Increasing Trends of Reduced Susceptibility to Antifungal Drugs Among Clinical Candida glabrata Isolates in Kuwait

Abstract

Among non-albicans Candida species, Candida glabrata is the leading cause of invasive infections in critically ill patients. It is intrinsically less susceptible to fluconazole/other azoles that limits therapeutic options. This study determined distribution of C. glabrata in clinical specimens and determined their susceptibility to fluconazole, caspofungin, and amphotericin B by E test. During 8-year period (2011–2018), 1,410 isolates were obtained from 1,410 patients including 600, 409, and 131 isolates from respiratory, urine, and bloodstream specimens, respectively. Proportion of C. glabrata isolates was nearly the same during the two 4-year periods. Demographic details were available from 731 patients and susceptibility data for 1,225 isolates. C. glabrata isolation from bloodstream, respiratory, and urine specimens was higher from elderly (>60 years) versus younger patients. More bloodstream and urine isolates were obtained from female patients, however, more respiratory isolates were recovered from male patients (p = <0.05). Resistance to all three drugs increased during 2015–2018 compared with 2011–2014 but was more pronounced for fluconazole (p = 0.001). More isolates with reduced susceptibility to fluconazole/amphotericin B were obtained from elderly patients versus younger subjects and urine versus respiratory samples (p = <0.05). Our data show increasing trends of reduced susceptibility to antifungals, particularly fluconazole, among clinical C. glabrata isolates in Kuwait. Most isolates with reduced susceptibility to fluconazole/amphotericin B were obtained from elderly patients and urine/respiratory samples with urinary tract appearing as the most favorable niche for antifungal drug resistance development. The study also highlights the need for continued surveillance and better antifungal drug stewardship to control resistance development in C. glabrata.

Introduction

The widespread use of antifungal agents among immunocompromised/immunosuppressed patients for prophylactic/empirical treatment has resulted in increased prevalence of infections caused by Candida species with reduced susceptibility to antifungal drugs.^1–3 Emergence of multidrug-resistant Candida auris is a recent classical example, which is now recognized as a significant bloodstream pathogen in many countries.^4–7 Although Candida albicans remains the most frequently isolated Candida species from blood and other clinical specimens worldwide, there is a gradual shift in favor of non-albicans Candida species.^2,8–10 Non-albicans Candida species tend to exhibit reduced susceptibility to one or more antifungal agents, thus posing therapeutic challenges that may lead to adverse clinical outcome.^5,6,10–15 Among non-albicans Candida species, Candida glabrata is the leading or second most common cause of candidemia and is intrinsically less susceptible to fluconazole and other azoles.^13,15–17 In many instances, resistance to fluconazole in C. glabrata isolates was also associated with reduced susceptibility to echinocandins.^15,18 This phenomenon of co-resistance with echinocandins has been described as a consequence of selection pressure caused by their increased therapeutic use in clinical practice in recent years.^12,15–20 In fact, because of the widespread use of echinocandins as first-line therapy, resistance rate to fluconazole has actually declined among invasive C. glabrata isolates in the United States.¹⁵ Echinocandins (caspofungin) were introduced in the therapy of invasive fungal infection in 2009 in Kuwait. Antifungal susceptibility data for six major Candida spp. isolates obtained from candidemia patients in Kuwait during 2006–2017 have recently shown that none of the 124 bloodstream C. glabrata isolates was resistant to caspofungin.²¹ It was, therefore, of interest to perform antifungal susceptibility testing of all clinical C. glabrata isolates (including isolates from invasive sites) cultured during 2011–2018 to determine resistance patterns to the three classes of antifungal drugs in Kuwait.

