Azole non-susceptible C. tropicalis and polyclonal spread of C. albicans in Central Vietnam hospitals

Introduction: Candida spp. are responsible for infections ranging from local to systemic, and resistance to antifungal first-line therapy is increasing in non-albicans Candida species. We aimed to determine the etiology of candidiasis and the antifungal resistance of Candida spp. isolated in Hue hospitals, Central-Vietnam. Methods: Species identification was performed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry supported by fungal internal-transcribed-spacer amplification and sequencing. Antifungal susceptibility testing was performed by disk diffusion method and minimum inhibitory concentrations of azoles, caspofungin, and amphotericin B against C. tropicalis were determined by broth microdilution. Polymorphism of erg11 gene associated with fluconazole resistance was carried out by polymerase chain reaction and sequencing. Multilocus sequence typing (MLST) was used for typing selected C. albicans isolates. Results: Overall, 196 Candida isolates were detected, mostly C. albicans (48%), followed by C. tropicalis (16%), C. parapsilosis (11%), C. glabrata (9%), C. orthopsilosis (6%) and to a lesser extent another eight species. High rates of resistance to fluconazole and voriconazole (18.8%) were observed in C. tropicalis with five isolates co-resistant to both agents. Y132F and S154F missense mutations in the ERG11 protein were associated with fluconazole-resistance in C. tropicalis (67.7%). Resistance to caspofungin was found in one isolate of C. albicans . MLST identified a polyclonal population of C. albicans with multiple diploid sequence types, and with few lineages showing potential nosocomial spread. Conclusions: Resistance to triazole agents should be considered in C. tropicalis infections in the studied hospitals, and surveillance measures taken to avoid Candida diffusion.


Introduction
Candida species are human commensals, commonly found on the mucosal surfaces of gastrointestinal and genitourinary tracts, skin, under fingernails, lung, and gut mycobiota [1]. However, Candida spp can act as opportunistic invaders capable of causing different diseases, ranging from commonly encountered superficial infections to systemic diseases in humans [2]. The switch from commensalism to pathogenesis in Candida spp. is influenced by both fungal and host factors [3], including immune status, co-morbidities, and underlying conditions, as well as exposure to certain medications and medical devices [4].
C. albicans represents one of the most common cause of hospital-acquired bloodstream infections (BSI), which leads to increased morbidity and mortality, prolongation of hospital stays, and increased hospital costs [5,6]. However, an increasing trend of invasive infections caused by non-albicans Candida (NAC) group has been observed, including several Candida species, such as C. tropicalis, C. parapsilosis, C. krusei, C. glabrata and C. stellatoidea [4,7,8]. In Vietnam, fungal opportunistic diseases are growing due to the increased number of immunocompromised patients [9,10]. Although physicians have dealt with Candida infections more frequently, there are limited systematic epidemiological data on Candida in the country [11][12][13], even less from Central Vietnam.
In the last few years, the frequency of resistance to antifungal therapy continued to increase, and data reported globally indicated higher levels of resistance from NAC species than C. albicans [14][15][16][17]. Among antifungal drugs, azoles are widely used to treat almost all superficial and deep mucosal and disseminated fungal infections caused by Candida spp. [18]. As a consequence, the extensive use of fluconazole led to the development of resistance, resulting in therapeutic failures [19], which makes the treatment of candidemia a huge challenge for physicians [20].
The most common mechanisms leading to Candida spp. resistant to fluconazole as well as other azole compounds include alteration of the target enzyme, the cytochrome P-450 lanosterol 14α-demethylase (ERG11), which is responsible for the synthesis of ergosterol. Mutations in the erg11 gene can alter the enzyme's structure and reduce its binding affinity for the drugs, making the fungal cells less susceptible to their effects [18]. Thus, monitoring for the presence of ERG11 mutations in Candida isolates can inform antifungal therapy decisions and guide the selection of alternative treatment options, such as echinocandins or polyenes, which are not affected by ERG11-mediated resistance. Understanding the molecular mechanisms of azole resistance can also aid in the development of new antifungal agents that target alternative pathways or proteins, thereby mitigating the impact of ERG11 mutations on antifungal therapy efficacy. The development of resistance to fluconazole and voriconazole can also occur through overexpression of efflux pump genes such mdr or cdr by reducing the intracellular concentrations of fluconazole and voriconazole [21].
The main purpose of this study was to investigate the etiology of candidiasis and the antifungal susceptibility of Candida spp. isolated from patients of the Hue University of Medicine and Pharmacy Hospital (HUMPH), and Hue Central Hospital (HCH) in Central Vietnam during October 2012 and June 2016. Moreover, molecular methods were utilized to investigate erg11 mutations in fluconazole-resistant isolates and to type selected C. albicans isolates.

