Etiology of invasive candidosis agents in Russia: a multicenter epidemiological survey

Abstract

A multicenter prospective epidemiological survey on the etiologic agents of invasive candidosis was conducted in Russia in the period of 2012–2014. Samples were collected from 284 patients with invasive candidosis and Candida species isolated by culture. The species were identified by DNA sequencing and MALDI-TOF mass-spectrometry. A total of 322 isolates were recovered, in which 96% of Сandida species belonged to six major species, namely, C. albicans (43.2%), C. parapsilosis (20.2%), C. glabrata (11.5%), C. tropicalis (9.6%), C. krusei (6.2%), and C. guilliermondii (5.3%). Most Candida species were isolated from blood samples (83.23%). Notably, the prevalence rate of C. albicans reduced from 52.38% to 32.79% (2012 vs. 2014) (P=0.01) whereas that of non-C. albicans increased from 47.62% (2012) to 67.21% (2014) (P<0.01). Species distribution differed among geographical regions; specifically, the prevalence rate of C. albicans as an etiologic agent of invasive candidosis in Siberian Federal region was significantly higher than that in other Federal regions. Results indicated a shift from C. albicans to non-C. albicans. Therefore, a detailed investigation on the contributing factors and appropriate treatment of invasive candidosis is needed.

Keywords

prevalence / Сandida species / invasive candidosis / nosocomial / etiology / MALDI-TOF mass-spectrometry / candidemia

Introduction

Candida is an extremely important pathogen and can cause severe life-threatening diseases [1–3], particularly invasive candidosis that is associated with high morbidity and mortality ranging from 25%–60% [4–6]. According to the Leading International Fungal Education study, the global burden of invasive candidosis is approximately 750 000 patients annually, with lethal outcomes of nearly 350 000 [7]. The prevalence rate of invasive candidosis in Russia is 11 840 cases per year, 8.29 cases per 100 000 of population. The highest prevalence rate of invasive candidosis is reported in Pakistan (38 795 cases, 21 cases per 100 000), followed by Brazil (28 991 cases, 14.9 cases per 100 000). The lowest incidence figures are reported in Austria (206 cases, 2.1 per 100 000) and Portugal (231 cases, 2.2 per 100 000) [7,8]. Candida species account for 8%–10% of all nosocomial bloodstream infections in the United States [9]. The attributable mortality rate of invasive candidosis is estimated to be>30%, with a crude mortality rate of>50% [10]. Over the last five decades, invasive candidosis has increased with the advances in medicine and the growing population of immunecompromised hosts [11]. Candida species are among the four top causes of healthcare-associated bloodstream infections [10,12]. The most prevalent non-C. albicans species among etiologic agents of invasive candidosis is С. glabrata in Northern Europe, USA, and Canada while С. parapsilosis in South Europe, Asia, and South America [10,13,14]. However, the detailed prevalence and species distribution of invasive candidosis etiologic agents in Russia has not been reported. Thus, the current study aims to address the research gap on the prevalence and species distribution of etiologic agents of nosocomial invasive candidosis in Russia.

Materials and methods

Candida strain isolation

Sample collection and diagnostics procedures were performed in accordance with the ESCMID guideline for the diagnosis and management of Candida diseases [15]. The samples were collected from 284 patients with invasive candidosis and who were hospitalized in 37 clinics (mostly, ICUs) of 6 Federal regions of Russia from 2012 to 2014. The samples collected were blood, peritoneal, pleural fluids, abscesses of deep tissues, cerebrospinal fluid, biopsy and autopsy of deep tissues, and bile and synovial fluid. Blood samples were cultured in BactAlert System (BioMerieux) and Oxoid Signal Blood Culture System (Oxoid) at 35 °C–37 °C for 10 days and plated on Sabouraud dextrose agar (SDA) for isolation of yeast colonies. Other clinical materials were cultured directly on SDA plates. The isolates for species identification were sub-cultured on SDA slants at 37 °C for 24 h (at 28 °C in the case of C. lipolytica).

Species identification

Candida species were identified by molecular-genetic analysis and MALDI-TOF mass-spectrometry.

Molecular-genetic analysis

Molecular-genetic identification was conducted by DNA sequencing methodology recommended by the Clinical and Laboratory Standards Institute documents (ММ18-А, CLSI) [16]. The yeast species were identified by nucleotide sequencing analyses of rDNA internal transcribed spacers (ITS1 and ITS2) and the 28s rRNA gene D1/D2 domain.