Materials and Methods

Isolation and identification of C. glabrata isolates

During the 8-year period (2011–2018), 1,410 C. glabrata isolates recovered from various clinical specimens obtained from 1,410 different patients were received in the Mycology Reference Laboratory, Department of Microbiology, Faculty of Medicine, Kuwait University for identification and antifungal susceptibility testing. The clinical specimens were taken from the patients after obtaining verbal consent as a part of routine patient care and diagnostic work-up at different hospitals across Kuwait and the results are reported on deidentified samples without revealing patient identity. Blood specimens were cultured in Bact T/Alert Blood Culture System (BD Diagnostics, Sparks, MD) and the culture bottles were incubated for up to 5 days at 37°C before they were considered as negative for yeast growth.²¹ Other specimens were cultured on Sabouraud dextrose agar (SDA) supplemented with chloramphenicol (50 mg/L) as described previously.²² The bloodstream isolates were also subcultured on SDA with/without additional supplements, as described previously.²³ Repeat isolates were also available for some patients and were used to ascertain the antifungal drug resistance patterns. Because this is a retrospective study that used clinical isolates obtained previously, it did not involve contact with patients and the results are reported on deidentified samples without revealing patient identity; ethical approval from the Ethical Committee of Health Sciences Center, Kuwait University, was not required.

Single colonies from each isolate were processed for phenotypic and/or molecular identification. Phenotypic identification was carried out by typical microscopic morphology, growth characteristics on Mast ID-CHROMagar Candida (Mast Diagnostics, Merseyside, United Kingdom), and Vitek2 yeast identification system (bioMérieux, Marcy l'Etoile, France), as described previously.²⁴ Molecular identification of 500 selected isolates was also carried out by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI TOF MS) and/or by PCR amplification of the internal transcribed spacer (ITS) region of rDNA, as described previously.^24–26 Further identity of the isolates that showed poor growth on SDA and/or on antifungal susceptibility (RPMI agar supplemented with 2 % glucose) medium was confirmed by sequencing of the ITS region and/or D1/D2 domains of rDNA by using panfungal primers, as described previously.^27,28

Antifungal susceptibility testing

In vitro susceptibility of C. glabrata isolates to antifungal agents was determined by E test (bioMérieux). The test was performed in accordance with the manufacturer's instructions and as described previously.²¹ Reference strains of C. albicans (ATCC 90028), Candida parapsilosis (ATCC 22019), and C. glabrata (ATCC90030 and CBS138) were used for quality control. Susceptibility breakpoints used for interpretation of susceptible, intermediate/susceptible dose-dependent and resistant strains were those recommended by the Clinical and Laboratory Standards Institute (CLSI)²⁹ and were as follows: fluconazole, susceptible dose-dependent <32 μg/mL and resistant ≥64 μg/mL; caspofungin, susceptible <0.12 μg/mL, intermediate 0.25 μg/mL, and resistant ≥0.5 μg/mL.^30,31 Because of lack of defined breakpoints for amphotericin B, isolates showing minimum inhibitory concentration (MIC) of <1.0 μg/mL were taken as wild-type and those with MIC ≥1 μg/mL as non-wild-type according to the epidemiological cutoff value.^30,31

Statistical analyses

Categorical variables are presented as absolute number. Statistical analyses were performed by using Fisher's exact test or chi-square test as appropriate and probability levels <0.05 by the two-tailed test were considered as significant. Statistical analyses were performed by using WinPepi software ver. 11.65 (PEPI for Windows, Microsoft, Inc., Redmond, WA).

