Evaluation of ID Fungi Plates Medium for Identification of Molds by MALDI Biotyper

MALDI-TOF mass spectrometry (MS) identification of pathogenic filamentous fungi is often impaired by difficulties in harvesting hyphae embedded in the medium and long extraction protocols. The ID Fungi Plate (IDFP) is a novel culture method developed to address such difficulties and improve the identification of filamentous fungi by MALDI-TOF MS. We cultured 64 strains and 11 clinical samples on IDFP, Sabouraud agar-chloramphenicol (SAB), and ChromID Candida agar (CAN2). We then compared the three media for growth, ease of harvest, amount of material picked, and MALDI-TOF identification scores after either rapid direct transfer (DT) or a long ethanol-acetonitrile (EA) extraction protocol.

ABSTRACT

MALDI-TOF mass spectrometry (MS) identification of pathogenic filamentous fungi is often impaired by difficulties in harvesting hyphae embedded in the medium and long extraction protocols. The ID Fungi Plate (IDFP) is a novel culture method developed to address such difficulties and improve the identification of filamentous fungi by MALDI-TOF MS. We cultured 64 strains and 11 clinical samples on IDFP, Sabouraud agar-chloramphenicol (SAB), and ChromID Candida agar (CAN2). We then compared the three media for growth, ease of harvest, amount of material picked, and MALDI-TOF identification scores after either rapid direct transfer (DT) or a long ethanol-acetonitrile (EA) extraction protocol. Antifungal susceptibility testing and microscopic morphology after subculture on SAB and IDFP were also compared for ten molds. Growth rates and morphological aspects were similar for the three media. With IDFP, harvesting of fungal material for the extraction procedure was rapid and easy in 92.4% of cases, whereas it was tedious on SAB or CAN2 in 65.2% and 80.3% of cases, respectively. The proportion of scores above 1.7 (defined as acceptable identification) were comparable for both extraction protocols using IDFP (P = 0.256). Moreover, rates of acceptable identification after DT performed on IDFP (93.9%) were significantly higher than those obtained after EA extraction with SAB (69.7%) or CAN2 (71.2%) (P = <0.001 and P = 0.001, respectively). Morphological aspects and antifungal susceptibility testing were similar between IDFP and SAB. IDFP is a culture plate that facilitates and improves the identification of filamentous fungi, allowing accurate routine identification of molds with MALDI-TOF-MS using a rapid-extraction protocol.

INTRODUCTION

From superficial infections to life-threatening invasive infections, filamentous fungi are involved in a large range of human diseases (1). Among them, invasive mold infections have been increasingly reported due to the expanded population of at-risk patients, i.e., those with immunosuppression, diabetes, or extensive burns (2, 3). Aspergillus fumigatus is still frequently involved, but because of the more intense and frequent immunosuppressive procedures and extensive use of antifungals, the epidemiology of medically important fungi is changing, and emerging species, including Fusarium spp., Scedosporium spp., and Mucorales are increasingly isolated from invasive infections (4–7). Moreover, there have been increasing reports of a large diversity of filamentous fungi encountered in respiratory samples of patients with cystic fibrosis (8, 9). In addition to Aspergillus spp. or Scedosporium/Lomentospora complex species, several unusual species, such as Rasamsonia argillacea, have recently emerged as clinically relevant molds (10).

In this context, rapid and reliable identification at the species level is crucial in terms of epidemiology, as well as for the optimal management of fungal infections (11). Indeed, the choice of antifungal therapy is driven by the identification of the fungus involved in the infection, as the susceptibility profile of filamentous fungi depends on the species (12, 13).

However, identification of filamentous fungi in clinical laboratories still frequently relies on morphological characteristics that, although they require specialized knowledge, are still subjective and time-consuming and are not always sufficiently discriminatory to enable correct identification at the species level (14). Indeed, cryptic species of the Aspergillus section Fumigati, e.g., Aspergillus fumigatus and Aspergillus lentulus, are not morphologically distinguishable, although they display distinct antifungal susceptibility profiles (12, 15, 16). Similarly, Rasamsonia strains are often misidentified as Paecilomyces or Penicillium (17, 18).

