Susceptibility of Filamentous Fungi to Voriconazole in Malaysia Tested by Sensititre YeastOne and CLSI Microdilution Methods

Background: Voriconazole is a trizaole antifungal to treat fungal infection. In this study, the susceptibility pattern of voriconazole against lamentous fungi was studied using Sensititre® YeastOne and Clinical & Laboratory Standards Institute (CLSI) M38 broth microdilution method. Methods: The suspected cultures of Aspergillus niger, A. avus, A. fumigatus, A. versicolor, A. sydowii, A. calidoutus, A. creber, A. ochraceopetaliformis, A. tamarii, Fusarium solani, F. longipes, F. falciferus, F. keratoplasticum, Rhizopus oryzae, R. delemar, R. arrhizus, Mucor sp., Poitrasia circinans, Syncephalastrum racemosum and Sporothrix schenckii were received from hospitals. Their identication had been conrmed in our lab and susceptibility tests were performed using Sensititre® YeastOne and CLSI M38 broth microdilution method. The signicant differences between two methods were calculated using Wilcoxon Sign Rank test. Results: Mean of the minimum inhibitory concentrations (MIC) for Aspergillus spp. and Fusarium were within 0.25 μg/mL-2.00 μg/mL by two methods except A. calidoutus, F. solani and F. keratoplasticum. Moreover, mean of MIC for S. schenkii were around 3.00 μg/mL by two methods. In contrast, mean of MIC for Rhizopus spp., Mucor sp., P. circinans and S. racemosum were ≥ 6.00 μg/mL by two methods. Generally, the MIC obtained by Sensititre YeastOne was one two-fold increase or decrease compared with the results obtained by CLSI method. The overall agreement between Sensititre YeastOne and CLSI methods to test susceptibility testing of voricaonazole was more than 70% except A. sydowii. The signicant differences between two methods


Background
Voriconazole is a potent new triazole drug with a broad spectrum of antifungal activity against many opportunistic fungal pathogens [1][2][3][4][5][6]. Previous studies reported that voriconazole can enhance clinical e cacy and coupled with lower toxicity [7]. It had prevented or delayed the mortality in infected animals [8,9].
To date, much work on susceptibility has been done on yeast, especially for the Candida species, but susceptibility data on moulds is still limited. This could be due to the lack of established breakpoints for moulds, cost of antifungal reagents and laborious laboratory procedures [10]. A commercial panel named Sensititre® YeastOne (Thermo Fisher Scienti c, Cleveland, United States) had been widely used in many routine microbiology laboratories recently [11,12]. It is a commercial colorimetric panel that contains dried serial dilutions of antifungal agents in a disposable tray [13]. The MIC from this panel is based on the visible colour change which caused by an oxidation-reduction indicator, Alamar Blue [14].
In this study, MICs of voriconazole against isolated moulds were determined. It will be bene cial to clinicians in monitoring and selecting appropriate therapy for patients. The present study had assessed the performance for in vitro susceptibility testing of voriconazole against several moulds in Mycology laboratory, Bacteriology Unit, Institute for Medical Research using Sensititre® YeastOne and compared with Clinical and Laboratory Standards Institute (CLSI) broth microdilution method M38 [15].

Ethic
Ethical review was conducted and approved by the Medical Research and Ethics Committee, Ministry of Health of Malaysia, Malaysia (NMRR-20-207-53607).
The lactophenol cotton blue wet mount was used to stain the mature colony with scotch-tape technique.
Culture medium RPMI-1640 (Sigma-Aldrich, St. Louis, United States) with glutamine and phenol red, without sodium bicarbonate and buffered with 0.165 mol/L 3-morpholinopropanesulfonic acid (MOPS) (Sigma-Aldrich, St. Louis, United States) at pH 7.0, was used as the basal medium. The procedure of preparing RPMI-1640 was following the procedures described in CLSI M38 [15].
For the reference broth microdilution testing, conidial suspensions were prepared as described in the CLSI M38 document [15]. The plates were incubated at 30°C, and MIC were de ned as the lowest drug concentration that prevents any discernible growth.

Sensititre YeastOne
A colorimetric microdilution method was performed using commercially available Sensititre YeastOne panels (Thermo Fisher Scienti c, Cleveland, United States) according to the manufacturer's recommendations. Colony-forming unit counts were a rmed each time by spread plate counts on potato dextrose agar. The range of concentration of voriconazole in the panel was 0.008 to 8 µg/ mL.