Results

A total of 1,410 clinical isolates from 1,410 patients (the first isolate from each patient) were included in the study. All isolates were identified as C. glabrata sensu lato by Vitek2 yeast identification system. The results obtained from growth on CHROMagar Candida medium were inconclusive as some isolates yielded light purple or white colonies; however, all isolates (n = 500) analyzed by molecular methods (MALDI TOF MS analyses, multiplex PCR for the three C. glabrata complex species, C. glabrata sensu stricto-specific uniplex PCR, and/or PCR sequencing of the ITS region or D1/D2 domains of rDNA) were identified as C. glabrata sensu stricto. They were obtained from diverse clinical specimens, such as blood (n = 131, 9.29%), urine (n = 409, 29.01%), respiratory specimens (n = 600, 42.56%; comprising sputum, n = 312 [22.13%]; endotracheal secretions, n = 223 [15.82%], and bronchoalveolar lavage, n = 65 [4.61%]), vaginal swabs (n = 53, 3.76%), and other miscellaneous specimens (n = 217, 15.38%) (Table 1). The total number of C. glabrata isolates was nearly same during the two 4-year periods (706 in 2011–2014 vs. 704 in 2015–2018). Although the isolation of C. glabrata from bloodstream was more frequent (74 vs. 57) during 2015–2018 compared with 2011–2014, the difference was not statistically significant (p = 0.120).

Table 1.

Specimen-Wise Distribution of Candida glabrata Isolates from 1,410 Cases During 2011–2018

Clinical specimen	2011	2012	2013	2014	2015	2016	2017	2018	Total cases (%)
Blood	12	14	14	17	12	13	17	32	131 (9.29)
Urine	40	36	54	62	75	54	34	54	409 (29.01)
Sputum/oral swab	52	70	77	20	30	35	14	14	312 (22.13)
Endotracheal secretions	21	28	21	23	29	38	32	31	223 (15.82)
Bronchoalveolar lavage	3	5	6	8	14	7	8	14	65 (4.61)
Wound/pus/tissue	5	4	3	7	9	9	6	14	57 (4.04)
Ear/nose/throat swab	5	3	6	3	3	0	3	5	28 (1.99)
Fluid/drain/aspirate	3	6	10	4	12	10	9	7	61 (4.33)
Vaginal swab	4	6	10	6	5	9	5	8	53 (3.76)
Rectal swab	3	4	6	2	0	0	1	0	16 (1.13)
Catheter exit site	0	1	1	0	2	0	0	2	6 (0.42)
Percutaneous gastrectomy site	2	4	1	3	1	7	3	5	26 (1.84)
Others	2	3	4	2	5	3	2	2	23 (1.63)
Total cases	152	184	213	157	197	185	134	188	1,410

The demographic details of patients infected/colonized with C. glabrata were available for 731 subjects. The distribution of C. glabrata isolates among different categories of patients and among the three major specimen types (bloodstream, urine, and respiratory specimens) are given in Table 2. Overall, more isolates (n = 448) were recovered from elderly (>60 years old) patients compared with younger (<60 years old) subjects (n = 283). In addition, more isolates were recovered from female (n = 408) than male patients (n = 323). The proportion of female patients was higher in older (272 of 448, 60.7%) compared with younger patients (136 of 283, 48.1%) (p = 0.001), whereas the proportion of male patients was higher in younger patients (147 of 283, 51.9%) compared with older subjects (176 of 448, 39.2%) (p = 0.001). More isolates were recovered from elderly (>60 years old) patients compared with younger (<60 years old) subjects from bloodstream (44 vs. 28), respiratory specimens (165 vs. 106) and urine samples (180 vs. 69), as expected. Consistent with the overall trend, most isolates from bloodstream (44 of 72) were also obtained from female patients. However, the distribution of male/female patients yielding C. glabrata isolates from respiratory and urine samples yielded significantly different results. Nearly 80% (195 of 249) of C. glabrata isolates from urine specimens were obtained from female patients, whereas only 35.1% (95 of 271) isolates from respiratory samples were obtained from female patients (p = 0.001) (Table 2).

Table 2.

