Performance of optimized McRAPD in identification of 9 yeast species frequently isolated from patient samples: potential for automation
© Trtkova et al; licensee BioMed Central Ltd. 2009
Received: 12 March 2009
Accepted: 10 November 2009
Published: 10 November 2009
Rapid, easy, economical and accurate species identification of yeasts isolated from clinical samples remains an important challenge for routine microbiological laboratories, because susceptibility to antifungal agents, probability to develop resistance and ability to cause disease vary in different species. To overcome the drawbacks of the currently available techniques we have recently proposed an innovative approach to yeast species identification based on RAPD genotyping and termed McRAPD (Melting curve of RAPD). Here we have evaluated its performance on a broader spectrum of clinically relevant yeast species and also examined the potential of automated and semi-automated interpretation of McRAPD data for yeast species identification.
A simple fully automated algorithm based on normalized melting data identified 80% of the isolates correctly. When this algorithm was supplemented by semi-automated matching of decisive peaks in first derivative plots, 87% of the isolates were identified correctly. However, a computer-aided visual matching of derivative plots showed the best performance with average 98.3% of the accurately identified isolates, almost matching the 99.4% performance of traditional RAPD fingerprinting.
Since McRAPD technique omits gel electrophoresis and can be performed in a rapid, economical and convenient way, we believe that it can find its place in routine identification of medically important yeasts in advanced diagnostic laboratories that are able to adopt this technique. It can also serve as a broad-range high-throughput technique for epidemiological surveillance.
Over the past decades, patients have increasingly been colonized and infected with a variety of yeast species mainly due to immunocompromising conditions as well as due to increased use of invasive techniques and devices (for reviews see [1, 2]). Therefore, clinical microbiology laboratories face an important challenge of rapid detection of pathogenic yeasts. However, accurate species identification is very much demanded in addition to mere detection, because susceptibility to antifungal agents, probability of resistance development and ability to cause disease vary in different species . Although there are several rapid diagnostic procedures available based mainly on PCR amplification of yeast DNA that have been developed to facilitate diagnosis, conventional cultivation techniques followed by identification of pure culture still dominate the field. A profound change can hardly be expected in the foreseeable future except for rapid detection of selected yeasts species in specific types of samples, blood in particular. This is mainly because only the identification techniques based on pure culture examination are able to identify the whole spectrum of potentially pathogenic yeast species reliably. Also, only cultivation techniques make antifungal susceptibility testing and strain typing for epidemiological purposes possible. However, diagnostic laboratories and clinicians can hardly be satisfied with the potential of routinely available identification techniques in this field because these are typically either (i) economical and easy to perform but time-consuming, or (ii) rapid but costly and/or requiring special equipment or expertise. For reviews on phenotyping- and genotyping-based systems see [4, 5].
We have recently proposed an innovative technique termed McRAPD (Melting curve of Random Amplified Polymorphic DNA) which has the potential to provide rapid and accurate pathogenic yeast identification grown in pure culture in an easy and economical way . Here we have evaluated the performance of optimized McRAPD on a broader spectrum of yeast species frequently isolated from clinical samples and also examined the potential of automated and semi-automated interpretation of McRAPD data for identification purposes. We believe that because of its advantages over conventional phenotypic approaches and its competitive costs, McRAPD can find its place in routine identification of medically important yeasts.
Crude colony lysates perform satisfactorily in McRAPD
Inter-run variability is very low, whereas inter-strain differences can be a source of considerable variability of McRAPD data in some species
Different genotypes can be recognized within species based on McRAPD data
Summary of discrepant identification results.
