Why maldi

Due to these differences in analytical processing, the numeric confidence for a given identification is not directly comparable between the two systems. Regardless of these differences, these platforms are equally accurate, specific, and reproducible They are both capable of identifying the vast majority of organisms commonly encountered in the clinical laboratory.

Over time, the platforms have gotten progressively better, with significant improvements in the software, interpretive rules, and databases. As a result, there is limited value in comparing results for any category of organisms using a retrospective review of the literature.

One of the difficulties that arises when identifying organisms based on traditional or molecular methods is that it can be difficult to discriminate among species that are phenotypically, biochemically, or even genetically similar. Depending on the organism, this may mean that similar species are grouped together e.

Thus, it provides a more reliable means of discriminating one species from another. This is especially beneficial for organisms in which an incorrect identification or lack of a species level identification could have a significant clinical impact. This could include species that are predictably resistant to specific antibiotics, those that have limited therapeutic options, and those in which clinicians base their therapeutic decisions on identification alone because susceptibility testing is not widely performed.

Among the organisms that are particularly difficult to identify to the species level using traditional methods, but readily identified by MALDI-TOF MS, are the coagulase-negative staphylococci and bacteria with complex nutritional requirements such as the nutritionally variant streptococci and organisms in the HACEK group Haemophilus, Aggregatibacter previously Actinobacillus , Cardiobacterium, Eikenella, Kingella.

Similarly, discrimination among species within the S. Given the importance of correctly identifying S. These fastidious gram-positive cocci, with non-specific colony morphology, are normal flora of the oral cavity but can cause invasive infections including endocarditis. Given that MALDI-TOF MS can be performed directly from blood culture bottles 38 , this method also has the potential to drastically reduce the time to diagnosis of invasive infection for these hard to identify organisms.

For example, reporting of skin and peri-prosthetic joint infections due to Staphylococcus lugdunensis , a member of the coagulase-negative staphylococci, appears to have increased due to the use of MALDI-TOF MS This has also been the case of urinary tract infections caused by Aerococcus urinae and other rare uropathogens Aerococcus urinae is a catalase negative, Gram-positive cocci that often forms pairs, chains, or triads and looks similar to streptococci not only on gram stain but also when grown on blood-agar.

Further, biochemical methods are frequently unable to discriminate Aerococcus species from each other or other Gram-positive bacteria Given the difficulty in accurately identifying A. As was the case described by von Rotz et al. Given that L. Upon identification, both patients were switched to appropriate antibiotics and recovered without further incident. Anaerobic organisms typically have slow doubling times. They are also not very biochemically active and often require a large amount of biomass for definitive identification using biochemical methods.

Thus, the length of time it takes to get to a final identification using traditional methods is largely spent on growing the organism, not on performing analytical testing. This can delay the time to diagnosis significantly. For yeasts, faster identification can significantly improve clinical outcomes.

In immunocompromised hosts, invasive yeast infections typically caused by C. Thus, as the antifungal susceptibilities are predictable based on the species, timely identification can reduce the time to appropriate empiric therapy.

This has been shown to lead to improved clinical care and reduction in the length of hospitalization 44, While the initial cost of the instrument is high, the cost savings on reagents and labor can offset the expenditure within a few years 4. It is anticipated that as the technology improves and more species are reliably identifiable, additional cost savings may be realized.

However, there are some exceptions. The inability to discriminate between related species can be due to the inherent similarity of the organisms themselves. This is likely because these may not be two species, but actually one, as has been suggested by taxonomists Other examples includes members of the B.

For inherently similar organisms, it is common to report to the group, complex or genus level. In cases where differentiation to the species level is clinically necessary, supplemental testing should be performed.

In the future, the addition of proteomic based approaches to the typical MALDI-TOF MS system may improve the discriminatory power of this method and make it possible to identify organisms at the strain or serotype level Another reason similar species may be incorrectly identified is a lack of sufficient spectra in the database.

