Edited by: John W. A. Rossen, University Medical Center Groningen, Netherlands
Reviewed by: Gianluca Picariello, Institute of Food Science (CNR), Italy; Alida Catharina Veloo, University Medical Center Groningen, Netherlands
*Correspondence: Qiwen Yang
Ying-Chun Xu
This article was submitted to Infectious Diseases, a section of the journal Frontiers in Microbiology
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Bloodstream infection is a major cause of morbidity and mortality in hospitalized patients worldwide, exhibiting a significant disease burden and negative economic impact (Klein et al.,
More recently, MALDI-TOF MS has revolutionized the identification of pathogens in clinical microbiology (Seng et al.,
In this study, we aimed at developing a simple, reliable, and accurate IH processing method for positive BCs in preparation for spectrometric analysis. We then compared its performance to the commercial Sepsityper™ kit, for direct identification of pathogens from positive BCs using MALDI-TOF MS (Bruker Daltonics, Bremen, Germany). The intention was to assess the possibility of fully integrating the new rapid method into the diagnostic routine of a microbiological laboratory.
The first stage of this study was the development of an in-house (IH) protocol (see below), for rapid direct identification of organisms in positive BCs using MALDI-TOF MS. This involved evaluation of the performance of the IH protocol vs. Sepsityper™ kit method, through inoculating various known bacterial and fungal species into BC bottles. In the second stage, we further tested positive clinical BCs using the IH protocol, and the results obtained by direct MALDI-TOF MS were compared with colony MS identifications.
Fifty-four blood samples (9 mL each), each from a healthy volunteer, were artificially spiked with 1 mL suspension of commonly isolated microorganisms with a final concentration of 104 CFU/mL. The organisms used were previously identified by partial 16S r
Organisms used in spiked samples.
2 | Partial 16S rRNA gene | |
2 | Partial 16S rRNA gene | |
2 | Partial 16S rRNA gene | |
2 | Partial 16S rRNA gene | |
2 | Partial 16S rRNA gene | |
2 | Partial 16S rRNA gene | |
2 | Partial 16S rRNA gene | |
2 | Partial 16S rRNA gene | |
2 | Partial 16S rRNA gene | |
Total Gram-negative | 18 | |
2 | Partial 16S rRNA gene | |
2 | Partial 16S rRNA gene | |
2 | Partial 16S rRNA gene | |
2 | Partial 16S rRNA gene | |
2 | Partial 16S rRNA gene | |
2 | Partial 16S rRNA gene | |
2 | Partial 16S rRNA gene & |
|
2 | Partial 16S rRNA gene & |
|
1 | Partial 16S rRNA gene & |
|
1 | Partial 16S rRNA gene & |
|
2 | Partial 16S rRNA gene & |
|
2 | Partial 16S rRNA gene | |
2 | Partial 16S rRNA gene | |
Total Gram-positive | 24 | |
2 | Internal Transcribed Spacer Regions | |
2 | Internal Transcribed Spacer Regions | |
2 | Internal Transcribed Spacer Regions | |
2 | Internal Transcribed Spacer Regions | |
2 | Internal Transcribed Spacer Regions | |
Total |
10 | |
2 | Partial 16S rRNA gene | |
Total Anaerobes | 2 | |
Overall | 54 |
Each organism was inoculated into 2 BC vials (BACTEC™ Plus Aerobic/F culture vial (PAV) and BACTEC™ Lytic/10 Anaerobic/F culture vial (LAV), Becton Dickinson). The BC bottles were incubated at 37°C in an automated BC machine (BACTEC™ FX400, Becton Dickinson) for 7 days until flagged positive. When the instrument indicated that a bottle was positive, Gram staining was performed and a drop was sub-cultured on agar plates, and incubated overnight at 37°C. In parallel with direct identification by MALDI-TOF MS (using two sample preparation method described below), bacteria grown on solid media were spotted onto the ground steel target plate for MALDI-TOF MS identification.
The broth from each of the positive BCs (artificially spiked) was simultaneously treated with two preparatory (extraction) methods (the Sepsityper™ kit vs. the IH protocol) prior to direct MALDI-TOF MS analysis. Detailed information of the two protocols are shown in Figures
Sample preparation from positive BCs using a Sepsityper™ kit according to the manufacturer's instructions for direct MALDI-TOF MS.
