Characterization and susceptibility of non-albicans Candida isolated from various clinical specimens in Lebanese hospitals

Background Invasive fungal infections have presented a challenge in treatment. In the past, it was known that the frontrunner in such infections is Candida albicans with little emphasis placed on non-albicans Candida species (NAC). Studies worldwide have shown a rise in fungal infections attributed to non-albicans Candida species. The aim of this study is to describe the epidemiology of NAC infections along with an overview of resistance in Lebanese hospitals. Methods This is a two-year observational multi-central descriptive study. Between September 2016 and May of 2018, a total of 1000 isolates were collected from 10 different hospitals distributed all over the country. For the culture, Sabouraud Dextrose Agar was used. Antifungal Susceptibility was evaluated by determining the Minimum Inhibitory Concentration (MIC) in broth (microdilution) of the different antifungal treatments. Results Out of the 1000 collected isolates, Candida glabrata, being the most isolated species (40.8%), followed by Candida tropicalis: 231(23.1%), Candida parapsilosis: 103(10.3%), and other NAC species at lower percentage. Most of these isolates (88.67%) were susceptible to posaconazole, 98.22% were susceptible to micafungin, and 10% were susceptible to caspofungin. Conclusion The change of etiology of fungal infections involving a significant increase in NAC cases is alarming due to the different antifungal susceptibility patterns and the lack of local guidelines to guide the treatment. In this context, proper identification of such organisms is of utmost importance. The data presented here can help in establishing guidelines for the treatment of candida infections to decrease morbidity and mortality. Future surveillance data are needed.


. Introduction
The incidence and burden of fungal infections is rising globally. Fungal infections are a major concern for clinicians because it is associated with high morbidity and mortality, mainly in critical and immunocompromised patients. Serious and invasive Candida infections are usually hospital acquired. In the hospital setting, Non-albicans Candida species (NAC) are more frequently isolated (1).
Invasive candidiasis includes a variety of infectious conditions caused by Candida species. Invasive candidiasis is a serious infection that causes high mortality and morbidity. In the United States (US), around 25,000 cases of invasive candidiasis are reported annually (2). The most common and studied form of invasive candidiasis is candidemia, especially in intensive care patients (3). It remains a challenge to estimate the global incidence of candidemia and this is due to many factors including diagnostic techniques as well as the lack of surveillance systems for fungal infections (4). New diagnostic techniques are developing including Polymerase chain reaction and specific rapid antigen. Nevertheless, positive predictive values of non-culture techniques remain low while negative predictive values are high. Therefore, clinical suspicion of invasive fungal infections in combination with Candida diagnostics should be used in patients care. However, the reported annual incidence of candidemia in the US is around 9 cases per 100,000 (5). Candida species rank as the fourth most common cause of hospital-acquired bloodstream infections, after coagulase-negative staphylococci (CNS), staphylococcus aureus, and enterococcus spp. (6).
Candida albicans is the predominant isolate from patients with invasive candidiasis worldwide (7). However, a new threat has emerged over the last few decades, as NAC are increasingly recovered from patients. The most reported species of NAC include C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei (8).
Collectively along with C. albicans, these species are responsible for over 90% of the cases of invasive candidiasis (9). The frequency of each species varies with geographic differences in different countries (10-14), the local hospital epidemiology within the same country (15-17), the different units within the same hospital, underlying patient characteristics, and the antimicrobial treatment strategies and protocols (18,19). Nevertheless, the clinical importance of NAC species lies in the potential antifungal resistance which can lead to treatment failure and its consequences.
Several studies (20-25) have estimated the incidence rates of candidemia in the Middle East and North Africa countries. Candidemia incidence rate was estimated to be the highest in Qatar, with a calculated rate of (15.4/100,000) (21) and the lowest in Iran (0.34/100,000) (20). In a study done by Koehler et al., European incidence of candidemia was estimated to be 79 cases per day, of which an estimated 29 patients might have fatal outcome at Day 30 (26). There was a higher proportion of Candida spp. other than C. albicans in the decade from 2010 till 2019 in population-based data (26).
Echinocandin and azole-resistance is increasingly reported in non-albicans Candida from cases of invasive candidiasis (27,28). Exceptional resistance to antifungals in some Candida species, such as in Candida auris, constitutes a major threat to patients and has a significant impact worldwide. Candida's ability to form biofilm represents a problem in the context of antifungal drug-resistance.
Lebanon is a small country in the Middle East Region where a prominent level of antimicrobial use has been documented (29). The current compiled antimicrobial susceptibility data have shed light on increasing bacterial resistance trends in this country, which were found to be comparable with data from some Eastern and Southern European countries (29). For that reason, it was important to understand the local epidemiology and subsequently to establish guidelines for the appropriate identification and treatment of such infections as well as for their prevention. This multicenter study aimed at describing the epidemiology and distribution of NAC species in the context of the global data, as well as identifying and determining the antifungal susceptibility profiles of 1000 NAC clinical specimens collected from various clinical infections.