In these cases, it is possible to get an incorrect species-level identification or no identification. For example, one study found that an misidentification was common for similar Trichophyton species Additionally, misidentification can occur when some members of a species complex are in the database, but others are not. For example, in a study by Body et al.

While there are cases where misidentifications such as these do not pose a clinical risk, in other instances there can be significant clinical impact.

For example, the inability to discriminate the subspecies of M. Database updates or user created libraries can usually overcome this problem, as has been the case for some anaerobes, including Bacteroides, Fusobacterium , and Lactobacillus species Alternatively, using back up methods such as sequencing can be an effective as long as the issue is known. In conclusion, the introduction of MALDI-TOF MS into the clinical laboratory has brought more timely and accurate identification of microorganisms with subsequent improvement in diagnosis and reduction in the time to appropriate therapy.

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Wolters, M. Wunschel, D. Methods 62, — Yaman, G. Yates, J. This is likely due to the amount of bacteria available after short incubation period Fig. Indeed, using fold dilutions of E. A lower amount of bacteria may, however, be identified using a different identification algorithm. Hsieh et al. This study opens new perspectives for the direct identification of low abundant bacteria located in mixed flora without the prerequirement of bacterial isolation and culturing.

The inoculums were prepared from a sample with a turbidity of 4. Protein extraction was performed by directly mixing the samples with formic acid on microplate. The results show that the quality of the spectrum and thus the performance of identification are largely dependent on the sample amount spotted on the microplate.

Usually, at least 6 h of incubation are required to get sufficient amount of material to obtain an efficient MALDI-TOF MS identification c Cumulative percentage of MALDI-TOF identification obtained from Gram-negative [ Escherichia coli 13 , Pseudomonas putida 2 , Klebsiella pneumoniae 2 , Enterobacter cloacae 1 ] and Gram-positive [ Staphylococcus epidermidis 13 , Staphylococcus aureus 9 , Streptococcus pyogenes 4 , Streptococcus pneumoniae 1 , Staphylococcus hominis 1 ] bacteria after short-time plating on agar during 2, 4, 6, and 8 h, respectively.

The number of samples for each bacterial species analysed is indicated in brackets. Fungal identification still largely relies on phenotypic traits. However, a few days are necessary to obtain mature fungi with phyalids phyalids are conidiogenous cells observed in a type of fungal asexual reproduction leading to the production of conidia.

This time delay may be important given the morbidity and mortality of fungal infections, especially common and life threatening among neutropenic patients.

In , Welham et al. Three fungal species, Penicillium spp. Since then, many studies have demonstrated the usefulness of the MALDI-TOF application for the identification of various fungal groups such as penicillia, aspergilla, Fusarium , Trichoderma , and dermatophytes.

However, until now, MALDI-TOF MS is mainly used for the routine identification of yeasts whereas further development has to be accomplished in database libraries and sample preparation protocols to implement this identification approach to other group a of fungi such as filamentous fungi and dermatophytes.

Marklein et al. In a study performed by van Veen et al. The suitability of the two commercially available MALDI-TOF MS systems, Bruker and Shimadzu, and their respective associated softwares and databases, Biotyper and Saramis, was tested for rapid species identification of yeasts in a clinical diagnostic approach Bader et al. Based on isolates that were contained in the respective database, no misclassifications were seen with Saramis and fewer misidentifications were reported by the Biotyper compared with classical approaches.

This library was challenged with clinical isolates. Three isolates gave no spectral score as no reference spectrum were included in the database library. Of the remaining clinical isolates, In summary, the use of MALDI-TOF MS for the identification of clinically relevant yeasts is rapid and accurate providing that the database is constructed with a comprehensive collection of accurately identified reference strains. Hettick et al. Using an extraction method similar to that used for Penicillium species, Hettick et al.

It was also pointed out that Aspergillus niger could not be distinguished from Aspergillus chevalieri. The authors concluded that the identification of Aspergillus spp. A study showed that different species of Aspergillus , including aflatoxigenic and nonaflatoxigenic spp.