Sample preparation from positive BCs based on Gram staining using the IH protocol for direct MALDI-TOF MS. SST, serum separating tubes; SDS, sodium dodecyl sulfate; RT, room temperature.
For direct MALDI-TOF MS analysis (using Sepsityper™ kit and IH protocol), duplicate 1 μL of extracted protein supernatant from the last centrifugation were applied to the polished steel target plate and once dried, were immediately overlaid with 1 μL α-cyano-4-hydroxycinnamic acid (HCCA) matrix (Bruker Daltonics, Bremen, Germany) solution before MALDI-TOF MS analysis. The final score used for interpretation for each sample was the higher one from two measurements. For MALDI-TOF MS analysis after BC subculture (standard method), a small portion of a single colony (after 24 or 48 h of incubation) was smeared onto the ground steel target plate using a wooden cocktail stick, and covered with 1 μL HCCA matrix solution immediately. Measurements were performed with the Bruker Biotyper MALDI-TOF MS system using FlexControl 3.3 and MALDI Biotyper V.3.3.1.2 software (Bruker Daltonics) as previously described (Zhou et al.,
Two sets of criteria were used to analyze the identification results. Firstly, all results were evaluated according to the manufacturer's values recommended for identification after culture on solid media (traditional or standard cut-off values). Briefly, a score of <1.7 was interpreted as “no” identification, a score of 1.7–2.0 as identification to genus level, and a score of ≥2.0 as identification to species level. The results (identification scores) were then further evaluated according to modified cut-off values as per the manufacturer's recommendation for Sepsityper™ kit protocol. For bacteria, scores of 1.8 and 1.6 were considered as acceptable identification to species and genus levels (Nonnemann et al.,
Based on these two criteria, the identification results were classified into the following categories: (i) correct species identification, (ii) correct genus identification, and (iii) misidentification or “no” identification.
In order to assess the utility of our IH protocol for routine clinical lab use, we analyzed its performance in clinical BCs. From June 1st to October 31st 2016, positive BCs for each bacteraemic episode in patients from Peking Union Medical College Hospital (PUMCH) were prospectively enrolled. Positive BCs from the same patient separated by more than a week were considered as different episodes. The positive BCs were Gram stained and sub-cultured for colony MALDI TOF MS identification. Furthermore, direct MALDI-TOF MS identification was performed but only using the IH protocol and the modified cut-off value interpretations. Discordant identification results between the direct MALDI-TOF MS result and the sub-cultured colony protocol, were resolved by molecular sequence (partial 16S rRNA gene,
When microscopy of Gram stain on the clinical BCs revealed more than one morphology, or subculture on solid media showed polymicrobial growth, direct MALDI-TOF MS profiles were compared to the database using the MALDI Biotyper MSP identification MIXED Method in addition to the Standard Method, both of which can be chosen in the MALDI Biotyper V.3.3.1.2 software. Use of the Mixed Method is recommended by the manufacturer for identification of organisms in polymicrobial BCs (Bruker, Personal communication).
A total of 54 spiked BC samples (44 bacteria and 10 yeasts) were tested as described above (Table
Of all the flagged positive BCs, accurate species, genus, mis/no-identification, was achieved in 35/52 (67.4%), 14/52 (26.9%), 4/52 (7.7%) from PAVs, and 32/40 (80.0%), 7/40 (17.5%) and 1/40 (2.5%) from LAVs respectively, using the Sepsityper™ kit. This is in comparison to 29/52 (55.7%), 7/52 (30.8%), 7/52 (13.5%), and 25/40 (62.5%), 11/40 (27.5%) and 4/40 (10.0%) using the IH protocol, respectively (Table
An overall increase in the identification rate was observed using the modified cut-off values irrespective of the method or culture vial deployed. Specifically, using the Sepsityper™ kit, accurate species identification increased from 67.4 to 82.7% for PAVs and from 80.0 to 95.0% for LAVs, compared to 55.7 to 84.6%, and 62.5 to 85.0%, using the IH protocol. Moreover, the IH method showed equal or even better performance in GP bacteria for both BC vials, and in GN bacteria for PAVs. For identification of GP bacteria in LAVs and
Based on the superior performance of the IH protocol in relation to Sepsityper™ kit, the performance of this protocol in clinical BCs was compared to the standard method (MALDI-TOF MS after subculture.) In five consecutive months, 301 positive BCs comprising 284 (94.4%) mono-microbial BCs (159 PAVs, 115 LAVs, and 10 BACTEC™ Myco/F Lytic Culture Vials (MLVs), and 17 (5.6%) polymicrobial BCs, were studied (Table
Direct organism identification by MALDI-TOF MS in clinical monobacterial blood cultures from patients.