Study population
Sampling was conducted during October 2012 and June 2016 from 163 patients in 10 departments at HUMPH (Dermatology, Endoscopy, Intensive Care, Internal Medicine, Obstetrics, Oncology, Ophthalmology, Otorhinolaryngology, Pediatrics, and Surgery) and in two departments at HCH (Hematology and Pediatric). Candida isolates were classified into four groups depending on the patient's status, including Candida colonization, cutaneous candidiasis, mucosal candidiasis, or systemic candidiasis.
Candida colonization is typically asymptomatic and it was classified through the isolation of Candida from clinical specimens, such as urine, sputum, stool, gastric, external ear, bronchoalveolar from patients without clinical signs and symptoms of candidiasis that recovered without antifungal therapy. In contrast Candida infections were classified by the presence of clinical signs and symptoms, such as fever, pain, inflammation, and discharge, along with the detection of Candida in relevant clinical specimens, such as blood, endotracheal aspiration, urine or tissue samples, and the patients were cured with antifungal therapy. Mucosal candidiasis included oral, vaginal, esophageal and cornea candidiasis. Onychomycosis and skin candidiasis patients belonged to cutaneous candidiasis. Systemic candidiasis included candidemia, peritoneal candidiasis, and pulmonary candidiasis.
Samples were first cultured onto Sabouraud Dextrose Agar medium plates (Oxoid Ltd, Basingstoke UK), yeast colonies were then subcultured in Brilliance Candida agar plates (Oxoid Ltd, Basingstoke UK) to screen mixed isolates. Seventeen samples had mixed isolates, of which 15 samples were two mixed isolates, and two samples were three mixed isolates. Therefore, 196 Candida isolates were collected from 177 samples. All Candida isolates were stored at -80 °C and subcultured in Sabouraud media for antifungal susceptibility testing.
Amplification conditions were as follows: initial denaturation at 94 °C for 5 min, followed by 30 cycles, each consisting of 30 sec at 94 °C for denaturation, 40 sec at 50 °C for annealing, and 50 sec at 72 °C for elongation, and a final elongation step of 10 min at 72 °C. The erg11a and erg11b amplicons, of 834 bp and 816 bp respectively, were then quantified, purified, and sequenced as described above. The nucleotide sequences were translated into amino acid sequences, aligned, and compared with a C. tropicalis ERG11 reference sequence (NCBI GenBank accession: AY942645) using Geneious 4.8.

Data analysis
The data were analyzed using Window SPSS 20.0. A p value less than 0.05 was considered to be statistically significant.

Ethical approval
This study was approved by the Ethics Committee of Hue University of Medicine and Pharmacy (code DHH2015-04-44).
In contrast, C. albicans (48.9%) was more represented in mucosal candidiasis compared to NAC spp. (25.5%) (p = 0.0007). No statistically significant differences between C. albicans and NAC spp. were found in cutaneous and invasive candidiasis (Table 1).
Among NAC colonization, 8 different species were detected as showed in Table 2, with C. albicans and C. tropicalis the most encountered species. Also, the distribution of Candida species by disease types is summarized in Table 2, with the dominance of C. albicans from mucosa diseases. On the contrary, NAC spp. showed a higher frequency of isolation from onychomycosis, and some species caused invasive candidiasis ( Table 2). Predominant Candida species from intensive care unit (ICU) patients (n = 27) were C. albicans (n = 13), C. tropicalis (n = 13) and C. parapsilosis (n = 1).

Antifungal susceptibility of Candida spp. isolates
The antifungal susceptibility results by disk diffusion method are shown in Table 3. C. albicans and C. parapsilosis isolates were susceptible to fluconazole. Resistance to fluconazole was found in C. tropicalis (12.5%) and C. glabrata (11.7%). C. tropicalis isolates were also resistant to voriconazole (18.8%) compared to the other Candida spp. One isolate of C. tropicalis was identified from blood and found to be susceptible to all antifungal drugs tested, with MIC values of 0.25 µg/mL for amphotericin B, 2 µg/mL for fluconazole, 0.008 µg/mL for itraconazole, 0.06 µg/mL for voriconazole, and 0.125 µg/mL for caspofungin.

Mutations in ERG11 protein associated with fluconazole resistance in C. tropicalis
The 67.7% of fluconazole-resistant isolates of C. tropicalis showed two mutations in the erg11 gene (A395T and C461T), corresponding to Y132F and S154F amino acids substitution in the ERG11 protein.

C. albicans MLST
Twenty-one C. albicans isolates selected from different sources and departments were typed by MLST as shown in Table 5 (Table 5). Nine clades plus 1 singleton were identified, with clade 5 (n = 5) and clade 1 (n = 3) the most common (Table 5).