Distribution of 731 Candida glabrata Isolates Obtained from Patients of Different Age Groups and According to Their Isolation from the Three Most Common Specimen (Bloodstream, Respiratory, and Urine) Types

Age range (years)	Total No. of patients analyzed			Bloodstream specimens			Respiratory specimens^a			Urine specimens
Age range (years)	No. of cases	Male	Female	No. of cases	Male	Female	No. of cases	Male	Female	No. of cases	Male	Female
<1 to 9	22	13	9	4	1	3	2	2	0	5	3	2
10 to 19	14	8	6	3	1	2	4	3	1	5	0	5
20 to 29	40	20	20	3	2	1	15	10	5	8	2	6
30 to 39	30	13	17	2	1	1	8	4	4	8	2	6
40 to 49	71	37	34	6	1	5	32	25	7	16	4	12
50 to 59	106	59	47	10	3	7	45	32	13	27	8	19
60 to 69	155	60	95	17	8	9	53	33	20	62	10	52
70 to 79	158	63	95	17	5	12	66	38	28	57	14	43
80 to 89	113	45	68	9	6	3	42	27	15	49	9	40
90 to 99	22	8	14	1	0	1	4	2	2	12	2	10
Total	731	323	408	72	28	44	271	176	95	249	54	195

Respiratory specimens included sputum (n = 103), endotracheal secretions (n = 118), and bronchoalveolar lavage (n = 50).

The antifungal susceptibility testing was performed on 1,225 available (185 isolates were lost during storage) C. glabrata isolates (including 594 isolates collected during 2011–2014 and 631 isolates collected during 2015–2018) and the comparative data are provided in Tables 3 and 4. Although the number of non-wild-type isolates for amphotericin B (MIC >1 μg/mL) was higher during 2015–2018 compared with 2011–2014 (11 of 631 vs. 4 of 594), the difference did not reach statistical significance (p = 0.119) (Table 3). For fluconazole, the number of resistant isolates (MIC >64 μg/mL) was much higher during 2015–2018 compared with the 2011–2014 period and the difference (73 of 631 vs. 15 of 594) was highly significant (p = 0.001) (Table 3). In addition, the number of isolates with MICs >32 μg/mL but <64 μg/mL was higher (46 of 631) during 2015–2018 compared with the number of isolates (9 of 594) during 2011–2014 (p = 0.001). Consistent with this trend, there was a shift toward higher fluconazole MICs among isolates collected more recently (227 of 631 isolates during 2015–2018 compared with 66 of 594 isolates during 2011–2014 with MICs of 16–48 μg/mL, p = 0.001). This was also indicated by higher mode (12 μg/mL vs. 4 μg/mL) and mean MIC values (15.16 ± 68.13 μg/mL vs. 6.95 ± 43.04 μg/mL) during 2015–2018 compared with 2011–2014 (Table 3). Although the number of isolates with reduced susceptibility to caspofungin (MIC >0.5 μg/mL) was also higher during 2015–2018 compared with 2011–2014 (9 of 631 vs. 5 of 594), the difference was not statistically significant (p = 0.424) (Table 3). Similarly, a higher percentage of fluconazole-resistant isolates were obtained from elderly (>60 years) patients compared with younger (<60 years) subjects (42 of 448 vs. 23 of 283); however, the difference was not statistically significant (p = 0.596).

Table 3.

Antifungal Susceptibility Profile of Candida glabrata Isolates

Antifungal agent	Year of testing	No. of isolates	MIC values (μg/mL) by E test
Antifungal agent	Year of testing	No. of isolates	≤0.125	0.19	0.25	0.38	0.5	0.75	1	1.5	2	3	4	6	8	12	16	24	32	48	≥64	Range	Geometric mean	p^a
Amphotericin B	2011–2014	594	373	102	69	29	13	4	2	1	0	0	0	0	0	0	0	0	1	0	0	0.002–32	0.1 ± 2.41	0.119
Amphotericin B	2015–2018	631	319	157	78	42	17	7	2	5	3	0	0	0	1	0	0	0	0	0	0	0.002–8	0.12 ± 0.40	0.119
Fluconazole	2011–2014	594	1	1	1	5	1	3	0	6	12	16	164	112	115	76	43	14	9	0	15	0.016–256	6.95 ± 43.04	0.001
Fluconazole	2015–2018	631	1	0	1	2	1	4	1	6	6	13	30	68	98	101	84	96	26	20	73	0.125–256	15.16 ± 68.13	0.001
Caspofungin	2011–2014	594	352	158	58	21	3	1	1	0	0	0	0	0	0	0	0	0	0	0	0	0.002–1	0.12 ± 0.09	0.424
Caspofungin	2015–2018	631	362	222	26	12	5	3	0	1	0	0	0	0	0	0	0	0	0	0	0	0.002–1.5	0.11 ± 0.09	0.424

Resistant isolates are given in bold and mode values are italicized.