23 | 0 | 0 | 1 | 22 | 23 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
1 | 0 | 0 | 1 | 0 | 1 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
8 | 0 | 1 | 6 | 1 | 7 | 87.5 | 1 | 12.5 | 0 | 0.0 | |
5 | 0 | 0 | 5 | 0 | 5 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
7 | 0 | 1 | 1 | 5 | 6 | 85.7 | 1 | 14.3 | 0 | 0.0 | |
1 | 0 | 0 | 1 | 0 | 1 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
1 | 0 | 0 | 0 | 1 | 1 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
Total |
46 | 0 | 2 | 15 | 29 | 44 | 95.7 | 2 | 4.3 | 0 | 0.0 |
5 | 0 | 1 | 3 | 1 | 4 | 80.0 | 1 | 20.0 | 0 | 0.0 | |
2 | 1 | 0 | 0 | 1 | 0 | 0.0 | 0 | 0.0 | 2 | 100.0 | |
13 | 0 | 0 | 10 | 3 | 11 | 84.6 | 0 | 0.0 | 2 | 15.4 | |
2 | 0 | 0 | 2 | 0 | 2 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
1 | 0 | 0 | 1 | 0 | 1 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
4 | 3 | 0 | 1 | 0 | 1 | 25.0 | 0 | 0.0 | 3 | 75.0 | |
2 | 0 | 0 | 0 | 2 | 0 | 0.0 | 0 | 0.0 | 2 | 100.0 | |
Total |
29 | 4 | 1 | 17 | 7 | 19 | 65.5 | 1 | 3.5 | 9 | 31.0 |
6 | 0 | 0 | 2 | 4 | 6 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
6 | 0 | 0 | 3 | 3 | 6 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
2 | 0 | 0 | 1 | 1 | 2 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
Total |
14 | 0 | 0 | 6 | 8 | 14 | 100.0 | 0 | 0.0 | 0 | 0.0 |
1 | 0 | 0 | 1 | 0 | 1 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
Total Other GP cocci | 1 | 0 | 0 | 1 | 0 | 1 | 100.0 | 0 | 0.0 | 0 | 0.0 |
2 | 1 | 1 | 0 | 0 | 0 | 0.0 | 1 | 50.0 | 1 | 50.0 | |
3 | 2 | 1 | 0 | 0 | 0 | 0.0 | 1 | 33.3 | 2 | 66.7 | |
1 | 0 | 1 | 0 | 0 | 0 | 0.0 | 1 | 100.0 | 0 | 0.0 | |
4 | 0 | 0 | 1 | 3 | 4 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
1 | 0 | 0 | 1 | 0 | 1 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
1 | 0 | 0 | 0 | 1 | 1 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
Total GP rods | 12 | 3 | 3 | 2 | 4 | 6 | 50.0 | 3 | 25.0 | 3 | 25.0 |
Total GP bacteria | 102 | 7 | 6 | 41 | 48 | 84 | 82.4 | 6 | 5.9 | 12 | 11.7 |
3 | 0 | 0 | 0 | 3 | 3 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
69 | 0 | 0 | 11 | 58 | 69 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
3 | 0 | 0 | 0 | 3 | 3 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
9 | 0 | 0 | 2 | 7 | 9 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
2 | 0 | 0 | 0 | 2 | 2 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
40 | 0 | 0 | 7 | 33 | 40 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
3 | 0 | 0 | 1 | 2 | 0 | 0.0 | 3 | 100.0 | 0 | 0.0 | |
Total |
129 | 0 | 0 | 21 | 108 | 126 | 97.7 | 3 | 2.3 | 0 | 0.0 |
11 | 2 | 1 | 6 | 2 | 8 | 72.7 | 1 | 9.1 | 2 | 18.2 | |
2 | 0 | 0 | 2 | 0 | 2 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