ID 32C identification
To see whether the strain clustering patterns resulting from McRAPD and conventional RAPD are consistent, McRAPD genotypes were color-coded by ground tint colors in the dendrogram of RAPD fingerprints using different color saturation for different genotypes (additional file 2: Dendrogram of RAPD fingerprints). Whereas McRAPD genotypes correlated very well with RAPD clustering in C. tropicalis, the correlation was limited in C. lusitaniae and no or almost no correlation was observed in C. albicans, C. krusei, and S. cerevisiae (no McRAPD genotypes were delineated in other species). This is mainly because of different data processing in conventional RAPD versus McRAPD. In RAPD, differences in overall amplification efficiency result in differences in intensity of banding patterns. Therefore, it is strongly recommended not to include weak bands into comparison of RAPD fingerprints, because these can appear or disappear in different amplification runs. Also, the relative intensity of strong bands cannot be reliably taken into account for comparison. That is why we used the band-based Jaccard coefficient for processing of RAPD fingerprints, which takes the position of a band into account but neglects its intensity. In contrast, raw fluorescence measured during melting in the McRAPD procedure truly reflects the relative representation of individual RAPD products (bands in electrophoresis) in the sample. Inter-sample and inter-run differences in overall fluorescence of samples are subsequently proportionally equilibrated during numerical normalization of melting data. Then, relative representation of individual RAPD products is reflected in the slope of a normalized curve or in the height of a peak in a derivative curve and this is taken into account during further evaluation.
McRAPD data can be used for automated species identification
Accurate identification rate achieved with different approaches to interpretation of McRAPD data.
Normalized curves + matching of derivative peaks
Visual matching of derivative plots
Matching of RAPD fingerprints
All species studied
Average melting temperatures of peaks in first derivative plots obtained in individual species/genotypes.
86.0 ± 0.22
82.9 ± 0.32
83.9 ± 0.27
87.6 ± 0.11
80.0 ± 0.44
82.0 ± 0.31
84.1 ± 0.10
85.3 ± 0.17
87.6 ± 0.12
79.9 ± 0.06
86.0 ± 0.34
82.0 ± 0.18
82.6 ± 0.30
87.6 ± 0.05
79.8 ± 0.36
83.9 ± 0.29
82.7 ± 0.27
85.0 ± 0.33
78.9 ± 0.24
82.7 ± 0.23
84.8 ± 0.50
83.0 ± 0.19
86.6 ± 0.11
84.1 ± 0.19
81.9 ± 0.12
81.2 ± 0.37
83.8 ± 0.12
79.5 ± 0.17
83.7 ± 0.23
82.1 ± 0.26
85.3 ± 0.22
87.1 ± 0.18
89.0 ± 0.36
82.8 ± 0.29
78.6 ± 0.19
85.5 ± 0.19
87.6 ± 0.19
89.2 ± 0.12
83.0 ± 0.22
78.6 ± 0.16
85.5 ± 0.18
83.9 ± 0.11
82.9 ± 0.25
85.5 ± 0.06
87.7 ± 0.10
89.1 ± 0.21
78.4 ± 0.07
85.4 ± 0.17
86.8 ± 0.15
89.1 ± 0.24
80.4 ± 0.28
82.3 ± 0.19
85.5 ± 0.10
86.9 ± 0.08
80.4 ± 0.23
81.6 ± 0.19
82.4 ± 0.19
80.7 ± 0.13
83.9 ± 0.13
85.7 ± 0.10
87.0 ± 0.09
85.2 ± 0.06
79.0 ± 0.14
82.8 ± 0.15
82.4 ± 0.12
84.7 ± 0.12
85.6 ± 0.11
86.4 ± 0.10
85.0 ± 0.16
86.0 ± 0.09
83.8 ± 0.19
88.3 ± 0.24
90.2 ± 0.16
85.1 ± 0.09
84.9 ± 0.16
82.8 ± 0.20
Therefore, we combined the two proposed approaches into one two-step approach. In the first step, the closest match was established between the McRAPD data of the unknown sample and a set of all the other McRAPD profiles in an automated way. Then, a derivative plot was checked for the presence of decisive peaks in the second step. When the examined peak was found to fit in the interval of average peak position ± 2 S.D., it was considered as matched to the average peak. If any of the average decisive peaks characteristic for the best matched species was missing in the examined strain, this best match was evaluated as incorrect identification and the second best match was further evaluated. If the automated identification suggested two very close matches with curves of different species, both concordant in decisive peaks with the examined strain, the characteristic peaks were evaluated and interpreted in favor of one of the matches. Performance of this two step approach was generally much better than the first-step approach alone, with overall accurate identification rate of 87%, varying between 72.7 and 100% in different species. Results of the evaluation are summarized in Table 2. Surprisingly, in C. tropicalis and C. pelliculosa, the two step approach showed lower sensitivity compared to the first-step alone. This indicates that in some cases the process of matching examined peaks with average decisive peaks ± 2 S.D. precludes correct identification. This is most likely because the interval defined by the position of a decisive peak ± 2 S.D. includes only 95% of the isolates of each genotype. Thus, some isolates among the remaining 5% can prove to match a closely positioned decisive peak of a different species, even if they in fact belong to the species originally suggested by first-step automated processing of McRAPD data.
