Rapid Identification Of Clinical Bacteria By MALDI-TOF MS And 16S RRNA Gene Sequence Analysis

PART Ⅰ Use of MALDI-TOF MS and 16 S r RNA gene sequence analysis for rapid identification of clinical bacteria that are difficult to identityObjective This study was performed to evaluate the analytical and practical performance of MALDI-TOF MS compared to the sequencing method and the Vitek 2Compact system for identification of clinical rare or difficult bacteria. The aim is to improve the identification accuracy of clinical rare or difficult bacteria, shorten the time of bacterial identification.Methods A total of 90 clinical isolates were collected in the First Affiliated Hospital of Anhui Medical University from January 2014 to December 2015.These bacteria were mainly isolated from blood, sputum, secretions, pleural fluid and urine. While using 16 S r RNA gene sequence analysis as the gold method, we compare the accuracy between MALDI-TOF MS and Vitek2 Compact. At the same time, the comparisons among MALDI-TOF MS, Vitek2 Compact and 16 S r RNA gene sequence analysis are implemented according to the identification time.Results For 90 isolates, 78 isolates(86.7%) were identified at the species level, 9isolates(10.0%) at the genus level and 3 isolates(3.3%) were unidentified by using MALDI-TOF MS; 90 isolates were amplified by 16 S r RNA gene sequence analysis successfully, 86 isolates(95.6%) were identified at the species level, 4 isolates(4.4%)were identified at the genus level; 61 isolates(67.8%) were identified at the species level, 11 isolates(12.2%) isolates at the genus level and 18 isolates(20.0%) were unidentified by using Vitek2 Compact. While 16 S r RNA gene sequence analysis was employed as the gold method, the correct identification rates for clinical rare or difficult bacteria by MALDI-TOF MS and Vitek2 Compact system were 85.6% and 57.8%,respectively, and there was statistical significance between the MALDI-TOF MS system and Vitek 2 compact system(χ2=90,P=0.000). 71.1%(64/90)of MALDI-TOF MS results were concordant with phenotypic results, 28.9%(26/90)of MALDI-TOF MS results were not concordant with phenotypic results. The time required for the identification of MALDI-TOF MS, Vitek2 Compact and 16 S r RNA sequence analysis is about 10min/strain, 8 ~ 12 h/strain and 10 h/ strain.Conclusion Compared to the Vitek2 Compact system, MALDI-TOF MS and 16 S r RNA gene sequence analysis can be the powerful tools for fast and accurate identification of clinical rare or difficult bacteria in the clinical microbiology laboratory.The identification time of MALDI-TOF MS was significantly shortened.MALDI-TOF MS can be one of the most important methods in clinical microbiology laboratory.PART Ⅱ Rapid detection of bacteria in patients with peritoneal dialysis-related peritonitis by using MALDI-TOF MS and Real-time PCRObjective This study was performed to evaluate the analytical and practical performance of MALDI-TOF MS of the 8-hour bacterial growth veil compared to the real-time PCR method and the routine blood culture method for identification of clinical bacteria causing peritonitis from peritoneal dialysis(PD) fluids.Methods(1) Application of blood culture system until the blood culture system is alarming. After plating of positive blood culture broth on three Columbia blood agars,the first Columbia blood agar was identified by Vitek 2 Compact after 18 ~ 24 h of incubation; the second Columbia blood agar was identified by MALDI-TOF MS using the 8-hour bacterial growth veil after 8 h of incubation; the third was identified by MALDI-TOF MS as positive control after 24 h of incubation.(2) Direct extraction of PD fluids DNA, the bacteria DNA were identified using real-time PCR. At the same time, the comparisons among rountine blood culture method, MALDI-TOF MS of the8-hour bacterial growth veil and real-time PCR are implemented according to the identification time.Results(1)For 80 cases, the positive rate was 68.8%(55/80) of blood culture method;In 55 cases of peritoneal dialysis fluid positive blood culture bottles, after 8h of blood agar inoculation, 55 cases were growing veil, 50 cases were identified at the species level and 5 cases were unidentified by using MALDI-TOF MS. The success rate was90.9%(50/55).89.7% of the Gram positive bacteria present had been correctly identified and 93.8% of the Gram negative bacteria present had been correctly identified. The consistency between the MALDI-TOF MS and the PD fluid culture method was 93.8%(75/80).(2) The positive rate of real-time PCR(69/80,86.3%) was higher than that of PD fluid culture(55/80, 68.8%).The difference had statistical significance(χ2=76,P=0.001). The consistency between real-time PCR and the PD fluid culture method was 82.5%(66/80). The time required for the identification of rountine blood culture method,MALDI-TOF MS and real-time PCR is about 24 ~ 48h/case, 10 ~20h/case and 2~3 h/case.Conclusion This study suggests that MALDI-TOF MS of the 8-hour bacterial growth veil can be an excellent technique for easy, rapid and accurate detection bacteria ofPD-associated peritonitis.While comparing with the PD fluid culture method, real-time PCR method can improve the positive rate of bacteria detection. Real-time PCR can be a rapid, accurate, sensitive and broad spectrum technique for rapid and accurate detection bacteria of PD-associated peritonitis.It is suitable for clinical diagnosis of peritonitis in a timely manner and rapid screening.And hence all these methods use as a diagnostic tool in future seem very promising.Researchers should select suitable method and weigh the advantages and disadvantages according to the need.