1 | 0 | 0 | 1 | 0 | 1 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
1 | 0 | 0 | 1 | 0 | 1 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
1 | 0 | 0 | 0 | 1 | 1 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
11 | 0 | 0 | 0 | 11 | 11 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
4 | 0 | 1 | 2 | 1 | 3 | 75.0 | 1 | 25.0 | 0 | 0.0 | |
1 | 0 | 0 | 0 | 1 | 1 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
Total Non-fermenting bacilli | 32 | 2 | 2 | 12 | 16 | 28 | 87.4 | 2 | 6.3 | 2 | 6.3 |
3 | 3 | 0 | 0 | 0 | 0 | 0.0 | 0 | 0.0 | 3 | 100.0 | |
2 | 2 | 0 | 0 | 0 | 0 | 0.0 | 0 | 0.0 | 2 | 100.0 | |
Total Other GN bacilli | 5 | 5 | 0 | 0 | 0 | 0 | 0.0 | 0 | 0.0 | 5 | 100.0 |
Total GN bacteria | 166 | 7 | 2 | 33 | 124 | 154 | 92.8 | 5 | 3.0 | 7 | 4.2 |
1 | 0 | 0 | 0 | 1 | 1 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
1 | 0 | 0 | 0 | 1 | 1 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
1 | 1 | 0 | 0 | 0 | 0 | 0.0 | 0 | 0.0 | 1 | 100.0 | |
2 | 0 | 0 | 0 | 2 | 2 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
Total Anaerobes | 5 | 1 | 0 | 0 | 4 | 4 | 80.0 | 0 | 0.0 | 1 | 20.0 |
1 | 0 | 1 | 0 | 0 | 1 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
1 | 1 | 0 | 0 | 0 | 1 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
4 | 0 | 1 | 3 | 0 | 4 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
2 | 0 | 1 | 1 | 0 | 2 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
1 | 1 | 0 | 0 | 0 | 0 | 0.0 | 0 | 0.0 | 1 | 100.0 | |
2 | 1 | 1 | 0 | 0 | 2 | 100.0 | 0 | 0.0 | 0 | 0.0 | |
Total Yeast | 11 | 3 | 4 | 4 | 0 | 10 | 90.9 | 0 | 0.0 | 1 | 9.1 |
Overall | 284 | 18 | 12 | 78 | 176 | 252 | 88.7 | 11 | 3.9 | 21 | 7.4 |
Only samples with discrepant results between direct and colony MS identification, and all VGS, were subjected to gene sequencing. A total of 20 such cases, including three
Among the 284 monomicrobial BCs, 102 were GP, 166 GN, 5 anaerobes, and 11 yeasts, representing 27 genera and 51 species or groups (Table
Of the 17 polymicrobial BCs, direct MALDI-TOF MS correctly reported a single species present in all the polymicrobial BCs, with 16/17 (94.1%) correctly identified to the species level and 1/17 (5.9%) to the genus level, under the Standard mode. However, using the MIXED Method, which the manufacturer recommends for suspected mixed organisms, two species were identified in 9/17 (52.9%) of all the episodes, among which 4/17 (23.5%) were accordant with colony MS results, and 5/17 (29.4%) were partially correct. The remaining cases with only one species identified, presented similar results to the Standard Method (Table
Direct identification by MALDI-TOF MS in clinical blood cultures containing more than 2 organisms.