Computer-aided visual matching of derivative plots shows excellent performance
Our results show that McRAPD offers a promising alternative to conventional phenotypic identification techniques. Surprisingly, simple visual inspection of derivative plots performed best among the approaches tested for interpretation of mere numerical McRAPD data. Its performance almost matched the performance of traditional RAPD fingerprinting. Compared to the automated processing developed and tested by ourselves, the time costs of simple visual evaluation were roughly equal when using a pre-made computer-aided plotting scheme. However, with a broader spectrum of yeast species and expanding database of McRAPD results, simple visual examination can become more time demanding and cumbersome. Therefore, it may be advantageous to test for a threshold score in automated matching which can guarantee flawless identification in the future. Then, the visual matching could be reserved for isolates failing to reach this score in automated matching. When looking at the accuracy of identification obtained in this study, this should be regarded critically in the light of the fact that all of the evaluations were based on an artificially assembled set of strains. However, because this set comprised almost 95% of species typically isolated from clinical samples, real performance in routine settings should not differ too much. An ongoing prospective study being performed by ourselves should prove this assumption.
When evaluating the future potential of McRAPD, we should first consider the main advantages and disadvantages of the RAPD technique itself. It is well-known that RAPD is highly sensitive not only to minor inter-strain differences, but also to minor differences in experimental conditions, which can result in different profiles, compromising intra- and interlaboratory reproducibility. There are many factors that can influence the appearance or disappearance of bands, including Mg2+ concentration, primer/template concentration ratio, Taq polymerase concentration and source, the model of thermal cycler etc. [15–18]. Since we aimed to use RAPD/McRAPD primarily not for strain typing but for species identification purposes, we optimised the amplification conditions in favour of low interstrain variability. This efficiently prevented problems with intralaboratory reproducibility, as clearly demonstrated in Figure 4 and discussed above. Of course, some problems may occur with interlaboratory reproducibility, mainly when using a different model of thermal cycler or a different Taq polymerase. However, machines using air heating and cooling of samples which secure rapid and accurate ramping and rapid cycling should be able to minimise this problem, as documented in our study using glass capillaries and RapidCycler 2 machine. Also, commercial polymerases with guaranteed performance are available globally. Therefore, we believe that these drawbacks can be at least compensated or even outweighed by the advantages of McRAPD. Firstly, RAPD itself is very easy and economical to perform, which makes it the second most widely used genotyping technique in yeast microbiology as illustrated by 92 citations in PubMed for "(RAPD OR AP-PCR) AND typing AND yeast" versus 139 for RFLP, 40 for PFGE, 30 for MLST, and 9 for AFLP. In addition, its usefulness for yeast species identification was documented by several groups independently [7, 19–23]. To the best of our knowledge, all of the other genotyping techniques are more laborious and less economical for the purpose of species identification. If there is a technology for melting analysis available, McRAPD is even easier and more economical to perform than RAPD, because it does not require gel electrophoresis. However, omitting the electrophoresis also means that a visual check of proper amplification is not possible. This can question the reliability of McRAPD results, because as in any PCR, RAPD amplification can also occur in negative controls, for reasons well documented earlier [24, 25]. Then, performance of DNA extraction can be another source of inadequate McRAPD performance, because it may not recover enough template DNA of adequate quality for amplification, opening the door for false RAPD amplification. However, this risk can be significantly reduced by applying the criterion of the relative value of fluorescence reaching a critical threshold, as used in this study. When a real-time cycler is used for amplification, a monitoring of fluorescence during McRAPD also allows for controlling the reliability of McRAPD data, because slow amplification of a specific sample as compared to standard samples clearly indicates improper performance, most likely because of the inadequate quality of template DNA. In this case, real-time amplification should reveal the failure of McRAPD even better than gel electrophoresis which can only demonstrate the end-point result of PCR amplification.