Over the last few years, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has emerged as a powerful microorganism identification tool. This technology is accurate, rapid, and easy to use, and now sufficiently widespread to identify bacteria or yeast in clinical microbiology laboratories (29, 30). However, despite its democratization, the identification of pathogenic filamentous fungi by MALDI-TOF MS is still difficult. Indeed, molds may grow inside the solid medium and are difficult to properly scrape off. Harvesting them is tedious and the spectra are often contaminated by agar, leading to low identification scores. In addition, long extraction methods are frequently needed to obtain a clean and significant spectrum, making identification of molds by MALDI-TOF difficult and time-consuming for routine use in clinical mycology or microbiology laboratories.

ID Fungi Plate (Conidia, Quincieux, France) employs a new medium developed to facilitate the identification of filamentous fungi by MALDI–TOF MS platforms. This culture plate includes a transparent membrane deposited on the agar surface, allowing the growth of molds but impassable by fungal hyphae, thus facilitating a clean harvest. We evaluated these new plates in routine practice in a clinical mycology laboratory and compared them with two other media classically used in mycology laboratories.

MATERIALS AND METHODS

Filamentous fungi isolates and clinical samples.

Suspensions of 62 filamentous fungi from our cryopreserved bank of clinical isolates and two reference strains (Aspergillus brasiliensis ATCC 16404 and Aspergillus fumigatus IHEM 3007) were subcultured. The isolates from our clinical bank, previously identified by MALDI–TOF MS (Microflex LT instrument from Bruker Daltonics GmbH, Bremen, Germany) or based on morphological features, are presented in Table 1.

TABLE 1.

Clinical strains and species used in this study for the MALDI-TOF identification assay and comparison of morphology and antifungal susceptibility

Species	No. of strains used for MALDI-TOF identification assay	No. of strains used for morphological comparison	No. of strains used for antifungal susceptibility comparison
Aspergillus	28	4	8
Aspergillus flavus/oryzae	7		1
Aspergillus fumigatus	10	2	4
Aspergillus japonicus	1		1
Aspergillus lentulus	1
Aspergillus nidulans	2	1	1
Aspergillus niger	4	1	1
Aspergillus terreus	1
Aspergillus ustus	1
Aspergillus versicolor	1
Fusarium	9	1
Fusarium equisety	1	1
Fusarium oxysporum	1
Fusarium petroliphilum	1
Fusarium proliferatum	3
Fusarium spp.	3
Mucorales	4	1	1
Lichtheimia spp.	1
Rhizomucor spp.	1
Rhizopus spp.	2	1	1
Penicillium	2
Penicillium spp.	1
Penicillium roqueforti	1
Dematiaceous	3	1
Chaetomium sp.	2	1
Fonsecaea nubica	1
Dermatophytes	4	1
Microsporum canis	1
Nannizzia gypsea	1
Trichophyton mentagrophytes	1	1
Trichophyton interdigitale	1
Others	12	2	1
Acremonium spp.	1
Purpureocillium lilacinum	2	1
Rasamsonia argillacea	2
Scedosporium/Lomentospora spp.	1
Schizophyllum commune	2
Sporothrix schenckii	1
Trichoderma spp.	1	1	1
Unidentified	2

The panel chosen was composed of fungi representative of a clinical microbiology laboratory, as well as much rarer and uncommon species. Moreover, 11 clinical samples (nine sputum and two nail samples) previously found to be positive for filamentous fungi, were selected, preserved at 4°C, and used retrospectively to inoculate the culture plates.

Fungal cultures.

Cryopreserved clinical and reference strains were subcultured on Sabouraud agar with chloramphenicol (SAB) (Becton, Dickinson GmbH, Heidelberg, Germany). A suspension of each strain was prepared in 3 ml pure water with 0.05% Tween 20. Three drops of suspension were then inoculated in parallel on ID Fungi Plates (IDFP), SAB, and ChromID Candida agar (CAN2) (bioMérieux, Marcy-l’Etoile, France). Clinical samples were used to inoculate the same media, using a dry swab for nail samples and a 10-μl inoculation loop for sputum. Plates were incubated at 27°C for one to eight days and checked daily until sufficient mycelium growth was obtained.

Culture and harvest parameters.

Growth intensity, ease of harvest, and the amount of material obtained were qualitatively evaluated by the same operator for each medium and reported on a form as follows. Growth intensity was characterized as rich, mild, or poor by a trained operator, according to a predefined abacus; harvest practicability was defined as easy when harvesting took <10 s, moderate when it took 10 to 60 s, or tedious when it took >60 s; and pellet size (i.e., the amount of material recovered from each medium) was characterized as invisible, small, or large by the same trained operator according to a predefined abacus.