However, the authors reported certain discrepancies due to the difficulties encountered to discriminate the spectra obtained with some of the analysed species. A database including the reference spectra of 28 clinically relevant Aspergillus species was engineered in a recent study by including species-specific fingerprints of both young and mature colonies of reference strains Alanio et al. The performance of the database was tested on clinical and 16 environmental Aspergillus isolates resulting in a This study has demonstrated that a complete fingerprint database including spectra from both young and mature fungal colonies makes MALDI-TOF a robust method for Aspergillus species identification regardless of the maturity of the tested isolates.

The identification of multiple Fusarium spp. In the study by Marinach-Patrice et al. Following updating of the BioTyper database with 13 strains of five Fusarium spp.

Only one Fusarium pseudonygamai isolate was misidentified and four Fusarium isolates were not identified due to absence of reference spectra in the database. The most important clinical fungal dermatophytes species, T. Except for one T. Similar to bacteria, absence of identification or misidentification of fungal species by MALDI-TOF MS analysis are essentially due to absence, mistakes or incomplete reference spectra in the database Table High quality MS spectra are usually easily obtained with both fungal hyphae and spores following the manufacturer's instruction or based on the recommendation of reference studies.

In the study of Bizzini et al. The spectra of several Penicillium spp. However, Valentine et al. Most of the studies showing that the MALDI-TOF MS identification is a powerful system for the characterization and identification of fungi have built and used their own reference spectra database and have developed their own sample preparation techniques.

There is thus still a lack of standardized extraction protocols regarding filamentous fungi. In addition, the spectral signal of filamentous fungi may be strongly influenced by the phenotype of the fungus including basidiospore, monokaryon, dikaryon, fruiting body, surface mycelium, strands and substrate mycelium. Moreover, vegetative mycelium grown on agar shows multiple zones that correspond to different ages or developmental stages.

These variations may thus influence the spectral reproducibility of the same isolate and a comprehensive database of filamentous fungi should include MS fingerprints of several different developmental forms to guarantee high yields and accuracy of identification as demonstrated by Alanio et al.

Given the accuracy of MALDI-TOF for bacterial identification, this technology might be directly applied to some clinical samples, such as blood, urine, cerebrospinal fluid, pleural fluid, peritoneal liquid, and synovial fluid. To circumvent this difficulty, large volumes are used for blood and urine and an additional enrichment by culture is available for blood see paragraphs below. Regarding cerebrospinal fluid, Nyvang Hartmeyer et al.

However, practically, bacterial identification from cerebrospinal fluid, strongly limited by the low bacterial load and the limited volume available, is yet not applicable in routine diagnostic laboratories. Bloodstream infection, septic shock, and endocarditis represent severe diseases with important mortality and morbidity.

Blood culture represents the best way to establish the etiology of such infections and to guide antimicrobial treatment. This is important since rapid and appropriate antimicrobial therapy is pivotal to reduce poor outcome Kollef,.

The rapid notification of the Gram stain result from positive blood culture has also a positive impact for adaptation of antimicrobial regimen Munson et al. Consequently, the precise identification of a microorganism isolated from positive blood culture early after Gram stain notification will likely help clinician to better adapt the antimicrobial therapy. As an example, the impact on the choice of the antibiotic will likely be significant when Gram-positive cocci are identified from blood cultures, as the antibiotic susceptibility of Enterococcus faecium is clearly different from that of alpha-hemolytic streptococci.

In practice, blood samples are inoculated into bottles containing broth media and incubated in automated instruments monitoring CO 2 concentrations released during bacterial growth. In our laboratory, Enterobacteriaceae , P. Such bacterial concentration might be adequate to allow accurate bacterial identification using MS.

However, the blood culture bottle fluid represent a complex solution with multiple nonbacterial proteins isolated from patient's blood and nutrient growth media. The preparation of a bacterial pellet from positive blood culture includes a differential centrifugation step to discard blood cells, an erythrocyte lysis step and a subsequent washing step to remove additional nonbacterial components.