1 | GP | 1.939 | 1.939 | |||
2 | GP | 2.067 | 2.067 | |||
3 | GP | 2.034 | 2.435 | |||
4 | GP | 1.938 | 1.938 | |||
5 | GP | 1.888 | 1.888 | |||
6 | GP | 2.15 | 2.15 | |||
7 | GP | 1.727 | 1.727 | |||
8 | GN | 2.143 | 2.587 | |||
9 | GN | 2.222 | 2.604 | |||
10 | GP, GN | 2.296 | 2.563 | |||
11 | GP, GN | 2.114 | 2.214 | |||
12 | GP | 2.045 | 2.045 | |||
13 | GP | 2.009 | 2.226 | |||
14 | GP | 1.842 | 2.132 | |||
15 | GP, GN | 2.138 | 2.477 | |||
16 | GP, GN | 2.407 | 2.563 | |||
17 | GP | 1.906 | 1.906 | |||
Overall, accurate species identification was achieved in 87.4% (139/159) of PAVs, 92.1% (106/115) of LAVs, and 80.0% (8/10) of MLVs (Table
In this study, we evaluated the performance of two extraction methods (Sepsityper™ kit vs. IH method) for MALDI-TOF MS direct identification of pathogens from blood culture (BC) vials. Using the modified cut-off values which is recommended for direct BC testing protocols (such as Sepsityper™ kit), a significant increase of about 15 and 25% was achieved in the accurate species identification rate in the MALDI Sepsityper™ kit and the IH method, respectively (Tables
A major difference between the current IH protocol and previous ones (Machen et al.,
Identification problems for GP bacteria were confined to
The IH protocol performed very well in the identification of GN bacteria, with almost all the
The newly designed IH protocol also performed better in the identification of anaerobes (80%) and yeasts (90%), than previously reported (Paolucci et al.,
Another interesting feature of the current study is the use of MALDI Biotyper MSP MIXED Method to identify organisms in polymicrobial infections. In most laboratories, Gram staining positive BCs remains a cornerstone in diagnostics until subcultures are available, but lacks high specificity. In this study, six of the 17 polymicrobial BCs contained both GP and GN bacteria, but only five showed concordant Gram staining results. In the majority of the cases, the most abundant organism detected by Gram staining was the one identified by MALDI-TOF–MS using the Standard Method (Table
Our study also revealed that irrespective of the type of BC bottle used, the identification accuracy of GN using the MALDI-TOF assay, is always better than that of GP bacteria. Studies in the performance of different BC bottles in the identification of different organisms, have yielded contrasting results. In this study, LAVs showed a significantly higher detection rate than the other two, which is in agreement to a previous study (Almuhayawi et al.,
In a laboratory setting, the IH method is far more cost effective than the Sepsityper™ kit, costing about $1.5 per sample compared to $7 for the Sepsityper™ kit (Caspar et al.,
Our study has some limitations. First, there is a possible selection bias as all data was from a single center with imbalance in group/species distribution. Second, we did not compare the performance of the IH protocol vs. Sepsityper™ kit in clinical BCs, which would be considered a weakness of the study. Further evaluation using the Sepsityper™ protocol needs to done for clinical samples. However, we compared the two protocols using simulated BCs and demonstrated the superior performance of the IH protocol. Therefore it made sense to compare the performance of the IH protocol to the routinely used MALDI-TO Ms colony method. Third, our protocol is somewhat more laborious and time-consuming compared to the Sepsityper™ kit and other IH protocols (Jakovljev and Bergh,
The quick identification of organisms in BCs even without antimicrobial susceptibilities could help clinicians make patient-tailored treatment more accurately, reducing the risk of potential development of resistance and possible side effects due to empirical broad-spectrum antibiotic therapy. In this respect, our study provides a novel sample preparation method for direct identification of pathogens from positive BCs with easy performance and low additional costs compared with the Sepsityper™ kit. The protocol exhibited an overall equal or even better performance than Sepsityper™ kit especially for yeasts, and showed better performance for GN bacteria than GP bacteria, and for LAVs than PAVs.
This study was carried out in accordance with the recommendations of Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Medical College Hospital ethics committee with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Medical College Hospital ethics committee.
MZ, QY, and YX conceived and designed the experiments, performed the experiments, analyzed the data, and wrote the paper. TK and FK revised the paper critically for important intellectual content. LS, RZ, MX, CL, SY, and YZ read and approved the final version of the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AV and handling Editor declared their shared affiliation.
The Supplementary Material for this article can be found online at:
matrix-assisted laser desorption ionization-time of flight mass spectrometry
viridans group streptococci
coagulase negative staphylococcus
Peking Union Medical College Hospital
in-house
blood culture.