MALDI-TOF MS sample preparation and MS acquisition.

MALDI-TOF MS was performed using a Microflex LT instrument (Bruker Daltonics GmbH, Bremen, Germany). Colonies were gently scraped using a sterile plastic tip and two preparation protocols were performed and analyzed in duplicate for each of the following methods: (i) the direct transfer to the target using formic acid method (DT), as previously described (19); and (ii) the ethanol/formic acid extraction (EA) (20). Briefly, the DT method consisted of directly smearing the colony onto the target, overlaid with 1 μl formic acid (70% vol/vol). The EA method consisted of first resuspending the fungal material, scraped from the plate with a calibrated 10-μl plastic disposable loop, in 300 μl pure water until the solution was cloudy (visual estimation). Sample were then mixed with 900 μl ethanol, centrifuged, and an equal volume of 70% formic acid (added to completely cover the pellet, with a maximum of 50 μl as recommended) and 100% acetonitrile was added to the pellet. One microliter of the supernatant was spotted onto the target and overlaid with 1 μl of matrix (α-cyano-4-hydroxy-cinnamic acid solution in 50% acetonitrile and 2.5% trifluoroacetic acid [Bruker Daltonics GmbH, Bremen, Germany]). After scraping the fungal colony off the plate, the DT process was approximately 2 min long for one strain versus 15 min for the EA process.

Spectra were submitted to two fungal libraries in a single run: (i) the Bruker Filamentous Fungi Library (Version 2.0) (364 MSP) and (ii) the NIH fungal library database (365 MSP) (21). A score between 0.00 and 3.00 was obtained, depending on the degree of similarity to a given spectra in one of the reference databases. The higher score of each duplicate was used. Scores were categorized as follows: score >2, highly reliable identification at the species level; score between 1.7 and 2, acceptable identification at the species level based on the recommendations of previous studies (22–24); and score <1.7, unreliable. Culture scraping failure resulted in spectra without peaks.

Morphological identification and antifungal susceptibility testing.

Macroscopic and microscopic identification and antifungal susceptibility were tested on 10 randomly chosen isolates cultured in parallel on SAB and IDFP to ensure that the entire mold diagnosis workflow was possible when subculturing on IDFP.

Voriconazole (VO), anidulafungin (ANI), amphotericin B (AB), and posaconazole (POS) MICs were evaluated using Etest strips (bioMérieux, Marcy I’Etoile, France). Itraconazole (IT) and isavuconazole (ISA) MICs were evaluated using other strips (Liofilchem, Roseto Degli Abruzzi, Italy).

Statistical analysis.

Statistical analyses were performed using XLSTAT statistical and data analysis solution v 2019.1 (Addinsoft, NY, USA, https://www.xlstat.com). Fisher’s exact test was used to compare the distribution of culture characteristics. Chi-square tests were used to compare the proportion of scores at >2 and >1.7. The Kruskal-Wallis test and multiple pairwise comparisons using Dunn’s procedure were used to compare identification scores. Student’s t test on matched samples was used to compare susceptibility results.

RESULTS

Among the 64 collection isolates, two were not identified by MALDI-TOF MS after subculture on the three media. Microscopy also failed to identify these two strains.

Among the 11 clinical samples directly inoculated onto the plates, seven (63.6%) could not be analyzed. Three were negative after culture on the three media of the study and four were positive for various fungi (yeasts and/or molds), resulting in the need for a subculture to proceed correctly. Finally, the results for 66 isolates could be correctly evaluated (60 clinical isolates from our cryopreserved bank, 2 reference strains, and 4 clinical samples).

Culture characteristics.

Growth intensity was not significantly different between the three plates (P = 0.592). IDFP enabled easy harvesting of 92.4% of the isolates versus 9.1% for SAB or CAN2. Moreover, the size of the pellet was large for 87.9% of the strains cultured on IDFP versus 22.7 and 15.2% for those cultured on SAB and CAN2, respectively (P < 0.0001) (Fig. 1).

FIG 1.

Distribution of cultural characteristics after culture on IDFP, SAB, or CAN2. IDFP, ID fungi plates (Conidia, Lyon, France); SAB, Sabouraud agar with chloramphenicol (Becton, Dickinson GmbH, Heidelberg, Germany); CAN2, ChromID Candida agar (bioMérieux, Marcy-l’Etoile, France).

Identification.