When comparing the McRAPD performance to its alternatives available in routine laboratories, we have clearly demonstrated that it performs better than conventional phenotypic identification techniques which are in addition much more time-consuming. In this study we do not provide any direct and extensive comparison to other approaches, except the limited comparison to the commercial assimilation set ID 32C. Among the 20 strains examined both by McRAPD and ID 32C, the results were concordant in 9 cases and McRAPD was superior to ID 32C in 4 strains of C. metapsilosis, whereas ID 32C was superior to McRAPD in 3 strains where McRAPD failed to suggest any identification. As already noted above, these cases of unmatched McRAPD profiles presumably represent a unique genotype represented by a single isolate among the strains included in our study. The database of standard McRAPD results is now very limited compared to ID 32C but can be expected to grow in future. This should help to resolve such cases. In addition, if McRAPD does not suggest any match or if there are any doubts about the match suggested, there is always an option of subsequent gel electrophoresis of the same sample that reveals a classical fingerprint. As clearly demonstrated in a dendrogram based on RAPD fingerprints of all strains included in the study (see additional file 2: Dendrogram of RAPD fingerprints), analysis of RAPD fingerprinting patterns always provided accurate identification except for 2 strains showing quite unique fingerprints (C. glabrata CCY 26-20-21 and C. guilliermondii I1-CAGU2-27, marked by arrows in the additional file 2: Dendrogram of RAPD fingerprints). Importantly, RAPD also identified correctly 2 of the 3 strains where McRAPD failed to suggest any identification. It should also be noted, that our study was performed with one single primer only. This primer showed very good performance with uniform melting profiles in most species, but also less uniform profiles in few other species. It can hardly be expected that one single primer can cover McRAPD identification of all medically important yeast species without problems. Thus, future studies may improve the performance of the McRAPD approach also by testing more primer systems and suggesting the best mixes. This was out of the scope of this study.
When comparing the routine processing of samples in McRAPD and ID 32C, both require pure culture of the respective yeast strain. Whereas ID 32C requires 1-3 colonies to achieve 2 ml of suspension medium showing turbidity of McFarland 2, sampling of a small fraction of one colony is enough for McRAPD as described in Materials and Methods. Concerning the time needed to achieve identification, McRAPD can be finished within 3.5 hours if simple DNA extraction is performed and a real-time cycler with high-resolution melting analysis option is available, whereas ID 32C can be read only after 24-48 hours reliably, as recommended by the manufacturer. Of course, both techniques can fail, e.g. with an unrecognised mixed culture. In such case, McRAPD repetition is completed within a few hours on the next day, whereas repeating ID 32C needs further 2 days. Concerning the labour time, McRAPD requires about 1.5 hours to process 10-20 samples, whereas ID 32C needs about 5 min to prepare a set for incubation and 1-3 min to evaluate the results per sample, i.e. about 1-2 hours to process 10-20 samples. Comparison of costs cannot be accomplished easily. Whereas McRAPD requires special and expensive instrumentation, ID 32C can be used in any cultivation laboratory without any special equipment. On the other hand, real-time PCR machines with high resolution melting option needed for McRAPD can be expected to gradually become a general must, at least in advanced routine microbiological laboratories. When comparing operation costs of both procedures, our experience shows that McRAPD can be quite competitive compared to ID 32C, however, market prices of materials and sets are always subject to change.
