmid sequencing and were compared. These plasmids were composed of an 83 470-bp IncI1 backbone carrying genes responsible for plasmid replication, transfer, maintenance and stability functions, as well as a 17 330-bp MRR for pEC006 and pEC007, and a 34 626-bp MRR for pEC008. Plasmid pEC006 was not transferable, thus truncation of the traI gene may explain the inability to conjugate. pEC008 harboured the blaTEM-1, rmtB, aacC2, tetA, floR and strAB genes as well as a class 1 integron cassette array (|dfrA12|orfF|aadA2|), which were interspersed with different mobile elements, including Tn2, Tn1721, Tn1722, Tn5393, ISCfr1, IS5057, ISCR1 and ISCR2, and three copies of IS26. The MRR of pEC008 may have resulted from transposition of Tn1722 into the plasmid backbone. Acquisition and rearrangement of MRRs demonstrated the accumulation of different resistance determinants.

Sharp instrument injuries among hospital healthcare workers in mainland China: a cross-sectional study.

OBJECTIVES: To determine the prevalence of sharp instrument injuries in hospital-based healthcare workers (HCWs) in mainland China and the contributing factors. DESIGN: Cross-sectional study. SETTING: The data were derived from public hospitals. PARTICIPANTS: A total of 360 hospitals were recruited in the study, including 289 general hospitals and 71 specialised hospitals. Among them, 194 are tertiary-level hospitals and 166 are secondary level. The study population finally consisted of 223 149 hospital HCWs. PRIMARY OUTCOME MEASURES: A questionnaire was designed based on the aim of the study. Profession of HCWs, workplace, circumstance and medical apparatus and instrument were covered in the survey. HCWs completed a self-administered questionnaire regarding details of sharp instrument injuries within the previous month. Prevalence estimates for the injuries were calculated for the overall HCWs and for subgroups according to profession, workplace, circumstance or instrument. RESULTS: Within the included HCWs, the prevalence of sharp instrument injuries was 0.08 per person-month. Only 4.6% of the HCWs reported to their hospitals after injury. The highest number of injuries occurred in nursing staff (10.3%). Injuries took place most frequently on general wards (44.5%). The circumstances that involved most frequent injuries include surgical needle insertion, removing an arteriovenous needle from a patient and recapping the needle. Single-use syringe caused more injuries incidents than other instruments. CONCLUSIONS: These results indicate that sharp instrument injuries have become a major occupational problem of HCWs in mainland China. Attentions need to be paid to the issue and strategies for preventing such injuries are needed.

Complete Sequence of pEC012, a Multidrug-Resistant IncI1 ST71 Plasmid Carrying bla CTX-M-65, rmtB, fosA3, floR, and oqxAB in an Avian Escherichia coli ST117 Strain.

A 139,622-bp IncI1 ST71 conjugative plasmid pEC012 from an avian Escherichia coli D-ST117 strain was sequenced, which carried five IS26-bracketed resistance modules: IS26-fosA3-orf1-orf2-Δorf3-IS26, IS26-fip-ΔISEcp1-bla CTX-M-65-IS903D-iroN-IS26, IS26-ΔtnpR-bla TEM-1-rmtB-IS26, IS26-oqxAB-IS26, and IS26-floR-aac(3)-IV-IS26. The backbone of pEC012 was similar to that of several other IncI1 ST71 plasmids: pV408, pM105, and pC271, but these plasmids had different arrangements of multidrug resistance region. In addition, the novel ISEc57 element was identified, which is in the IS21 family. The stepwise emergence of multi-resistance regions demonstrated the accumulation of different resistance determinants through homologous recombination. To the best of our knowledge, this is the first study to identify a multidrug-resistant IncI1 ST71 plasmid carrying bla CTX-M-65, rmtB, fosA3, floR, and oqxAB in an avian E. coli ST117 strain.

[One Carrier of Robinsoniella peoriensis in China].