A comparison of the extraction protocols showed the proportion of scores >2 to be not significantly different after EA or DT following culture on IDFP or CAN2 (P = 0.256 and 0.292, respectively). For culture on SAB, more scores >2 were obtained after EA (P = 0.032). However, a comparable proportion of scores above 1.7 (i.e., acceptable identification) was obtained after EA or DT for cells cultured on the three media (P = 1.000 on IDFP and SAB, and P = 0.088 on CAN2) (Table 2).

TABLE 2.

Distribution of MALDI Biotyper scores depending on the culture plate and extraction protocol^a

Score	IDFP DT		IDFP EA		SAB DT		SAB EA		CAN2 DT		CAN2 EA
	No.	%	No.	%	No.	%	No.	%	No.	%	No.	%
Score ≥2	42	63.6	49	74.2	23	34.8	36	54.5	34	51.5	27	40.9
Score between 1.7 and 2	20	30.3	14	21.2	23	34.8	10	15.2	22	33.3	20	30.3
Unreliable score	3	4.5	3	4.5	20	30.3	15	22.7	8	12.1	8	12.1
Spectra without peaks	1	1.5	0	0.0	0	0.0	5	7.6	2	3.0	11	16.7

^aIDFP, ID fungi plates (Conidia, Lyon, France); SAB, Sabouraud agar with chloramphenicol (Becton, Dickinson GmbH, Heidelberg, Germany); CAN2, ChromID Candida agar (bioMérieux, Marcy-l’Etoile, France); DT, direct transfer-formic acid method; EA, formic acid-acetonitrile extraction method.

After DT, the proportion of scores >2 was significantly higher for cultures grown on IDFP than on SAB (63.7% versus 34.8%, P = 0.001). The trend was similar when we compared IDFP to CAN2, but the difference did not reach statistical significance, possibly due to the small number of samples (63.7% versus 51.5%, P = 0.214). Similarly, the proportion of scores >1.7 for cultures grown on IDFP was significantly higher than those grown on SAB (93.9% versus 69.7%, P < 0.001), and the trend was similar when comparing IDFP and CAN2, but the difference did not reach statistical significance (84.8%, P = 0.153). Considering exclusively scores >1.7, the scores were significantly higher for cultures grown on IDFP than SAB after DT (P = 0.008). Values were not significantly different between cultures grown on IDFP or CAN2 after DT (P = 0.287) (Fig. 2).

FIG 2.

Distribution of scores above 1.7 by culture plate and extraction protocol. Red plus signs correspond to means. Lines through boxes correspond to medians. Scores were significantly higher after EA than after DT on IDFP and SAB (P = 0.021 and 0.016, respectively). Scores after DT were significantly higher on IDFP than on SAB (P = 0.008). Scores after EA were significantly higher on IDFP than on SAB or CAN2 (P = 0.033 and 0.005, respectively). NS, nonsignificant differences.

After EA, the proportion of scores >2 was significantly higher for cultures grown on IDFP than SAB or CAN2 (P = 0.026 and P < 0.001, respectively). We observed the same trend for the proportion of scores >1.7 (P < 0.001 with CAN2 and SAB). The average of scores above 1.7 was significantly higher for cultures grown on IDFP than SAB (P = 0.033) or CAN2 (P = 0.005) (Fig. 2).

In addition, the proportion of acceptable identifications was significantly higher after DT for molds grown on IDFP than after EA for those grown on SAB (P < 0.001) or CAN2 (P = 0.001).

Morphological identification and susceptibility testing.

Comparative macroscopic and microscopic observation of 10 fungi cultured on either SAB or IDFP showed the duplicate cultures to have the same color, structure, and fructification on both media after the same period of culture.

We tested the MICs of 10 fungi to ANI, VO, POS, AB, ISA, and IT after culture on IDFP or SAB. The results were not significantly different depending on the culture plate (P = 0.22).

DISCUSSION

The integration of MALDI-TOF MS within routine clinical laboratories has revolutionized medical microbiology, but its use to identify filamentous fungi is still jeopardized by practical difficulties. Here, we demonstrate that the use of the ID Fungi Plate (Conidia), a culture medium designed specifically for this purpose, facilitates and improves the identification of filamentous fungi by MALDI-TOF MS.