The p values were calculated for the difference in rate of resistance during the two periods (2015–2018 vs. 2011–2014).

MIC, minimum inhibitory concentration.

Table 4.

Comparison of Antifungal Susceptibility Profile of Candida glabrata Isolates Obtained from Different Clinical Specimens During 2011–2014 and 2015–2018

Antifungal drug/specimen	2011–2014					2015–2018
Antifungal drug/specimen	No. of isolates tested	MIC range (μg/mL)	GM ± SD	Resistant, n (%)	No. of isolates tested	MIC range (μg/mL)	GM ± SD	Resistant, n (%)	p^a
Amphotericin B
Blood	47	0.002–0.5	0.10 ± 0.1	0	74	0.002–0.75	0.12 ± 0.15	0
Urine	152	0.002–1.5	0.09 ± 0.16	2 (1.3)	131	0.004–8	0.13 ± 0.75	6 (4.6)
Sputum	187	0.002–0.75	0.1 ± 0.12	0	192	0.003–0.5	0.1 ± 0.1	0
Endotracheal aspirate	77	0.002–1	0.08 ± 0.15	0	129	0.016–1.5	0.13 ± 0.18	1 (0.7)
BAL	19	0.016–0.75	0.11 ± 0.15	0	43	0.002–1	0.11 ± 0.16	1 (2.3)
Vaginal swab	25	0.002–32	0.14 ± 6.36	1 (4)	27	0.008–0.5	0.12 ± 0.13	0
Others	87	0.002–0.5	0.08 ± 0.09	1 (1.1)	135	0.002–1.5	0.12 ± 0.21	3 (2.2)
Fluconazole
Blood	47	0.75–256	6.35 ± 36.58	1 (2.1)	74	3–256	17.29 ± 71.81	9 (12.2)
Urine	152	0.19–256	7.28 ± 49.07	8 (5.2)	131	0.125–256	16.61 ± 82.41	23 (17.5)	0.001
Sputum	187	0.38–256	6.63 ± 26.05	2 (1.1)	192	1–256	13.49 ± 45.2	6 (6.5)	0.017
Endotracheal aspirate	77	2–256	7.99 ± 48.9	4 (5.1)	129	0.38–256	15.7 ± 76.17	16 (12.4)
BAL	19	2–32	7.46 ± 7.3	0	43	1.5–256	12.2 ± 38.24	1 (2.3)
Vaginal swab	25	1.5–32	7.01 ± 7.68	0	27	0.75–256	16.6 ± 77.7	4 (14.8)
Others	87	0.125–32	6.08 ± 5.36	0	135	0.25–256	14.68 ± 64.82	14 (10.3)	0.001
Caspofungin
Blood	47	0.003–0.38	0.11 ± 0.07	0	74	0.002–0.5	0.1 ± 0.07	1 (1.3)
Urine	152	0.008–0.5	0.12 ± 0.08	1 (0.6)	131	0.006–1.5	0.13 ± 0.15	5 (3.8)
Sputum	187	0.002–0.5	0.12 ± 0.07	1 (0.5)	192	0.008–0.38	0.11 ± 0.07	0
Endotracheal aspirate	77	0.016–1	0.13 ± 0.12	1 (1.2)	129	0.008–0.5	0.11 ± 0.08	2 (1.5)
BAL	19	0.064–0.75	0.14 ± 0.14	1 (5.2)	43	0.016–0.75	0.12 ± 0.1	1 (2.3)
Vaginal swab	25	0.002–0.19	0.08 ± 0.05	0	27	0.032–0.25	0.14 ± 0.05	0
Others	87	0.004–0.5	0.12 ± 0.08	1 (1.1)	135	0.008–0.38	0.11 ± 0.06	0

The p values were calculated for the difference in rate of resistance during the two periods (2015–2018 vs. 2011–2014). Only p values <0.05 are shown.