The results varied according to the bacterial pellet preparation protocol and the type of microorganism present in blood cultures. Similar to MALDI-TOF identifications from pure microbial isolates, several hypotheses have been suggested to explain discordant results obtained from blood cultures: 1 the close relatedness of the different species especially within Streptococci, notably within S. In addition, the presence of capsules in different species S.

Adapted from Carbonnelle et al. In one study Ferreira et al. This poor performance is attributed to the relatively low load of fungi observed in positive blood culture and to the presence of residual blood protein which co-migrates during the MALDI-TOF assay, which impairs the performance of the diagnostic algorithm.

To circumvent this detrimental effect, a reference database of fungi obtained from blood culture spiked with fungi was established to obtain correct identification at the species level Marinach-Patrice et al.

The impact of the broth on the spectral quality and thus on the rate of identification has been analysed in several studies. In this system, charcoal is used to inactivate antimicrobial agents present in the patient's blood. The importance of the protein extraction method was compared with the so-called intact cell method, which consist in the direct deposition on MALDI plate of bacterial pellet obtained from positive blood culture.

In our laboratory, we now use this approach on a routine basis, with a turnaround time estimated to be about 1 h. However, to be efficient and have such a low turnaround time, there is a need to prioritize identification of bacteria isolated from blood cultures over other routine applications of the MALDI-TOF.

Thus, this activity somehow delays other microbial identifications, as shown in Fig. Importance of the organization of a working day to optimize time to results. Urgent samples such as blood cultures are directly processed while colonies identification from agar cultures are processed by batch.

In conclusion, the application of MALDI-TOF identification to microorganism pellets obtained from positive blood culture allows a rapid identification of microorganisms growing in blood culture which is important for the management of bloodstream infections. Using two consecutive centrifugation steps low speed to remove leukocytes and high speed to collect the bacteria , Ferreira et al. When investigating a simplified protocol in our laboratory for the identification of E.

However, the yield was poor with lower bacterial load Fig. Given the huge amount of urine processed on a daily basis, the low value of early identification and the requirement of bacterial isolation in pure culture for antibiotic susceptibility testing, it appears that the MALDI-TOF on urine is not cost-effective and not efficient enough to be implemented directly on urine samples. Indeed, as most urinary isolates are E. Escherichia coli identification yield in urine samples.

Five milliliters of urine samples positive by microscopy were centrifuged to concentrate and collect the bacteria. The graph shows the identification yield according to the bacterial load per mL. Problems 1 and 2 , due to intrinsic bacterial properties, may only be circumvented by the development of specific extraction protocols and problems 3 and 4 might be prevented by an adequate quality program. The performance of the extraction step and of the MALDI-TOF mass spectrometer may be checked by routinely testing a few selected bacterial strains, for which spectra are available in the database.

This control should ideally be done in parallel with and without a specific extraction step. We thus implemented in our laboratory routine internal quality controls that test the quality of the extraction step on two different bacterial species E.

To set-up this quality control, we first investigated the reproducibility of the extraction step see Fig. Then, we routinely tested once a week both bacterial species. As score values were always above 2 and to obtain a better expression of the quality of the extraction, we decided to report the proportion of conserved peaks detected, considering a peak as present only when its intensity was above Indeed, the rate of detection of conserved peaks reflects not only the quality of the sample but also the protein yield and the spectral quality Fig.

Escherichia coli and Staphylococcus aureus quality controls fingerprints were compared with a set of conserved peaks 81 and 80, respectively. MALDI-TOF results may also be impaired by inadequate deposit of the sample on the microplate and by poor cleaning of the microplate between runs. Inadequate deposit of samples is relatively rare when starting from bacterial colonies and the learning curve is rapid with most laboratory technicians being already experts in depositing appropriate amount of bacteria after only a few training days.

However, erroneous identification may occur due to well inversions, especially when large series are processed and when stress is increased by human resources shortage. Bruker commonly propose to use trichlorofluoroacteate TFA or guanidium to clean microplates between usages. This protocol, initially proposed by Bruker, which mainly uses ethanol and mechanical cleaning of target plates, is, however, insufficient to properly clean MALDI-TOF microplates.