. . Samples and study population
A total of 1,000 clinical samples including urine, vaginal swabs, sputum, blood, cerebrospinal fluid (CSF) and miscellaneous samples were collected prospectively from all patients having a positive fungal culture and presenting to 10 hospitals located in different geographic areas of the country between September 2016 and May of 2018 according to standard procedures. More than one clinical sample from the same patient with the same identification and same susceptibility profile were considered duplicates, and therefore only the first isolate was included. All clinical samples were inoculated on Sabouraud dextrose agar (Oxoid, Basingstoke, UK) to which 50 µg/ml of Gentamycin was added to suppress the growth of bacterial contaminants. Inoculated plates were incubated at 37 • C for 72 hours aerobically, extended incubation was performed when needed. Isolates were identified by conventional methods using microscopic examination using KOH preparation, colonial morphology, and carbohydrate assimilation method using the API 20C Aux system (bioMerieux-Vitek, Hazelwood, Mo.).

. . Antifungal susceptibility testing
Antifungal Susceptibility testing was evaluated by determining the Minimum Inhibitory Concentration (MIC) in broth .
/fpubh. .  Antifungal solutions were standardized based on assays of the lots of antifungal powders. Antifungal stock solutions were prepared at concentrations of at least 1280 µg/mL or ten times the highest concentration to be tested, whichever was greater.
The antifungal agents tested were: Amphotericin B, Micafungin, Caspofungin, Anidulafungin, Voriconazole, Fluconazole, and Posaconazole). Antifungal powders were dissolved depending on the chemical properties of each one. Some were dissolved in DMSO diluted in RPMI (Amphotericin B, Ketoconazole, Itraconazole, Posaconazole, Voriconazole). The concentrations to be tested were based on the breakpoint concentrations and the expected results for the quality control strains. Based on previous studies, the following drug concentration ranges were used: amphotericin B, 0.0313 to 16 µg/mL; flucytosine, 0.125 to 64 µg/mL; ketoconazole, 0.0313 to 16 µg/mL; itraconazole, 0.0313 to 16 µg/mL; fluconazole, 0.125 to 64 µg/mL; and new triazoles, 0.0313 to 16 µg/mL.
Quality control strains included C. parapsilosis ATCC 22019, C. albicans ATCC 90028, and C. krusei ATCC 6258. RPMI 1640 medium was used as a Synthetic Medium for susceptibility testing. Zwitterion buffers were used to buffer the media to a pH of 7.0 ± 0.1 at 25 • C. All organisms were sub-cultured from sterile vials onto Sabouraud Dextrose Agar.

. . Data analysis and interpretation
Patients' privacy and Identities were not revealed, all data were coded for that purpose. Statistical analysis was performed using SPSS version 20. Descriptive statistics such as frequency and percentage of Candida species were calculated.

. . Ethical clearance
All ethical deliberations and responsibilities were appropriately addressed, and the study was conducted after the approval of the Institutional Review Board (IRB) of the Lebanese American University. (IRB# LAU.SOM.RH1.26/Apr/2016).
Among the 48 candidemia cases, 66.7 % had C. glabrata. Similarly, C. glabrata grew in 9 specimens among the 10 CSF specimens. Similarly, in the miscellaneous group (mostly abdominal and skin infections) the most common pathogens IE, Insufficient Evidence that the organism or group is a good target for therapy with the agent, ND, Not Determined (for statistical significance purposes, MIC90 was not determined when the number of isolates was lower than 10. were C. kefyr, Candida glabrata, C. tropicalis and C. parapsilosis ( Figure 1). Candida auris was not isolated in any of the specimen.
Susceptibility profile: Table 2 shows Candida spp. Isolates susceptibility to various antifungals. C. glabrata isolates were highly 100% susceptible to Anidulafungin, and Amphotericin B, 98.5 % susceptible to micafungin, but none was susceptible to Fluconazole ( Table 2). C. tropicalis isolates were 100% susceptible to Anidulafungin and Voriconazole and 99.6% to Amphotericin B. Only 4.3 % of C. tropicalis were susceptible to Fluconazole and 3.9 % to Pozaconazole. C. parapsilosis isolates were 100 % susceptible to Micafungin, Voriconazole, Anidulafungin and Amphotericin B. Only 6.8% were susceptible to Fluconazole and none to Pozaconazole. Multidrug resistance was not seen among any of the pathogens cultured. The data showed that the isolates found in blood and CSF were mostly C. Glabrata and C. tropicalis. These species had the highest pattern of resistance.

