In Vitro Susceptibility of Talaromyces Marneffei In Malaysia: Comparison of Yeast and Mycelial Phases

Talaromyces marneffei is an etiologic agent of talaromycosis. It can cause serious complications and death in immunocompromised patients, particularly in acquired immunodeciency syndrome (AIDS) patients. This infectious disease is endemic in Southeast Asia including Malaysia. To date, published reports on the antifungal susceptibility prole of T. marneffei is very limited. The objective of this study is to determine the minimum inhibitory concentration (MIC) of T. marneffei in yeast and mycelial phases in Malaysia. In the year 2020, 27 clinical strains of T. marneffei were received from various hospitals in Malaysia. The identication was carried out using microscopic, macroscopic and molecular methods. Following that, the susceptibility of each isolate in both yeast and mycelial form to thirteen common antifungals was performed according to the broth microdilution in Clinical & Laboratory Standards Institute (CLSI) M38 method. The antifungals tested were anidulafungin, micafungin sodium, caspofungin diacetate, 5-uorocytosine, amphotericin B and terbinane hydrochloride, posaconazole, voriconazole, itraconazole, ketoconazole, ravuconazole, clotrimazole and isavuconazole. The geometric mean of all antifungals other than anidulafungin, micafungin sodium, caspofungin diacetate and 5-uorocytosine against T. marneffei mould (mycelial) were >2 μg/ml. However, the geometric mean of all antifungals against T. marneffei yeast was <2 μg/ml. Our in vitro data suggests promising activities of amphotericin B, terbinane hydrochloride, posaconazole, voriconazole, itraconazole, ketoconazole, ravuconazole, clotrimazole and isavuconazole against yeast and mould phases of T. marneffei.

The inhalation of spores was considered as the route of transmission of T. marneffei [4][5][6]. T. marneffei mostly causes mild and localized infections in patients with normal immunity, but it can cause severe disseminated infections in immunocompromised patients [7]. Even though T. marneffei is frequently detected in HIV patients; however, it also had been reported in non-HIV or immunocompromised patients recently [8][9][10]. The mortality rate is 75% in those with delayed diagnosis and administration of antifungal therapy [11,12].
This pathogen is dimorphic. This fungus grows as yeast at 37°C; while it grows as a mould at 25°C [13].
The yeast form is pathogenic as it can produce proteins or toxins that can evade the immune defence of the host [14]. The most common treatment of talaromycosis is amphotericin B [15]. In addition, itraconazole, posaconazole and terbina ne were also reported to be effective in treatment [13,16,17].
However, the susceptibility pattern of Malaysian isolates of T. marneffei is rarely reported. Therefore, in this study, we report the susceptibility pattern of Malaysian T. marneffei which might be useful to the treatment of talaromycosis.

Materials And Methods
Ethical review was conducted and approved by the Medical Research and Ethics Committee, Ministry of Health of Malaysia, Malaysia (NMRR-20-207-53607).

(i) Isolate
Twenty-seven clinical isolates of T. marneffei on potato dextrose agar (PDA) were received from Malaysian hospitals in the year 2020. Their identitis were con rmed by both macroscopic and microscopic methods. Internal transcribed spacer (ITS) region of the nuclear rDNA was ampli ed with PCR and detected with direct DNA sequencing to determine the species [18]. The mycelial and yeast form of T. marneffei were obtained as mentioned by Sar et al [19].

(ii) Clinical & Laboratory Standards Institute (CLSI) methods
Since no existing guidelines were available for susceptibility testing of T. marneffei, the minimum inhibitory concentration (MIC) was performed according to the broth microdilution in CLSI M38 [20]. The antifungals tested were the echinocandin anidulafungin, micafungin sodium, caspofungin diacetate, 5uorocytosine, amphotericin B and terbina ne hydrochloride, posaconazole, voriconazole, itraconazole, ketoconazole, ravuconazole, clotrimazole and isavuconazole. Each microdilution well contained 100 µl of antifungal. The nal concentrations of antifungals were ranged from 0.0313 to 16.0 µg/ml for each antifungal.
Following that, 100 µl of diluted inoculum suspension was added to the well. The inoculum sizes for yeast and conidia mould were 0.4 x 10 4 to 5 x 10 4 colony-forming unit (CFU)/ml. The mould mixture was then incubated at 25°C for 96 h while the yeast mixture was incubated at 37°C for 72 h [13].
(iii) Quality control Each test included two reference strains; A. avus ATCC 204304 and A. fumigatus ATCC 204305 to ensure that the MIC obtained was within the reference range.
(iV) Data analysis A descriptive analysis was performed using geometric mean (GM) values. For each antifungal test, the MIC 50 and MIC 90 were calculated. MIC 50 was de ned as 50% of the isolates were inhibited; whereas MIC 90 is the MIC at which 90% of the isolates were inhibited. Comparisons between results from the yeast and mycelial phases were evaluated by the Wilcoxon test using SPSS 20.0. P-values less than 0.05 were considered statistically signi cant.