MALDI-TOF mass-spectrometry

Autoflex speed TOF/TOF (Bruker) with software MALDI Biotyper 3.1 was used. Proteins were extracted from yeasts by ethanol and formic acid. The obtained mass-spectrum profiles were compared with the data base for species identification. The protein extraction of test-standard Escherichia coli was used as a calibrator [17].

Statistical analysis on epidemiological characteristics

The prevalence rate of Candida species was analyzed as percentage occurrence of each species. The rate of Candida species distribution in blood and other samples were analyzed using chi-square test, while the rate of species distribution in different years and the rate of three common Candida species distribution in different geographical regions were analyzed using ANOVA. Statistical analyses were performed using GraphPad Prism 5. A P value of<0.05 was considered statistically significant.

Results

Candida species prevalence

The results of species identification by MALDI-TOF mass-spectrometry and DNA sequencing were identical for 100% of yeast isolates. Among 322 strains from patients with invasive candidiasis, the following 14 Candida species were revealed: C. albicans– 139 (43.2%), C. parapsilosis– 65 (20.2%), C. glabrata– 37 (11.5%), C. tropicalis– 31 (9.6%), C. krusei– 20 (6.2%), C. guilliermondii– 17 (5.3%), C. lusitaniae– 4 (1.3%), C. pararugosa– 2 (0.6%), C. dubliniensis– 2 (0.6%), C. inconspicua– 1 (0.3%), C. bracarensis– 1 (0.3%), C. utilis– 1 (0.3%), C. kefyr– 1 (0.3%), and C. lipolytica– 1 (0.3%). The mass-spectrum profiles of six main Candida species are presented in Fig. 1.

Among all Candida species revealed, C. albicans was the most common species (43.2%). Meanwhile, non-C. albicans isolates accounted for 56.8% of etiologic agents of invasive candidosis. The prevalence rate of six main Candida species isolates, namely, C. albicans, C. parapsilosis, C. glabrata, C. tropicalis, C. krusei, and C. guilliermondii, accounted for 95.97%. The isolation rate of the predominant species, namely, C. albicans, was significantly higher (P<0.01) than that of other non-C. albicans species. The prevalence rate of C. guilliermondii with the lowest isolation rate among the main six species was also significantly higher than the rates of the eight other rare isolate species (P<0.01) (Fig. 2).

Candida species distribution in blood and other clinical samples

A total of 322 Candida species were isolated from 284 patients with invasive candidiasis and who were hospitalized in 37 clinics (mostly, ICUs) of 6 Federal regions of Russia from 2012 to 2014. Candida species were isolated from blood (268), peritoneal (24), pleural (6), cerebrospinal (6) and synovial (1) fluids, deep abscesses (8), biopsy and autopsy tissues (5), and bile (4).

A total of 83.23% of the strains identified were isolated from blood. The rate of C. albicans isolation from other samples was significantly higher (P<0.01) than that of C. albicans isolation from blood. Meanwhile, a large number of strains of C. parapsilosis and C. tropicalis were isolated from blood. C. glabrata, C. krusei, and C. guilliermondii had same prevalence in blood and other clinical samples. All the eight other rare isolate species were from blood (Fig. 3).

Species distribution in different years of investigation

We compared the prevalence rate of six main invasive candidosis agents isolated from 2012 to 2014 (Fig.3). The change in species distribution in three successive years revealed that: (1) the prevalence rate of C. albicans reduced from 52.38% to 32.79% (2012 vs. 2014) (P=0.01); (2) the prevalence rate of non-C. albicans increased from 47.62% (2012) to 67.21% (2014) (P<0.01); (3) the prevalence rate of C. parapsilosis and C. guilliermondii significantly increased from 2012 to 2014 (P<0.01); and (4) the prevalence rate of C. glabrata (16.4% vs. 13.2%), C. tropicalis (10.6% vs. 8.2%), and C. krusei (8.2% vs. 6.1%) insignificantly changed (Fig. 4).

Candida species distribution in different geographical regions of Russia

The multicenter study of the etiology of invasive candidosis included medical centers of six Federal regions: Southern, Central, North-Western, Volga, Ural, and Siberian. Specific features of species spectra were found in several geographical regions (Fig. 5). In all the six Federal regions, the isolation rate of C. albicans was significantly higher (P<0.01 or P<0.05) than the isolation rates of C. parapsilosis and C. glabrata.

For C. albicans as an etiologic agent of invasive candidosis, the isolation rate in Siberian and Ural Federal regions was significantly higher (P<0.05) than in North-Western and Southern Federal regions (P<0.05). Furthermore, Siberian Federal region showed no C. parapsilosis and C. glabrata, whereas Central, Ural, and Volga Federal regions presented nearly similar occurrence rates of C. parapsilosis and C. glabrata (Fig. 5).