Thus, it should be fair to say that both approaches are roughly comparable, McRAPD being more rapid with a potential for future improvements. Since ID 32C offers the most extensive set of assimilation tests among commercially available yeast identification systems, it can be expected that other phenotyping approaches will show inferior performance. Thus, the need of special instrumentation and skills should be the only obstacle for general acceptance of McRAPD in routine diagnostic laboratories. Generally speaking, those laboratories being able to adopt McRAPD will be also able to adopt other genotyping techniques. Then, such techniques, Multi Locus Sequence Typing (MLST) in particular, should be the main competitors of McRAPD. Although MLST is more demanding concerning instrumentation, skills and labour, it has the advantage of unmatched interlaboratory reproducibility, enabling global epidemiology. However, it can hardly be expected that MLST can present an economically affordable alternative for routine identification and prospective epidemiological surveillance in near future. It can rather be expected that its use will be limited to retrospective epidemiological studies. Thus, McRAPD offers a promising choice for routine identification of pathogenic yeast species; in case of failure, it could be supplemented by other techniques, the best of which appears to be single-locus sequencing in our opinion.
1. Crude colony lysates provide an economical, rapid and reliable alternative to elaborate DNA extraction techniques for the purposes of McRAPD when performed by skilled personnel.
2. Our optimized McRAPD protocol shows excellent intralaboratory reproducibility and is able to delineate specific genotypes in some of the species studied.
3. Computer-aided visual matching of first derivative plots shows best performance among the approaches tested for interpretation of mere numerical McRAPD data. Its performance almost matched the performance of traditional RAPD fingerprinting and was comparable to the performance of the ID32C commercial system.
4. We believe that because of its advantages over conventional phenotypic identification approaches and competitive costs McRAPD can find its place in routine identification of medically important yeasts in advanced diagnostic laboratories being able to adopt the technique. It can also serve as a broad-range high-throughput technique for crude epidemiological surveillance.
The 9 yeast species most frequently isolated from clinical samples in our settings, namely representing 94.3% of yeast species isolated from patient samples at our department, were included into the study. Among these, 7 more common species, i.e. Candida albicans (56.2%), C. glabrata (12.6%), C. krusei (8%), C. tropicalis (7.7%), Saccharomyces cerevisiae (3.1%), C. parapsilosis (2.5%), and C. lusitaniae (2%) were represented by at least 35 isolates each, whereas the less frequently isolated species C. guilliermondii (1.3%) and C. pelliculosa (1%) were represented by at least 15 isolates each. A few isolates of C. orthopsilosis and C. metapsilosis were also included into the study later, when described as cryptic species of C. parapsilosis . See also additional file 4: Listing of clinical isolates and reference strains included in this study. The strains were stored in 20% BBL Skim Milk Powder supplemented with glycerol (BD, Franklin Lakes, New Jersey, USA) at -70°C until used.
All of the isolates were identified using conventional phenotypic identification techniques, i.e. evaluation of micromorphology on rice agar and evaluation of biochemical properties using in-house prepared assimilation and fermentation tests  followed by interpretation using the identification key according to Fragner . Selected isolates were also identified using the ID 32C commercial set (bioMérieux, Marcy l'Etoile, France) in accordance with manufacturer's instructions.