Discussion
During the course of this study, we determined the etiology and the antifungal resistance of Candida spp. that have been isolated from hospitalized patients in two hospitals in Hue, Central Vietnam between October 2012 and June 2016. Among the 196 isolates, a large variety of Candida species was detected, with C. albicans, being the most prevalent followed by C. tropicalis, C. parapsilosis, C. glabrata, C. orthopsilosis, and C. krusei, as also previously reported from other countries [7,17,29,30]. Moreover, C. digboiensis, C. blankii, and C. norvegensis species were detected in Vietnam for the first time; the last two rarely detected in other countries [31][32][33].
C. albicans remains the most common Candida species reported worldwide [6,7,16,17,30,34], while the distribution relative to other species varies in different geographical areas with C. glabrata the most frequent species in Northern Europe and the USA [6], C. parapsilosis in Italy, Spain and Brazil [6,29], and C. tropicalis in Asian countries [17,30,35], especially in tropical southeast Asian countries [13,[36][37][38]. In this study, C. albicans was dominant in mucosal diseases and it was the most isolated species in invasive candidiasis, even if no a statistically significant difference was observed compared to NAC. The presence of NAC species (C. tropicalis, C. parapsilosis and C. orthopsilosis) was higher in Candida colonization.
C. albicans isolates were susceptible to all antimycotics tested; only one isolate was resistant to caspofungin. We found remarkable resistance of C. tropicalis to fluconazole and voriconazole (18.8%) and also to a lesser extent to itraconazole (6.2%). Our results are in line with studies conducted in other Asian countries such as China (2011-2021) [17], Asia-Pacific region (2016) [37], Australia (2017) [38], and Japan (2019) [16]. The isolates of C. tropicalis in our hospitals were less resistant than those in Ho Chi Minh and Ha Noi cities of Southern and Northern Vietnam, respectively [37]. C. tropicalis is reported as one of the four major Candida species responsible for candidemia worldwide [39]. It has been described as the first species in Vietnam [12]. It has increased dramatically in the last years due to the development of resistance to fluconazole [34,40], especially in Asia than in North America or Europe [35,41], resulting in higher mortality compared to C. albicans [40]. Nevertheless, some studies have indicated that fluconazole resistance has been increasing in C. tropicalis in Europe [42,43]. Fluconazole is usually used to treat systemic mycosis in Vietnam [44], particularly in ICU [11], where impaired immunity of patients and prolonged stay in hospitals facilitate the development of invasive mycoses. When comparing disk diffusion and broth microdilution methods in testing C. tropicalis, there was a good concordance for voriconazole. Conversely, compared to disk diffusion testing, a greater level of resistance to fluconazole was determined by broth microdilution in the case of C. tropicalis isolates that displayed susceptibility-dependent doses and were classified as resistant by broth microdilution. In nearly 70% of C. tropicalis, fluconazole-resistance was associated with ERG11 protein missense mutations Y132F and S154F, in accordance with previous studies [45,46]. Regarding resistance to C. glabrata, our results were similar to reports from European countries [42,43] and Japan [16].
Although few numbers of isolates of C. krusei and C. guilliermondii were isolated in this study, we found that C. krusei was susceptible to voriconazole and caspofungin, while C. guilliermondii was susceptible to caspofungin.
In 2018, C. tropicalis isolated from ICUs of our hospitals showed a higher fluconazole resistance (data not shown). Thus, these hospitals should be aware of possible Candida treatment failures because of a noticeable resistance to azole observed in NAC isolates (C. tropicalis, C. glabrata). Moreover, MLST highlighted a polyclonal nature of selected C. albicans isolates, belonging to several DSTs and clades, with few detected in subsequent years within the departments, suggesting their possible nosocomial spread.
Some studies have indicated that clade 1 was the most common C. albicans clade in Asiatic countries [47][48][49], with other dominant clades varying by countries; clades 6 and 17 in China [49], clade 4, 12 and 18 in Korea [48], clades 3, and 17 in Taiwan and Thailand [50,51]. Thus, the current study described the circulation of "Asian clades" (clades 1, 3, 4, 5, 12 and 17 and 18) in Central Vietnam hospitals, including the presence of isolates from clade 5, DST 2933 and DST 768, previously detected in Japan [52]. A better understanding of the risk factors associated with nosocomial transmission of Candida infections by healthcare personnel will significantly contribute to limiting their spread.

Conclusions
Our results highlighted a variety of Candida species in Hue hospitals, with Candida albicans prevailing in the majority of cases. The study underscores that healthcare professionals in Hue hospitals should be vigilant in managing Candida colonization and infections, particularly in light of the emergence of fluconazole resistance in C. tropicalis and C. glabrata. Rational use of azole drugs and improved surveillance can help prevent Candida infections and reduce their spread in healthcare settings, ultimately improving patient outcomes.