| Objective:1.The detection situation of hypervirulent klebsiella pneumoniae(hvKP)strains in the Affiliated Hospital of Chengde Medical College from November 2020 to October 2021 was analyzed.2.To evaluate the ability of matrix-assisted laser desorption /ionization time-of-flight mass spectrometry(MALDI-TOF MS)SARAMIS software to identify hypervirulent Klebsiella pneumoniae(hvKP)from classical Klebsiella pneumoniae(c KP)rapidly.3.To compare the clinical diagnostic efficacy of MALDI-TOF MS technique,the string test and their combined application to distinguish hvKP from c KP.Methods:1.A total of 199 Klebsiae non-repeated pneumonia strains(KP)clinically isolated from our hospital from November 2020 to October 2021 were collected according to Vitek 2.0 identification,and then identified again by VITEK MS(VITEK Mass Spectrometry,VITEK Mass spectrometry).The peg-344,rmp A and iuc A virulence genes were amplified by PCR.peg344,rmp A and iuc A were all positive at the same time as the diagnostic criteria to distinguish hvKP from c KP.The prevalence of hvKP strain in our hospital was analyzed.2.After virulence gene detection of peg-344,iuc A and rmp A,90 strains of Klebsiella pneumoniae(47 strains of hvKP and 43 strains of c KP)in our hospital were randomly selected as retrospective study objects.A screening model was established for rapid identification of hvKP and c KP,and string test was conducted to identify the hypermucoviscous phenotype.3.Preliminary experiment of optimization research index parameters:Formic acid-acetonitrile protein extraction method was used for pretreatment of the strain and then tested on the VITEK MS and collected by bio Merieux Vitek MS mass spectrometry system.20 parallel control targets were set for each sample.The protein profiles of the two groups of bacteria were collected and then introduced into SARAMIS mass spectrometry for modification,amplification and comparative analysis.The protein peaks with obvious differences were screened out by visual observation,and the parameters of the ratio of peak area to intensity(A/I)which was the lowest cumulative CV at the peaks with differences were selected as the best research indexes,which provided the basis for the establishment of the standard for rapid detection of hvKP by MALDI-TOF technology in subsequent tests.4.The establishment of hvKP screening indexes: Firstly,the protein peak maps of the two groups of strains(47 hvKP strains and 43 c KP strains)were collected according to the pre-experimental method,and the A/I values at different mass charge ratios in the above maps were calculated respectively,and the A/I values of the two groups of bacteria were imported into SPSS 26.0 software for statistical analysis and P<0.05 was considered as statistical difference.Then,the receiver operating characteristic curve(ROC)was used to evaluate the clinical diagnostic effect of the above statistically different A/I values.Finally,the combined application of research indicators with diagnostic value was selected,and compare the clinical diagnostic efficacy of single indicators and multiple indicators in distinguishing hvKP from c KP.5.Verification of hvKP screening indexes: Another 109 strains were verified by blind method to evaluate the diagnostic efficacy of each index in distinguishing hvKP from c KP.Results:1.Prevalence of hvKP strain in our hospital: In this study,peg344,rmp A and iuc A gene positive strains were used as the standard for identification of hvKP.Finally,79 hvKP strains were screened from 199 strains.The strains were mainly distributed in intensive care(22.8%),geriatrics(11.4%),hepatobiliary surgery(10.1%)and neurology(7.6%).The strains were mainly derived from respiratory tract specimens in 51 cases(64.6%),abscess in 15 cases(19.0%)and blood in 9 cases(11.4%).2.The test results of virulence gene: Peg-344,rmp A and iuc A virulence gene analysis showed that hvKP accounted for 39.7%(79/199)of all strains.In the screening model,the detection rate of hvKP and c KP strain positive were 89.4%(42/47)and 10.6%(5/47)in positive string test.The detection rate of hvKP in negative string test was 14.0%(6/43)and that of c KP was86.0%(37/43).The sensitivity and specificity of string test for identifying hvKP and c KP strains were 89.4%(42/47)and 86.0%(37/43)respectively.3.Optimize the pre-experimental results of the research index parameters: At the same mass charge ratio of hvKP and c KP groups,the protein peaks with obvious difference of A/I value were 4154±5,4185±5,4366±5 and 4765±5,respectively,With peak intensity or area as the parameter alone,The same strain was tested for 20 times in parallel,and the CV of the A/I was highest than other parameters,with the fluctuation range of 57%～88.2% and 54.5%～95.1%,respectively.The ratio of peak area to intensity(A/I)was taken as the parameter,and the cumulative CV was lowest(7%-21.2%).Therefore,A/I was taken as the reliable research index parameter.4.Establishment of hvKP screening index:At the same mass charge ratio of hvKP and c KP groups,the protein peaks with obvious difference of A/I value were 4154±5,4185±5,4366±5 and 4765±5,respectively.The A/I value of hvKP group was 49.94±2.94,38.34±5.34,55.56±5.04 and42.39±3.47,respectively.The A/I values of the c KP group were 45.79±2.52,44.40±4.72,58.55±5.07,44.64±3.65,respectively,and the differences between the hvKP and c KP groups were statistically significant(P <0.01).The results of ROC curve showed that the area under the ROC curve(AUC)of the protein peak A/I value at 4154 mass charge ratio was 0.892.When the critical point of A/I value was 47.87,the Yuden index and the diagnostic efficiency were the highest,and the sensitivity and specificity were 91.5% and 86.0%,respectively.The AUC values of A/I of other protein peaks were all less than 0.5 which indicating low diagnostic value.The location of the mass charge ratio of 4154 when A/I value ≥47.87 combined with positive in string test was hvKP,while A/I value < 47.87 and negative in string test was c KP,the sensitivity and specificity were 93.6%and 86.0%,respectively.When the two indexes were combined,the sensitivity to identify hvKP showed an increasing trend compared with that of each index alone,but there was no statistical difference.5.Verification of hvKP selected index: Each index was used to blind detect another 109 strains.According to the A/I value of the mass charge ratio 4154,the results showed that the sensitivity and specificity of hvKP and c KP were 87.5% and 85.7%,respectively.The sensitivity and specificity of string test for identifying hvKP and c KP strains were 81.3% and 84.4%,respectively.Conclusions:1.The detection rate of hvKP strain in our hospital was 39.7%.The hvKP strains in our hospital are mainly distributed in the department of intensive care,geriatrics,hepatobiliary surgery and neurology.This study revealed the epidemic characteristics of hvKP in our hospital,and provided a scientific basis for its origin,prevention and treatment,as well as clinical rational empirical medication.2.Mass spectrometry can distinguish hvKP rapidly and accurately.The A/I value of the mass charge ratio 4154 was higer than 47.87 which may be a potential indicator for predicting hvKP.In this research,the protein peak A/I value of mass spectrometry was proposed as a parameter to identify hvKP and c KP for the first time,and providing a new idea for MALDI-TOF MS to screen hvKP rapidly.3.High sensitivity and specificity can be obtained to distinguish hvKP and c KP based on MALDI-TOF MS technique,the string test and their combined application.When the two indexes were combined,the sensitivity to identify hvKP showed an increasing trend compared with that of each index alone,but there was no statistical difference. |
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