. Discussion
Fungi are increasingly recognized as important pathogens in critically ill and immunocompromised patients (32)(33)(34)(35)(36). The incidence of invasive candidiasis has increased over the past decade due to the increasing prevalence of immunosuppressive therapy, invasive surgical procedures, and use of indwelling medical devices (13). In addition, the increased use of broad-spectrum antibiotics leads to changes in the microbiome, shifting the balance toward fungi and more resistant strains of bacteria (37). Antifungal susceptibility is not uniform among different candida species, and some species are innately resistant while others acquire resistance to the first line of antifungals, Fluconazole and Echinocandins (38,39). Because of this increase in resistance, candida speciation and Surveillance of Candida infection has become a must for every country as well as each hospital. Accordingly, the Clinical and Laboratory Standards Institute (CLSI) has recently adopted species-specific minimum inhibitory concentration (MIC) breakpoints for Candida species and recommends speciation and antifungal susceptibility of candida species isolated from sterile sites and causing invasive fungal infections. High rates of morbidity and mortality are associated with invasive Candida infections. The rate of mortality from candidemia is about 30%, while directly attributable mortality is between 19 and 24% (40, 41). Treating these infections requires antifungals that are expensive, and this is considered a burden in our country. Table 3 summarizes the most common species in different countries around the world. Looking at the most common species in Lebanon, C. tropicalis was dominant in Lebanon with percentage ranging from 20 % to 45 % in some studies (56,57). However, our study showed that C. Glabrata was the most common pathogen detected in all sites.
. /fpubh. .        In a study done in one region in Lebanon on 93 Candida isolates, C. glabrata was the most common, followed by C. parapsilosis, and C. tropicalis which is similar to our results (64).
Invasive Candidiasis is a major healthcare problem associated with high mortality and cost. According to the country's susceptibility pattern described above, non-albicans species are increasing and are associated with reduced antifungal susceptibility. Thus, Echinocandins are the drug of choice in empirical treatment for these patients with risk factors for invasive candida infection. However, according to the literature de-escalation and the use of oral therapy are acceptable strategies to follow in the management of such patients. Voriconazole is also an acceptable alternative if the patient did not receive prior azoles therapy whether prophylaxis or therapeutic. Clearly, this data sheds light on proper management of patients with fungal infections. However, patients with vaginal infection who have C. glabrata need further studies and consideration of treatment since oral medications might not be the best choice as seen in our data. In addition, CNS infections should be treated with amphoteric B not Echinocandins because of lack of concentration in the CNS (65).
Newer technologies such as Maldi-tof-MS and molecular techniques are considered the most reliable for microbial identification. However, sugar fermentation-based techniques are still reliable and commonly used for yeast identification. In a study by Arastehfar (66), API 20C AUX correctly identified 83.7% of yeast isolates. Another study Using sequencing as a standard technique for NAC identification, 78.9% of the isolates were correctly identified by API 20C AUX while the Vitek 2 YST ID Card system yielded 71.8% and Bruker and Vitek proteomic techniques yielded 90.1% and 80.3% of correct identification (67). These studies, in addition to many others, show a high accuracy of yeast identification of sugar fermentation-based methods and support their use for yeast identification.
Invasive Candida infections has high mortality and the yield of culture remains low. Mucocutaneaous Candida infection and colonization have a high positive predictive correlation with invasive infection. Thus, any patient with risk factors of invasive candidiasis should be empirically or preemptively treated before susceptibility pattern in determined. This is why it is important to know the epidemiology and resistance patterns in order to direct our treatment properly especially in the ICU and in immunocompromised patients.
The importance of such studies is obvious. It can help in establishing guidelines of treatment for such infections. However, this should be complemented by continuous proper surveillance system to interpret the dynamic changes of the epidemiology. For example, it is important to note that lately Candida auris was reported in one of the tertiary centers in our country but not in others. Moreover, further studies about the epidemiology from animals and environmental candida species are needed as part of the One Health approach to decrease morbidity and mortality associated with this infection.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
The studies involving human participants were reviewed and approved by the Institutional Review Board (IRB) of the Lebanese American University (IRB# LAU.SOM.RH1. 26 informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.