Indeed, we found that the proportion of acceptable identifications and identification score values were higher after culture on IDFP than on SAB. The difference in terms of identification score was less obvious when IDFP was compared to CAN2, but CAN2 is not suitable for subculturing filamentous fungi, as this medium was not initially designed to culture molds and their morphological aspect can be altered on this plate. Furthermore, IDFP provided a clear-cut benefit for fungal harvesting relative to both SAB and CAN2.

Sample preparation protocols leading to a generous amount of clean material and improved databases are two key factors for performing reliable fungal identification by MALDI-TOF. Although a larger pellet is not necessary to obtain good-quality spectra and identifications, we observed that the spectra obtained with IDFP were of higher quality (not shown) when comparing spectra profiles of the different culture conditions and provided more specific peaks, which probably led to the better identification scores.

Using their homemade reference spectra library and an ethanol-acetonitrile extraction protocol, Ranque et al. demonstrated the improvement of identification of clinical strains of filamentous fungi (mostly for non-Aspergillus species) by MALDI-TOF MS over conventional methods (14). Also, Riat et al. demonstrated that identification is possible directly from solid isolation media, but they emphasized that this approach requires fungal material free of culture medium (25). However, due to the ability of molds to grow into the medium, this is particularly tedious and consequently time-consuming to achieve using the usual culture plates. This specific aspect of culturing molds triggered Bruker’s recommendations to use an overnight subculture in liquid media before identification by MALDI-TOF MS when DT and EA are unsuccessful. However, this supplemental step delays the diagnosis and is not adapted to routine and rapid mold identification. IDFP is the first plate-based medium that proposes a solution to facilitate the harvest of fungal material with the same time course as other subculture media, such as SAB plates.

Interestingly, we did not find any added value of using the long extraction (EA) versus short DT protocol with IDFP. A similar proportion of acceptable identification at the species level was obtained after DT or EA (93.9 and 95.5%, respectively). However, Zvezdanova et al. obtained significantly better results using classical culture media following mechanical lysis of the molds and subsequent protein extraction than by directly smearing the sample on the MALDI-TOF MS target (26). Moreover, a preparation protocol similar to EA or involving a bead-beating step is generally recommended by authors and providers of filamentous fungi libraries (21, 27). With IDFP, the DT protocol was sufficient to reliably identify filamentous fungi by MALDI-TOF MS, leading to significant time savings.

In this study, spectra were compared to those in two different libraries in a single experiment: the NIH library was used as a complement to the Bruker database to enhance the rate of acceptable identifications. Our aim was to evaluate the media (and not the libraries) in the setting of our routine clinical laboratory practice. Thus, we choose to compare only spectra obtained after DT or EA, which are the recommended protocols in the first line when using Bruker databases. We did not use the mechanical method recommended by the provider of the NIH library, which could have led to more “best-match” identifications from this library. It was important to verify that the new plate allowed the identification of molds based on macroscopic and microscopic features, as microscopic examination is still commonly used in medical mycology. We observed similar morphological aspects and susceptibility profiles for fungi cultured on either SAB or IDFP. These results suggest that IDFP could be used as a single subculture plate, replacing the usual SAB media, for example.

One remaining question was whether IDFP is suitable in the first line for clinical samples, but the high failure rate with clinical samples directly inoculated onto the identification media in this study did not allow us to clearly address this point. This issue has to be addressed in a study with more clinical samples prospectively inoculated on IDFP to evaluate this plate as a primary culture medium. Indeed, the use of such a medium as a first-line culture plate may have particular time savings. However, most clinical samples transmitted to clinical mycology or microbiology laboratories are negative for filamentous fungi and thus IDFP would be useless for such samples. In addition, colony isolation and subcultures are frequently needed for polymorphic cultures, which are frequent when dealing with respiratory samples of patients with cystic fibrosis (9, 28), and the use of IDFP in the first line in this context would also be of limited value. Furthermore, IDFP is almost 12 times more expensive than SAB, and five times more expensive than CAN2. Given these considerations and for maximum cost efficiency, IDFP is, in our opinion, more suitable for second-step subculture and mold reisolation, when needed, than for direct use on clinical samples.

The advantages of identification by MALDI-TOF MS are well established for yeast, bacteria, or molds, for which technical issues have been an obstacle for routine laboratory use of this technology. Here, we found that the use of the new ID Fungi Plate as a single-subculture plate both facilitates and improves the identification of filamentous fungi. This simplification and optimization may allow routine use and rapid identification of molds by MALDI-TOF MS in clinical laboratories, which is of increasing importance for appropriate patient management.