BAL, bronchoalveolar lavage; GM, geometric mean; SD, standard deviation.

Comparative antifungal susceptibility data of C. glabrata isolates recovered from different clinical specimens during 2011–2014 and 2015–2018 are given in Table 4. The non-wild-type isolates for amphotericin B (MIC >1 μg/mL) were mostly obtained from urine specimens and their number was higher during 2015–2018 compared with 2011–2014 (6 of 631 vs. 2 of 594); however, the difference was not statistical significant (p = 0.150) (Table 4). The mean MIC values of fluconazole for C. glabrata isolates were two to three times higher from all clinical specimens during 2015–2018 compared with isolates from 2011–2015 (Table 4). Consequently, more fluconazole-resistant isolates were obtained during 2015–2018 compared with 2011–2015 from all specimen types and the differences were also statistically significant (p < 0.05) for urine, sputum, and other miscellaneous specimens (Table 4). Although C. glabrata isolates with reduced susceptibility to caspofungin were distributed across various clinical specimens during 2011–2014, most (7 of 9) of the isolates during 2015–2018 were obtained from urine and endotracheal secretions (Table 4). Only three multidrug-resistant isolates were obtained; one isolate in each 4-year period with reduced susceptibility to fluconazole and amphotericin B and one isolate with reduced susceptibility to fluconazole and caspofungin during 2015–2018. Repeat isolates recovered from some patients yielded the same results as the first isolate, as expected.

Discussion

Although C. glabrata does not form pseudohyphae, it exhibits reduced susceptibility to azole antifungal agents and also has several virulence attributes that are required for a successful colonizer and a fungal pathogen.^18,32,33 Among non-albicans Candida species, C. glabrata is the second most important cause of vulvovaginitis, candidemia, and invasive candidiasis in susceptible patients in many countries.^{9,12,13,15,33–37} C. glabrata is also among the four most frequently isolated Candida spp. from candidemia patients in Kuwait.²¹ The echinocandins were introduced in 2009 to treat invasive fungal infections in Kuwait. In this study, 1,225 of a total of 1,410 clinical isolates obtained from diverse clinical specimens from 1,410 patients collected during an 8-year period (representing all C. glabrata isolates collected during 2011–2018) were tested to see whether resistance to fluconazole is also associated with reduced susceptibility to echinocandins, a phenomenon recognized recently in some countries.^{12,15–20,38}

All randomly selected isolates (n = 500), chosen from a total of 1,410 C. glabrata sensu lato strains included in this study, were identified as C. glabrata sensu stricto by a combination of molecular methods. Candida nivariensis and Candida bracarensis, two other phenotypically similar species belonging to C. glabrata complex have never been isolated in Kuwait despite the fact that extensive efforts were made for their detection.²⁴ Although C. glabrata is more commonly associated with urogenital infections,^32–34,37 the largest proportion of our isolates (600 of 1,410 or 42.5%) were obtained from respiratory specimens including 65 isolates from invasive samples. Although clinical details of these patients were not available that could help to differentiate colonization from infection, our data are in line with the increasing role of C. glabrata as a respiratory pathogen.^12,35,36 Our data also showed that the role of C. glabrata in candidemia in Kuwait is increasing as its isolation from bloodstream was more frequent (74 of 704, 10.5%) during 2015–2018 compared with 57 of 706 (8.1%) during 2011–2014; however, the difference did not reach statistical significance (p = 0.120).