OBJECTIVES: To study morphological feature, biochemical characteristic and antibiotic resistance of Robinsoniella peoriensis (R.peoriensis) strain. METHODS: The clinical R.peoriensis strain was isolated from ananus swab samples being screened in ICU. Biochemical characteristic of the strain was completed by fully automatic microbial identification and drug susceptibility analysis system (BioMérieux, Marcy-l'éE toile, France) with the ANC Vitek2. Antibiotic susceptibility in vitro was performed using agar dilution. RESULTS: The organism was found to be positive to saccharose, beta-galactopyr anosidase indoxyl, alpha-arabinosidase and so on, while negative to the others. The susceptibility test in vitro showed that this strain was resistant to clindamycin, rifampicin, moxifloxacin, while sensitive to vancomycin, metronidazole and tetracycline. CONCLUSIONS: R.peoriensis is positive to many biochemical products such as saccharose. The clinical isolate of R.peoriensis strain is resistant to clindamycin, rifampicin and moxifloxacin.

[Nosocomial diarrhea in intensive care unit: other than Clostridium difficile].

OBJECTIVE: To investigate the incidence and clinical features of non-Clostridium difficile (C. difficile) associated nosocomial diarrhea in intensive care unit (ICU) caused by Klebsiella oxytoca and Clostridium perfringens. METHODS: The faeces of 102 patients with non-C. difficile associated nosocomial diarrhea in ICU of West China Hospital, were collected during April to November, 2012. The target bacterial genes were detected by PCR amplification and sequencing, including toxic gene pehX of Klebsiella oxytoca, species-specific 16S rRNA gene and toxic gene cpa and cpe of Clostridium perfringens, species-specific 16S rRNA gene with mapA and toxic gene hipO of Campylobacter jejuni. Clinical features of the patients with positive results were summarized. RESULTS: Among 102 patients with non-C. difficile associated nosocomial diarrhea, 4 patients (3.9%) were detected with toxic Klebsiella oxytoca while 4 patients (3.9%) were detected with toxic Clostridium perfringens. No toxic Campylobacter jejuni was detected. Most of the patients had severe underlying diseases and poor final outcome, accepted potent antibiotics which disturbed intestinal flora obviously.. CONCLUSION: Non-C. difficile associated nosocomial diarrhea in ICU caused by Klebsiella oxytoca is and Clostridium perfringens is associated with severe diseases and poor outcome, but the incidence in our hospital is relatively low in our hospital.

[Clonal relatedness of bla(OXA-58)--carrying Acinetobacter baumannii clinical isolates].

OBJECTIVE: To investigate the clonal relatedness of local bla(OXA-58)-carrying Acinetobacter baumannii clinical isolates. METHODS: Non-duplicated isolates of Acinetobacter baumannii were collected in West China Hospital and verified by recA sequencing. Acquired bla(OXA-58) gene and natural bla(OXA51/66) genes were detected by PCR. Strain typing for bla(OXA-58)-carrying Acinetobacter baumannii isolates was performed by Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR), multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). RESULTS: A total of 115 Acinetobacter baumannii isolates were verified by recA gene and bla(OXA-51/6)6 detection. Among them, nine (7.8%) isolates carry bla(OXA-58) with reduced susceptibility to imipenem (MIC > or = 2 mg/L) were observed. ERIC-PCR fingerprints of nine bla(OXA-58)-carrying isolates were highly similar. MLST revealed that eight isolates were ST95 and one isolate was ST75. PFGE showed that eight isolates with the same sequence type were of the same fingerprint types, which were of two closely-related subtypes. CONCLUSION: In West China Hospital, some Acinetobacter baumannii isolates with reduced susceptibility to carbapenem carried bla(OXA-58). The major spread way of bla(OXA-58)-carrying isolates was clonal dissemination.

Epidemic plasmid carrying bla(CTX-M-15) in Klebsiella penumoniae in China.