Such accurate identifications obtained after plate cleaning could rarely correspond to wells where some material was still present Fig. Thus, a systematic control of the microplates should be done and the cleaning protocol adapted when necessary.

Noteworthy, disposable microplates are now also available for Bruker users. To identify possible technical problems and to recalibrate the MS apparatus, we routinely use in Lausanne the calibration control proposed by Bruker. This control called BTS consists of lyophilized E.

Finally, in the future, external quality control should be implemented. Appropriate maintenance Fig. S1 is also essential to warrant accurate bacterial identification. S1c and d. Carbonization of bacteria embedded in the matrix material following each laser pulse is also a source of concern, as the laser source may be soiled Fig.

With the Bruker instrument, the level of dirt present on the laser source may be indirectly estimated according to the number of shots needed to obtain a correct identification. Despite adequate maintenance and correct procedures, some microbial groups will repeatedly be misidentified due to poor content of some databases. Thus, it appears critical not only to implement a quality control program targeting routine procedure but also to incrementally improve the quality of the database.

The MALDI-TOF MS approach represents a new tool that has the potential to replace conventional identification techniques for a majority of routine isolates encountered in clinical microbiology laboratories. These studies showed that the MALDI-TOF technique has a high accuracy for most microbial identifications and performed equally as well as or better than conventional techniques.

For instance, van Veen et al. Similarly, Bader et al. The most striking differences between MALDI-TOF technique and conventional identification methods are observed in the estimated time and costs required for sample identification. The expensive prices of MS instruments are comparable with other common bacteriology laboratory equipment such as automated blood culture and 16S sequencing devices but the running costs are significantly cheaper than those of conventional identification methods.

Compared with conventional identification methods, MALDI-TOF has been shown to confer in most cases a significant gain of both technician working time preanalytical procedure to prepare samples and turnaround time automated analytical procedure to obtain results. The time needed for bacterial identification from intact cells was 6—8. When an extraction step is required, Bizzini et al.

The time effectiveness gained with MALDI-TOF identification compared with classical identification approaches is even more accentuated when several isolates are analysed in parallel. As reported by Cherkaoui et al. The MALDI-TOF identification procedure from single yeast colonies on the agar plate was generally completed within 10 min per isolate and within 3 h for 96 samples.

In contrast, the identification of germ tube-negative Candida species by phenotypic methods can require incubation periods of up to 72 h, a significantly longer turnaround time compared with MALDI-TOF. Molecular approaches have been or are currently under development to provide efficient identification of yeasts with a more rapid and reliable efficiency than classical phenotypic methods.

Taxonomy is the systematic classification of organisms based on their phenotypic, genetic and phylogenetic characteristics. Thus, various phenotypic approaches morphology, biochemical reactions, and sugar assimilation have been used by microbiologists to classify microorganisms. However, genome analysis through sequencing of bacterial genes or of the entire genome currently represents the gold-standard of microbial taxonomy, although it should always be confronted to phenotypic traits in a polyphasic approach.

Relatively few biomarkers 5—10 peaks are usually required for the identification of microbial isolates at the species level, whereas a much larger number of reproducible peaks is needed for subspecies identification Dieckmann et al.

Microbial typing and thus microbial characterization at the subspecies level required very different sample preparation and analytical procedures Murray,.

As mentioned previously in this review, accurate sample preparation is generally unnecessary for microorganism identification but for strain typing and subspecies identification, a rigorous optimization of testing parameters appears to be crucial.

The challenge is to obtain a sufficient number of reproducible markers with specificities below species-level specificity Rupf et al. For instance, the sample preparation procedure whole cell or protein extraction , the protein concentration, the type of matrix, the sample : matrix ratio, the concentration of acid added to the matrix and the growth medium are examples of technical parameters that can have a significant influence on the MALDI-TOF spectral profile of biomarkers Vargha et al.