Quality controls.
Each test had included two reference strains; A. avus ATCC 204304, A. fumigatus ATCC 204305, in order to detect any alteration in the antifungal agent should the MIC obtained fail to fall within the reference range.

Agreement between two methods
The 2-fold differences and the level of agreement between the two methods were calculated for each species/drug/ combination as the proportion of the Sensititre YeastOne colour endpoints determined for each strain that fell within ±1 twofold dilutions of the corresponding MICs of the CLSI method.

Analysis of data
The signi cant differences between two methods were calculated using Wilcoxon Sign Rank test. The tests were performed for species that have more than three isolates. Any p values less than 0.05 were considered as statistically signi cant.
Meanwhile, the mean MICs of voriconazole for Fusarium spp. were slightly higher than most of the Aspergillus spp. The minimum and maximum MIC mean for Fusarium was 0.75 µg/ mL and > 8.00 µg/mL respectively. Some of F. solani and F. keratoplasticum have high MIC (≥ 8.00µg/mL) compared with F. longipes and F. falciferus which have lower MIC (≤ 2.00µg/mL). In general, Sensititre MIC 50 and MIC 90 were one two-fold dilution higher or lower than those of the CLSI. Finally, the percentage of agreement between two methods were perfect (100%) and it was higher compared with Aspergillus spp.
All of the Rhizopus spp. had given the similar results when tested by Sensititre or CLSI method. The mean MICs of voriconazole for Rhizopus spp. were higher than Aspergillus spp. and most of the Fusarium spp. However, the exact MICs were not able to be determined by Sensititre as it had exceeded the tested range. Therefore, the MIC 50 and MIC 90 were not able to be determined for Rhizopus spp. On the other hand, the percentage of agreement between two methods were perfect and achieved 100.00%.
In addition, the mean MICs of voriconazole against Mucor sp., P. circinans, S. racemosum and S. schenkii were higher than most of the Aspergillus spp. and Fusarium spp. too. The MICs by Sensititre method were one double dilution different than those of the CLSI method, except S. schenkii which recorded similar value. One of the Mucor sp. and P. circinans were found unable to be inhibited by the highest tested concentration of voriconazole by both methods. However, the percentage of agreement for both methods to determine MIC against S. schenkii (77.78%) was lower than Mucor sp., P. circinans, and S. racemosum (100.00%).