Discussion

Important data on epidemiology of invasive candidosis was obtained during the prospective multicenter study conducted in six Federal regions of Russia in the period of 2012–2014. The etiological agents isolated were 14 Candida species, in which the main Candida species isolated included C. albicans (42.86%), C. parapsilosis (20.19%), C. glabrata (11.49%), C. tropicalis (9.63%), C. krusei (6.52%), and C. guilliermondii (5.28%); while the rare Candida species isolated were C. lusitanie, C. dubliniensis, C. pararugosa, C. bracarensis, C. kefyr, C. lipolytica, C. utilis, and C. inconspicua at 4%. The finding that C. albicans is the most isolated Candida species among all isolates is affirmed by many studies [8,10]. For example, a European multicenter study showed that C. albicans is the most common species (54.4%), followed by C. glabrata (14.5%) and C. parapsilosis (14.1%) [18]. Similarly, in a Belgium multicenter study, C. albicans is the most frequently found (50.4%), followed by C. glabrata (27.3%) and C. parapsilosis (9.8%) [19]. In a French multicenter study, Candida albicans is also the most frequent species (55.8%), followed by C. glabrata (14.1%) and C. tropicalis (10%) [20,21]. These results also concur with the prevalence of Candida species causing candidemia in Asia, which recorded C. albicans as the leading (41.3%) isolate causing candidemia [12]. However, the prevalence rate of non-C. albicans varies considerably given that they found C. tropicalis (25.4%), C. glabrata (13.9%), and C. parapsilosis (12.1%) as the main species [12,22].

Most of the isolates in the current study were obtained from blood, which is not in agreement with the authors of Reference 22, who observed high isolation from samples other than blood. They further noted that blood cultures become positive in a minority of patients with deep-seated candidosis and often only late in the course of infection. Furthermore, in Singapore hospitals, a few of the sample isolates are from blood [23]. Given that most samples in the present study were from ICU, high systemic infection could be attributed to catheter use. In Reference 8, candidemia is diagnosed using blood cultures. Diagnosis remains a challenge to clinicians and microbiologists. Thus, half of all the cases of invasive Candida infections go undetected. In the present study, C. parapsilosis was the second most common species. Its prevalence increased from 9.8% in 2012 to 27.8% in 2014 (P<0.05) with the decrease in C. albicans. Similar results on species distribution of etiologic agents of invasive candidiasis are found in Spain, wherein the rates are 45.4% and 54.6% for C. albicans and non-C. albicans, respectively; C. parapsilosis is the second most common species (24.9%) in Spain and China [8,21,24,25]. On the contrary, a surveillance of candidemia in North America revealed that C. glabrata is the second most common etiologic agent (27%) [13]. Similar results are obtained wherein C. parapsilosis is the second most common species of Candida recovered from blood cultures obtained from patients in Europe (12%) and the Asia-Pacific region (17%). The prevalence rate of C. parapsilosis increases from 14% to 20% in Latin America over a period of 4 years [9,26]. The shift of species from C. albicans to non-C. albicans is mainly attributed to long use of antifungal drugs, such as azoles [22].

Geographic differences in species distribution of etiologic agents of invasive candidiasis have been revealed in References 8 and 27. In the current study, in Siberian and Ural Federal regions, C. albicans was a more common etiologic agent of invasive candidiasis (75% and 57.8%, respectively) than in other Federal regions; its prevalence rate was significant higher (P<0.05) than that in North-Western and Southern Federal regions. The variation in prevalence rate of Candida species in relation to geographical location has also been confirmed in Reference 12. The present findings confirmed the importance of local epidemiology studies on nosocomial mycoses to conduct effective antifungal stewardship [16]. For example, Siberian Federal regions results showed rare non-C. albicans isolates, which indicated better use of azoles compared with Central, Ural and Volga Federal regions that had nearly similar prevalence rates of C. parapsilosis and C. glabrata. This result also indicated potential dangerous natural selection owing to azole resistance owing to C. glabrata isolate [23].

In conclusion, this study successfully addressed the knowledge gap on the detailed prevalence and species distribution of etiological agents of invasive candidosis in Russia. In summary, C. parapsilosis was the second most common species. Its prevalence rate increased from 9.8% in 2012 to 27.8% in 2014 (P<0.05) with the decrease in C. albicans from 52.38% to 32.79% within the same period. Blood as a clinical sample source showed high prevalence (83.23%) of the total Candida species isolates. The distribution patterns of Candida species within Russia significantly differed. Thus, this type of study can be repeated in cycles of a period to monitor the prevalence rate of Candida species for improved management of invasive candidosis.

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