The choice of analysis solutions used to process mass spectra can have a significant impact on the power of discrimination and thus on the ability to distinguish closely related isolates.

Maximizing reproducibility is also critical for accurate microbial characterization. Several similarity coefficients can be used to determinate level of similarities. The chosen similarity coefficient affects the reproducibility and the discriminatory power of the method. Several studies have demonstrated that the Pearson coefficient appears to be more adequate for the correct classification of microbial isolates. A study by Giebel et al. Using optimal sample preparation and MALDI conditions for discrimination at the strain level and by using the Pearson coefficient, Vargha et al.

For instance, members of the A. In some cases, the identification of multiple or single unique subspecies biomarkers have been used to discriminate closely related microbial isolates exhibiting highly similar mass signatures. For instance, five unique and conserved biomarkers ions were identified in environmental E.

Similarly, several Listeria monocytogenes serotypes could be separated using discriminating peaks Barbuddhe et al. Despite an increased level of complexity required for microbial subspecies classification, several published studies support the observation that MALDI-TOF MS represents a new promising technological approach for the classification of clinical and environmental isolates.

Dieckmann et al. They found that out of three matrix mixtures, SA produced the most informative spectra by providing a significant increase of high molecular mass peaks with important subspecies specificity. In addition, simple clustering of mass data from bacterial fingerprints did not initially provide a clear discrimination of the strains at the subspecies level and a bioinformatic approach recently published by Teramoto et al.

The approach is a new phylogenetic classification method based on ribosomal protein profiling by MALDI-TOF MS using the bioinformatics-based method for rapid identification of bacteria published by Demirev et al. Using this approach, the result of the classification of several P. The determination of serotypes of Shiga toxin-producing E.

The prototype spectra were generated by removing masses with low discriminative significance, which is a process comparable with the generation of super-spectra proposed by the Saramis software. The generation of prototype spectra allowed a reduction of incorrect assignments down to 0. Unlike restriction fragment length polymorphism analysis, this analytical methodology could not achieve a differentiation below the serotype level.

The typing of several microorganisms, such as Staphylococcus and Listeria species, for epidemiological studies require the use of various conventional techniques such as pulsed-field gel electrophoresis PFGE , amplified fragment length polymorphism analysis and multilocus sequence analysis MLSA. These gold-standard techniques provide accurate classification of microorganism but suffer from important time and cost investments.

In addition, these methods are technically relatively complex and have to be usually performed by experienced technicians. Thus, compared with conventional antimicrobial susceptibility test methods or gene sequencing techniques, these studies have demonstrated that MALDI-TOF MS represents a fast and cheap approach to accurately differentiate S.

However, Barbuddhe et al. Similarly, Fujinami et al. Hazen et al. Overall, the ultimate goal would be to use MALDI-TOF for a rapid prospective typing at the time of identification, which should significantly benefit to hospital epidemiology and to infection control measures that have to be applied to prevent dissemination of pathogens.

These studies demonstrate that for several microbial species, minor changes in standardized procedures such as improved algorithm and user-friendly softwares applied in routine diagnostics will allow the use of MALDI-TOF MS for rapid and inexpensive microbial typing. This could significantly improve the approaches currently used to monitor epidemiological outbreaks and pathogen surveillance.

This explains why MALDI-TOF MS can be successfully used in clinical diagnostic laboratory for microbial identification starting from subcultures on agar plates and broth media but also directly from positive blood cultures and to a lesser extent from clinical samples such as urine.

The application of MALDI-TOF at the subspecies level in typing is promising but still needs further improvement including instrument sensitivity, database quality, and postrun analysis methods. Overall, a MALDI-TOF MS will be soon present in most diagnostic laboratories as, despite the significant cost of the instrument and for maintenance, running costs and consumables are much lower than those for other conventional methods, rendering this technology a worthy quantum leap tool.

We thank Myriam Corthesy for technical support. Nature : — Google Scholar. Clin Microbiol Infect 17 : — Anal Chem 47 : —