Discussion
Voriconazole is a new broad-spectrum triazole antifungal agent with fungicidal activity against Aspergillus spp. [1]. It has good bioavailability and well tolerated by humans [16]. Previous clinical studies of invasive aspergillosis have provided encouraging results [17,18]. Voriconazole was demonstrated well absorbed following oral administration and was highly effective in preventing or delaying mortality in an experimental model of pulmonary aspergillosis [9]. The initial results from animals' trials also suggested it was effective in disseminating the Aspergillus infection [8,19]. The survival was proved greater with voriconazole treatment compared to itraconazole treatment [9]. Moreover, the superiority of voriconazole to amphotericin B for the treatment of invasive aspergillosis also has been reported [20,21]. In May 2002, voriconazole had been approved by the Food and Drug Administration for the treatment of invasive aspergillosis [5].
The breakpoints of voriconazole against mould have not been determined by CLSI [15]. However, European Committee on Antimicrobial Susceptibility Testing Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST) has determined breakpoints for voriconazole against A. fumigatus where susceptibility ≤ 1 mg/L and resistance > 1 mg/L [22]. The voriconazole MICs for Aspergillus showed no signi cant difference among species [23]. This is parallel with our nding except for A. calidoutus. In addition, the ratio of Sensitititre MIC 50 to MIC 90 for A. niger and A. avus in this study were one two-fold dilution higher than Linares et al. [24]; however, the CLSI ratio for A. fumigatus and A. niger were same. On the other hand, the ratio of CLSI MIC 50 to MIC 90 of A. niger [9] were same with the result in this study; however, its ratio for A. fumigatus and A. avus were lower. Furthermore, the CLSI mean and MIC 90 for A.
fumigatus, A. niger and A. avus reported by Espinel-Ingroff were one two-fold dilution higher than our nding [25].
Fusarium spp. are resistant in vitro to many antifungal compounds [26]. The management of fusariosis is not well de ned; therefore, susceptibility test might be useful in choosing the suitable antifungal therapy [26]. The satisfactory response for voriconazole against fusariosis was 45% [27]. Voriconazole MICs for Fusarium spp. were higher than other genus including Aspergillus spp. [23,29]. Arikan et al. suggested that it might due to the use of 100% growth reduction endpoint instead of 50% growth reduction endpoint [23]. The MIC of Fusarium spp. was usually ranged 1 to 4 µg/mL [2,23,29]. However, among several tested Fusarium spp., F. solani was found the most resistant species to various drugs including amphotericin B, itraconazole, posaconazole and voriconazole [30]. Both of their MIC 50 [31,32]. The treatment of zygomycosis is problematic and frequently associated with suboptimal therapeutic outcomes [33]. Voriconazole possesses no meaningful activity against Rhizopus strains [34][35][36]. This is parallel with our nding. Instead, posaconazole and amphotericin B were found active and potent against Rhizopus [36,37]. The MIC from the combination of both of these drugs were lower than those from single drug [36]. Therefore, this combination could be tested in our future study.
Similar to Rhizopus spp., voriconazole has shown no reliable activity against Mucor spp. [34,37]. As mucormycosis is less common than aspergillosis and the course is progressively rapid; therefore, the effectiveness of antifungal treatment in small case studies is di cult to evaluate. Since Mucorales are resistant in vitro to many antifungals [38], its treatment with uconazole, ucytosine, ketoconazole, echinocandins, itraconazole and voriconazole were reported not effective in many cases [39][40][41][42][43]. On the other hand, data on the antifungal susceptibility of Mucorales spp. are limited, and MIC testing remains investigational [44]. The means of voriconazole MIC were higher than 32 µg/ mL [34,37] and the MIC 90 was even > 64 µg/mL [37]. These MIC were much higher than the nding in this study. However, more samples are needed to determine the accuracy of the result.
Poitrasia circinans is fall under the order Mucorales. Both Sensititre and CLSI methods had shown that voriconazole had inactive activity against this mould. The result of this sample was similar with Mucor sp.
Meanwhile, S. racemosum is an opportunistic pathogen and rarely caused infection in human [40]. Thus, research related with its susceptibility testing was limited. Chowdhary et al. reported that the MIC 50 and MIC 90 were 8 and 16 µg/mL respectively by CLSI method [45]. This result was four-fold higher than the CLSI result obtained in this study.
Sporotrichosis is a subacute or chronic infection which caused by the dimorphic fungus Sporothrix schenckii [46]. The antifungal drugs which commonly used are itraconazole for cutaneous or lymphocutaneous xed forms [47], and amphotericin B for disseminated cases [48,49]. However, these antifungal drugs are not always e cient and may lead to chronicity and disseminate in immunocompromised patients [50].  [51,52,50]. However, both of the CLSI MIC 50 and MIC 90 in this study were lower than these reported ndings.
Although the broth microdilution methods had improved the level of interlaboratory agreement of antifungal MIC endpoints; however, these procedures are laborious, inconvenient, labour intensive and ine cient for the clinical laboratory [13,53,54]. This is due to the main disadvantage of the M38 method is the need to prepare microdilution plates, which is time-consuming and impractical for routine use in clinical microbiology laboratories [55]. Sensititre® YeastOne is an adapted susceptibility system of the microbroth CLSI method based on the M27-A3 standard for yeasts and has been extensively evaluated for yeasts. It has been approved by the U.S. Food and Drug Administration (FDA) for Candida species but not for mould yet [56].  [11,13,14,54].
Based on in vitro susceptibility pattern in this study, voriconazole seems to have reliable activity against most of the species except A. calidoutus, F. keratoplasticum, R. oryzae, R. delemar, R. arrhizus, Mucor sp., P. circinans, S. racemosum and S. schenkii as their mean were more than 1 µg/mL. However, the correlation between MIC results and treatment outcome is not well de ned [26,58]. More data from clinical trials with voriconazole are required for this purpose. Despite voriconazole was shown welltolerated in human [58]; however, the doses have to be monitored to minimize its side effects such as visual disturbances, skin rashes, elevations in several hepatic enzyme levels, headache, nausea and vomiting, diarrhoea and abdominal pain [4].
To our knowledge, this was the rst study to compare the susceptibility of voriconazole against Malaysian moulds using both CLSI and commercial Sensititre YeastOne methods.