Results
T. marneffei was initially isolated from blood (n=23), pleural uid (n=1), tracheal aspirate (n=1) and skin biopsy specimen (n=2). All samples were obtained from HIV-infected patients and their common clinical manifestations were fever and cough.
The geometric mean of MIC, MIC 50 and MIC 90 of T. marneffei are listed in Table 1. Most of the MICs were fallen within the expected range for each antifungal. In addition, triazoles were found to have lower MIC against most of the strains as compared with echinocandins.
The antifungals which demonstrated active against both mycelial and yeast phases of T. marneffei were terbina ne hydrochloride, posaconazole, voriconazole, itraconazole, ketoconazole, ravuconazole, clotrimazole and isavuconazole as their geometric mean of the MIC were <0.10 µg/ml. However, the GM MICs of anidulafungin, micafungin sodium, caspofungin diacetate for the mycelial forms of T. marneffei strains were more than 10 times higher than their yeast forms.

Discussion
T. marneffei has become one of the most common opportunistic pathogens in Southeast Asia, southern China and northeastern India [21]. It is a primary cause of morbidity and mortality in HIV-infected and other immunosuppressed patients who live in endemic areas including Malaysia [22]. Disseminated T. marneffei infection is associated with a high mortality rate in patients whose treatments were delayed [23].
Amphotericin B belongs to the polyene class of antifungals. It is fungicidal and approved by the United States Food and Drug Administration (FDA) to combat a broad range of fungal infections [24]. It was suggested to be used as initial treatment for patients with talaromycosis or severe stage of talaromycosis [25,26]. Therefore, amphotericin B was frequently included in the susceptibility testing. The MIC of amphotericin B against yeast and the mycelial form of T. marneffei was typically low. However, some MIC results were reported in high value. For instance, MIC ≤2 µg/ml of yeast form was reported by both Liu et al. [17] and Lei et al. [27]; however, MIC ≥ 24µg/ml in yeast and mycelial form were reported by Sekhon et al.
[28] and Sar et al. [19] respectively. Some of the yeast and mycelial phases of isolates in this study also showed MIC ≥ 24µg/ml, which correlates to the results mentioned above.
Azoles antifungals can directly inhibit the enzyme lanosterol 14-α demethylase from converting lanosterol to ergosterol. This leading to the increase in the cell membrane permeability and nally lead to cell death [29]. A recent study reported talaromycosis was successfully treated with a reduced dose of itraconazole instead of using amphotericin B or voriconazole [30]. Furthermore, the use of itraconazole is frequent particularly in lower-income countries as it has better tolerability, availability, less toxicity and is economical [11,[31][32][33]. Several in vitro studies had reported its active activities against yeast and the mycelial form of T. marneffei. The MIC of T. marneffei yeast and mycelial were ≤ 0.5 µg/ml [13,27,34,35]. The nding of this study also proposes that itraconazole has potential active activity against both mycelial and yeast phases of T. marneffei since the MIC 90 for both phases were <0.5µg/ml.
Voriconazole is a potent broad-spectrum triazole antifungal with satisfactory safety for combating various fungal infections [36]. The active activities of voriconazole against T. marneffei were mentioned by and Lau et al. [13] and Ouyang et al. [34] It was suggested to be used in the treatment of patients who could not tolerate amphotericin B and itraconazole [34,37]. The MIC of voriconazole was the lowest reading compared with uconazole, itraconazole, terbina ne and amphotericin B in Liu et al. [17] This is parallel to our study since voriconazole achieved the lowest MIC 90 against both phases of T. marneffei.
However, many patients were still died after treated with amphotericin B, itraconazole and voriconazole [8,[38][39][40]. In the year 2017, Lau reported that T. marneffei demonstrated high susceptibility against Posaconazole [13]. Posaconazole is bene cial in salvage therapy of various severe and refractory invasive fungal infections [41][42][43]. Similar to this study, the MIC reading of posaconazole was generally lower than 0.1µg/ml for both yeast and mycelial form.
Besides itraconazole, ketoconazole was suggested to be used in the treatment of mild to moderately severe talaromycosis [26]. This is due to the MIC being less than 0.5µg/ml from their study [26]. In addition, Kantipong and Walsh reported that the oral lesions caused by T. marneffei were resolved by the treatment of ketoconazole [44]. The results of the present study also supported these previous ndings as the MIC 90 for both phases of T. marneffei was <0.5µg/ml.