Previous studies have shown that the prevalence of C. glabrata and its role in human disease is predominantly dependent on patient characteristics. This species is isolated more frequently from elderly patients (>60 years) as compared with younger subjects.^{12,16,32,33,39} The risk factors associated with greater frequency of C. glabrata colonization/infection in elderly patients are not fully understood. History of treatment with antifungal drugs, comorbidities, and underlying conditions among susceptible patients, ability of C. glabrata to persist under stressful conditions, and its preference to colonize certain anatomical sites (such as oral cavity of denture wearers) may be contributing factors to this association.^32,33,40,41 Our results are consistent with these observations as most (448 of 731, 61.3%) C. glabrata isolates for which patient details were available were also isolated from elderly (>60 years old) patients. An interesting observation of our study was that the greater number (408 of 731, 55.8%) of C. glabrata isolates and nearly 80% of urine isolates were obtained from female patients. This was also reflected in higher number (44 of 72, 61.1%) of candidemia cases in female patients and most (25 of 44, 56.8%) of these bloodstream infections occurred in elderly (>60 years old) women. It is probable that vagina (and perhaps also the female urinary tract) that provides an additional niche for C. glabrata colonization and infection in women^33,37 increases the risk of candidemia in female patients. On the contrary, Arastehfar et al.⁴² recently reported no difference in the prevalence of candidemia owing to C. glabrata between male and female patients in neighboring Iran.

Antifungal resistance among non-albicans Candida species is on the rise and recent emergence of multidrug-resistant C. glabrata is a cause of growing concern.^{15–20,38,43} In a recent study based solely on bloodstream isolates of six major Candida species isolated over a 12-year period (2006–2017) in Kuwait, we recently reported that 17 of 189 (9%) C. glabrata isolates were resistant to fluconazole.²² However, when the data were analyzed for two 6-year periods (i.e., 2006–2011 and 2012–2017), it became apparent that the rate of resistance had increased from 4.1% in the first-half to 12.2% in the latter half of the study.²¹ The antifungal susceptibility data presented in this study are more broad based as they provide information about the occurrence and distribution of C. glabrata in different clinical specimens and are also correlated with patient characteristics such as their age and gender. The overall prevalence of resistance to fluconazole among 1,225 C. glabrata isolates was 7.2% but was much higher (11.6%) among isolates obtained during 2015–2018 compared with the rate of 2.5% among isolates collected during 2011–2014 (p = 0.001).

Our results are contrary to the data reported recently from a large study involving invasive C. glabrata isolates from diverse geographical locations.¹⁵ Although fluconazole resistance was recorded in 8.1% of C. glabrata isolates obtained during the entire study period (2006–2016), resistance rates of 8.6%, 7.6%, 10.1%, and 5.6% were recorded during different subperiods of 2006–2008, 2009–2011, 2012–2014, and 2015–2016, respectively.¹⁵ More importantly, resistance rate that peaked at 10.1% during 2012–2014 declined nearly twofold to 5.6% during 2015–2016.¹⁵ This decline has been attributed to the increasing use of echinocandins over fluconazole as first-line therapy for invasive candidiasis in most industrialized countries.^15,44,45 Early treatment with echinocandins followed by fluconazole therapy for Candida spp. isolates susceptible to the latter drug have also proven to be cost-effective in addition to improving survival.⁴⁶ However, in many other countries, including Kuwait, fluconazole is still widely used for several reasons (such as cost, drug safety profile, ease of administration, antifungal availability, etc.).^47,48 Another reason that has probably contributed to the continued use of fluconazole is owing to very low rate of resistance to this drug among the three other major Candida spp. isolates in Kuwait.^21,49,50 These factors seem to have contributed to the increasing trend of resistance of C. glabrata to fluconazole in recent years in Kuwait. Although not statistically significant, another interesting observation of our study was the isolation of a greater number of fluconazole-resistant isolates from elderly patients compared with younger subjects (42 of 448 vs. 23 of 283, p = 0.596). Again, our findings are contrary to the data reported from the United States. Although the incidence of invasive candidiasis increased steadily with increasing age of the patients, the rate of resistance to fluconazole was lowest in isolates from >70-year age group, whereas the highest rates (11.8% resistant) were seen in the 20- to 49-year age group.^15,51