Crude colony lysates described earlier as suitable for amplification were prepared by simple toothpick technique . Briefly, a part of colony grown on SGA plate was picked up by a micropipette tip at latest one day after inoculation and transferred into 5 μl of freshly prepared lysing solution (1 M sorbitol, 5 mM MgCl2, 2 mM dithiothreitol, 12 U of Zymolyase, all from Sigma-Aldrich, St. Louis, Missouri, USA). The mixture was incubated for 30 min at 37°C and centrifuged (10,000 g for 5 min). The supernatant was transferred into a new tube, diluted with TE buffer to 300 μl and stored at -20°C until used. For comparison and reference, YeaStar Genomic DNA Kit (Zymo Research, Orange, California, USA) was also used for DNA extraction in selected strains following manufacturer's recommendations. Briefly, 1 ml of yeast submerged culture (approx. 1.5 × 107 cells) grown in YPG (1% of each yeast extract, peptone and glucose) in an Erlenmeyer flask shaken at 30°C was spun down and the pellet was subjected to enzyme lysis in 120 μl of YD Digestion Buffer (containing RNase A and Zymolyase) for 1 hour at 37°C. Then, 120 μl of YD Lysis Buffer and 250 μl of chloroform were added, mixed and spun down again. The aqueous supernatant was then loaded onto a fast spin-column, spun down, and the impurities were washed away using DNA Wash Buffer. Finally, DNA was eluted by 60 μl of water.
PCR reaction was performed in a glass capillary in a total volume of 10 μl consisting of 0.5 μM primer ACGGGCCAGT , 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 0.1% Triton X-100, 2 mM MgCl2, 200 μM of each dNTP, 2.5 U of Taq polymerase Unis (Top-Bio, Prague, Czech Republic), 250 μg/ml BSA and LCGreen dye at 1× concentration (Idaho Technology Inc., Salt Lake City, Utah, USA). Either 1 μl of crude colony lysate or 1 μl of DNA extracted using the YeaStar Genomic DNA Kit was added into the reaction. Amplification was performed in a Rapid Cycler 2 apparatus (Idaho Technology Inc., Salt Lake City, Utah, USA) applying an empirically optimized protocol of initial denaturation at 95°C, 5 min, followed by 45 cycles of denaturation at 95°C for 5 s, annealing at 48°C for 10 s, and extension at 72°C for 40 s, with ramping 1°C/s, followed by final extension at 72°C for 5 min.
Analysis of McRAPD data
AD1,2 was absolute distance between isolates No. 1 and 2
f1(t) was normalized fluorescence of isolate No. 1 measured at temperature t
f2(t)was normalized fluorescence of isolate No. 2 measured at temperature t
A matrix of relative distances was assembled for the isolates included into each comparison. Then, the matrix of relative distances was used to calculate tree data for a cladogram using the UPGMA method and Phylip software [28, 29]. PhyloDraw 0.8 software [30, 31] was used for cladogram construction.
For additional analysis, plots of the first negative derivation of fluorescence depending on temperature were also prepared based on melting data normalized previously. To delineate the melting peaks better, smoothing of data was performed using the HR-1 analysis software as recommended by the manufacturer. In some cases this smoothing resulted in truncation of the left and/or right end of the derivative curve. This process was carefully observed to prevent any loss of potentially discriminatory peaks at both ends of the derivative curves. To prevent excessive simplification and loss of informative data, smoothing was performed only if it undoubtedly resulted in a distinct amelioration of peaks' discrimination.
Electrophoresis and analysis of banding patterns
After melting analysis was performed, each sample was also subjected to gel electrophoresis in 2% agarose gel at 5 V/cm for 3 hours. The gels were stained by ethidium bromide added into them during preparation at the final concentration of 1 μg/ml and resulting banding patterns were photographed. Comparison of fingerprints was performed using GelCompar II software (Applied Maths, Sint-Martens-Latem, Belgium) applying the Jaccard coefficient at 1.5% positioning tolerance. Dendrograms were constructed using the UPGMA algorithm.
Ministry of Health (NR8365-4/2005), Czech Republic, supported this work. Dr. Mine Yücesoy from Dokuz Eylül University, Izmir, Turkey and Dr. Jozef Nosek from Comenius University in Bratislava, Slovakia kindly gifted some of the strains. Technical assistance of Mrs. Jana Novotna, Mrs. Jitka Cankarova, and Mrs. Ivana Dosedelova is highly acknowledged.
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