OBJECTIVE: To investigate the local epidemiology of Klebsiella penumoniae carrying bla(CTX-M-15) in southern China and to characterize the genetic environment of bla(CTX-M-15). METHODS: PCR and DNA sequencing were used to detect and characterize the genetic contexts of bla(CTX-M-15). The clonal relatedness of isolates carrying bla(CTX-M-15) was determined by pulse-field gel electrophoresis. Conjugative plasmids carrying bla(CTX-M-15) were obtained by mating and were further subject to restriction analysis and replicon typing. RESULTS: A total of 47CTX-M-15 ESBL-producing isolates of K. pneumoniae were collected from nine hospitals in China from October 2007 to October 2008. Isolates were clustered into various clonal groups. The local spread of bla(CTX-M-15) was mainly mediated by one major conjugative plasmid as determined by S1-PFGE and restriction analysis. A 90-kb plasmid belonging to incompatible group FII was the major carrier of bla(CTX-M-15) in K. pneumoniae. Except bla(TEM-1), the resistance genes such as bla(SHV), bla(DHA-1), bla(OXA-1), qnrB, qnrS, aac(3)-II, and aac(6')-Ib were not found in the plasmid. In the comparing of conjugative gene sequence, it is 100% identical with the plasmid pKF3-94, which was found in K. pneumonia from Zhejiang province of china previously. CONCLUSIONS: bla(CTX-M-15) was prevalent in K. pneumonia of southern China. The dissemination of bla(CTX-M-15) appeared to be due to the horizontal transfer of a 90-kb epidemic plasmid.

PCR versus serology for diagnosing Mycoplasma pneumoniae infection: a systematic review & meta-analysis.

BACKGROUND & OBJECTIVES: Diagnosis for Mycoplasma pneumoniae usually relies on serological tests. PCR technology has some advantages but also limitations. The optimal selection for these tests still needs discussion. This paper reviews the overall diagnostic accuracy of PCR versus serological assays for diagnosis of M. pneumoniae infections and to identify factors associated with heterogeneity of results. METHODS: MEDLINE and Embase databases were searched. Articles meeting the selection criteria were retrieved for data collection and analysis. Studies were assessed for methodological quality using QUADAS. Hierarchial summary receiver operating characteristic (HSROC) model was used to estimate summary ROC curve. RESULTS: Initial meta-analysis showed a summary estimate of sensitivity (SEN) 0.62 (95% CI, 0.45-0.76), and specificity (SPE) 0.96 (95% CI, 0.93-0.98). Subgroup analyses were performed to identify factors associated with heterogeneity. For different gene targets, reference standards, subjects (children or adults) and different PCR types, these aspects can generate results of heterogeneity. The 16s rDNA target and adult subjects and real-time PCR may have better test results for PCR. INTERPRETATION & CONCLUSIONS: Commercial PCR tests generated consistent results with high specificity but a lower and more variable sensitivity. The findings suggest commercial PCR tests having superiorities in diagnosing M. pneumoniae infections but still cannot replace serology. PCR plus serology could be good screening tests for reliable and accurate diagnosis of M. pneumoniae.

[Study of detection and homology analysis of the 16S rRNA methylase in 374 enterobacteriaceae clinical isolates].

OBJECTIVE: To investigate the prevalence of genes encoding 16S rRNA methylase and the spreading path of these genes in 374 clinical isolate of enterobacteriaceae. METHODS: The genes encoding 16S rRNA methylase in 374 clinical isolate of enterobacteriaceae was detected with PCR. The sequence homogeny analyses were carried out with the NCBL BLAST program and enterbacterial repetitive intergenic consensus PCR (ERIC-PCR). The conjugation experiments were used to determine the transference of 16S rRNA methylase in vitro. RESULTS: In 374 clinical isolates, methylase genes were detected in 24 strains (6.41%), including 10 armA gene positive strains and 10 rmtB gene positive strains, and 4 strains with both genes positive. Highly homogenous strains were confirmed by ERIC-PCR. 45.8% (11/24) of the conjunction experiments for these strains were positive. CONCLUSION: The aminoglycoside resistance in enterobacteriaceae was concerned related to 16S rRNA methylase. The 16S rRNA methylase transferred either by clone or by plasmids horizontal spreading.