Terbina ne is an allylamine antifungal. It is fungicidal and able to inhibit enzyme squalene monooxygenase from synthesizing the sterol of the fungal cell membrane. As a result, the cell membrane will unable to grow and the fungus will be lysed eventually [45]. Terbina ne was one of the most effective antifungals with in vitro activity showing the lowest MIC values, which supports ndings of other studies. Similar to our ndings, McGinnis et al. reported the geometric mean of MIC for terbina ne against T. marneffei was as low as itraconazole [16]. Moreover, the terbina ne MIC was reported as ≤ 2 µg/ml [17]. Hence, the researchers suggested terbina ne might be effective against T. marneffei [16].
Echinocandin is a noncompetitive inhibitor of the formation of the enzyme 1,2-β and 1,6-β glucan synthase. It disrupts glucan and results in fungal cells being unable to maintain their shape and rigidity and hence leading to fungal cell lysis. In this study, echinocandin displayed very low activity in vitro against the mycelial form of T. marneffei. It indicates these echinocandin antifungals might have reduced activities against T. marneffei. Studies conducted by Lau et al. [13], Liu et al. [17] and Lei et al. [27] reported echinocandin was comparatively lower or no activity against T. marneffei yeast and mould. In contrast, Odabasi et al. [46] reported anidulafungin exhibits potential activity against the mycelial form of T. marneffei as the MIC was ≤2 µg/ml. However, the nding in this study indicated that echinocandin might be active against the yeast form but inactive against the mycelial form of T. marneffei.
Currently, no guidelines are available for the susceptibility testing of T. marneffei. Even though CLSI M38 and M27 are references for broth dilution antifungal susceptibility testing of mould and yeasts respectively; however, they have been unevaluated for T. marneffei yet [20,47]. Recent studies reported the susceptibility test using 10 4 CFU/ml yeast inocula in their studies [17,48]. Hence, the variation of different inoculum sizes when comparing the results with mould inocula can be avoided. However, no result for T. marneffei mould form was reported by them [17,48], thus the signi cant differences of MIC resulting from yeast and mould were unable to be compared. Therefore, this study used the same inocula size for both phases of T. marneffei to facilitate this comparison.
The growth form to be used for the test still remained unclear. Several studies reported the MIC using both mycelial and yeast form [13,19,26]; while others utilized the mycelial form only [35]. In the present study, some of the mycelial forms demonstrated greater MIC against antifungals. This phenomenon was also observed by Lau et al. [13] and Sar et al. [19]. This could be due to the production of red pigment, which exclusively appears when T. marneffei is grown as mould. The red pigment was reported to be very important for the production of citrinin [49], where it is hepatotoxic and nephrotoxic to humans [50].
To our knowledge, this is the rst study of susceptibility testing against Malaysian clinical isolates of yeast and mycelial phases of T. marneffei using the CLSI method. To emphasize, this is the rst study to determine the susceptibility pattern of T. marneffei against ravuconazole, isavuconazole and clotrimazole. In contrast, there are several limitations to this study. The sample size of the isolate was small within the study period and thus an accurate MIC 50 and MIC 90 were di cult to be determined. In addition, the results were unable to be interpreted as susceptible or resistant as there are no o cial breakpoints for T. marneffei according to the CLSI method. However, these results are bene cial and could contribute to limited antifungal database in Malaysia.

Conclusion
In conclusion, our data suggest all antifungals used in this study might possess potential antifungal activities against yeast T. marneffei; and therefore, could be useful in the clinical management of talaromycosis. In contrast, all antifungals except anidulafungin, micafungin sodium, caspofungin diacetate and 5-uorocytosine were shown inactive against the mycelial form of T. marneffei as the geometric means were >2.0µg/ml. More samples are required in future studies to ensure these data are reliable and useful to patients.