The resistance to echinocandins among Candida species is uncommon except in C. glabrata.^15,17,33,43 Infections with C. glabrata respond differently to three echinocandins in vivo with/without resistance conferring mutations in FKS genes.⁵² Furthermore, spontaneous mutational frequency and mutation rates in FKS genes vary with the three echinocandins in C. glabrata.⁵³ Although resistance to echinocandins among C. glabrata isolates in Kuwait is still low compared with fluconazole, it is showing an upward trend as 9 of 631 isolates during 2015–2018 were resistant to caspofungin compared with only 5 of 594 during 2011–2014. Of interest, 7 of 9 caspofungin-resistant isolates were obtained from urine and endotracheal secretions. Although data on treatment history was not available, it is reasonable to assume that low level exposure to echinocandins in these anatomical niches may have contributed to the development of echinocandin resistance, as was recently described for Candida tropicalis.⁵⁴ Although echinocandin resistance in C. glabrata is often associated with cross-resistance to azole antifungals yielding multidrug-resistant strains,^19,43 only one caspofungin-resistant isolate in our study exhibited cross-resistance to fluconazole. The widespread use of echinocandins and azoles for prophylaxis may be a significant underlying factor contributing to increasing role of C. glabrata as a bloodstream pathogen including breakthrough infections.^1,17,20

The number of non-wild-type isolates for amphotericin B was also higher during 2015–2018 compared with 2011–2014 that were mostly obtained from urine specimens. The urine isolates appear to exhibit reduced susceptibility not only to amphotericin B (9 of 15, 60% isolates with reduced susceptibility to amphotericin B were recovered from urine samples) but also to fluconazole (31 of 106, 35.2% fluconazole-resistant isolates were recovered from urine samples) and caspofungin (6 of 14, 42.9% caspofungin-resistant isolates were recovered from urine samples). Although the molecular basis of resistance to fluconazole and echinocandins among C. glabrata isolates from Kuwait has not been studied in detail, reduced susceptibility to amphotericin B, as reported in few other studies,^55,56 has been attributed to mutations in ERG2 and ERG6 genes involved in ergosterol biosynthesis.⁵⁷

Our study has a few limitations. (1) Since this is a retrospective study, demographic information pertaining to age, gender, underlying conditions and so on was not available for all patients. (2) The treatment history of patients yielding resistant isolates and outcome were not available. (3) The antifungal drug susceptibility testing was performed by E test and not by the reference broth microdilution method and susceptibility testing for another echinocandin, micafungin, was not performed.

In conclusion, this study provides information on occurrence and distribution of C. glabrata in different clinical specimens in Kuwait and analyses the impact of therapeutic use of caspofungin, fluconazole, and amphotericin B in clinical practice on its susceptibility profile over an 8-year period. Increasing trends in the rate of resistance to antifungal drugs, particularly fluconazole and caspofungin are important findings. Predominance of C. glabrata in elderly age group and its greater association with female patients was also apparent. Three multidrug-resistant isolates of C. glabrata were also identified and their emergence in Kuwait is a cause of concern. The study underscores the need for continued surveillance and implementation of antifungal drug stewardship policies to limit drug resistance development in this important fungal pathogen.

Footnotes

Acknowledgments

The technical support provided by Sandhya Vayalil and Omar Al-Musallam is thankfully acknowledged. Authors are also thankful to clinical microbiologists and supporting staff of different hospitals for sending yeast isolates to Mycology Reference Laboratory for identification and antifungal drug susceptibility testing.

Disclosure Statement

No competing financial interests exist.

Funding Information

No specific funding was obtained for this study.

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