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 significant difference among species [23]. This is parallel with our finding except for A. calidoutus. In addition, the ratio of Sensitititre MIC50 to MIC90 for A. niger and A. flavus 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 MIC50 to MIC90 of A. niger [9] were same with the result in this study; however, its ratio for A. fumigatus and A. flavus were lower. Furthermore, the CLSI mean and MIC90 for A. fumigatus, A. niger and A. flavus reported by Espinel-Ingroff were one two-fold dilution higher than our finding [25].
Fusarium spp. are resistant in vitro to many antifungal compounds [26]. The management of fusariosis is not well defined; 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 MIC50 and MIC90 of voriconazole against F. solani were recorded > 8.0 µg/mL. A similar result also obtained by Alastruey-Izquierdo et al. where their MIC was ranged 4–16 µg/mL [26]. Interestingly, their findings are similar with our observations except one of the samples had low MIC which recorded 0.50 µg/mL.
Besides Aspergillus and Fusarium, Rhizopus can also cause severe and fatal infections in immunocompromised patients [31, 32]. The treatment of zygomycosis is problematic and frequently associated with suboptimal therapeutic outcomes [33]. Voriconazole possesses no meaningful activity against Rhizopus strains [34–36]. This is parallel with our finding. 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 difficult to evaluate. Since Mucorales are resistant in vitro to many antifungals [38], its treatment with fluconazole, flucytosine, ketoconazole, echinocandins, itraconazole and voriconazole were reported not effective in many cases [39–43]. 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 MIC90 was even > 64 µg/mL [37]. These MIC were much higher than the finding 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 MIC50 and MIC90 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 fixed forms [47], and amphotericin B for disseminated cases [48, 49]. However, these antifungal drugs are not always efficient and may lead to chronicity and disseminate in immunocompromised patients [50]. Several studies have searched and tested for alternatives including voriconazole; however, the MIC50 and MIC90 obtained were varied among them. By using CLSI method, MIC50 was reported 32, 16 and 8 µg/mL; while the MIC90 was reported 32, > 16 and 16 µg/mL by Marimon et al., Rodrigues et al. and Córdoba et al. respectively [51, 52, 50]. However, both of the CLSI MIC50 and MIC90 in this study were lower than these reported findings.
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 inefficient 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].
The level of agreement for the voriconazole between Sensititre and CLSI methods were inconsistent throughout publications. For example, Wang et al. and Mello et al. had found 100% agreement of Sensititre with the CLSI reference method for the voriconazole when they tested on all the Aspergillus spp [55, 57]. In contrast, Castro et al. reported that the overall agreement between Sensititre and CLSI methods for voriconazole was only 82.5% [13]. Moreover, the phenomenon of Sensititre® YeastOne test tended to increase or decrease the MIC by only one dilution when compared with the reference test also had been reported by Siopi et al., Castro et al., Sanchez Sousa et al. and Guinea et al. This phenomenon was consistent with the finding in this study [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 defined [26, 58]. More data from clinical trials with voriconazole are required for this purpose. Despite voriconazole was shown well-tolerated 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 first study to compare the susceptibility of voriconazole against Malaysian moulds using both CLSI and commercial Sensititre YeastOne methods. Moreover, the research pertaining susceptibility pattern of A. versicolor, A. sydowii, A. calidoutus, A. creber, F. keratoplasticum, Mucor sp., S. racemosum, P. circinans and S. schenkii are still limited especially in Malaysia. In contrast, there are several limitations in this study. The sample sizes of some isolates were small and MIC50 and MIC90 were unable to be determined. In addition, the MICs were still not able to be interpreted as susceptible or resistant there are no official clinically correlated breakpoints for moulds according to CLSI method. However, these results could contribute to its limited antifungal database in Malaysia.