Infection DOI 10.1007/s15010-013-0570-4

CLINICAL AND EPIDEMIOLOGICAL STUDY

Epidemiology of candidemia in Qatar, the Middle East: performance of MALDI-TOF MS for the identification of Candida species, species distribution, outcome, and susceptibility pattern S. J. Taj-Aldeen • A. Kolecka • R. Boesten • A. Alolaqi • M. Almaslamani • P. Chandra • J. F. Meis • T. Boekhout

Received: 15 August 2013 / Accepted: 2 December 2013 Ó Springer-Verlag Berlin Heidelberg 2013

Abstract Introduction Bloodstream infections (BSIs) due to Candida spp. constitute the predominant group of hospitalbased fungal infections worldwide. A retrospective study evaluated the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of BSI Candida isolates. The epidemiology, risk factors, demographic features, species distribution, and clinical outcome associated with candidemia in patients admitted to a single tertiary-care hospital in Qatar, were analyzed. Methods A single-center, retrospective analysis covering the period from January 1, 2004 to December 31, 2010 was performed. Molecular identification used sequence analysis

of the D1/D2 domains of the large subunit ribosomal DNA (LSU rDNA) and the ITS1/2 regions of the rDNA. MALDI-TOF MS-based identification of all yeast isolates was performed with the ethanol/formic acid extraction protocol according to Bruker Daltonics (Bremen, Germany). The susceptibility profiles of 201 isolates to amphotericin B, itraconazole, fluconazole, voriconazole, anidulafungin, caspofungin, posaconazole, and isavuconazole were tested using CLSI standard broth microdilution method (M27-A3 and M27 S4) guidelines. Statistical analyses were performed with the statistical package SPSS 19.0. Results A total of 187 patients with 201 episodes of candidemia were identified. Candida albicans was the most

S. J. Taj-Aldeen (&)
A. Alolaqi Mycology Unit, Microbiology Division, Department of Laboratory Medicine and Pathology, Hamad Medical Corporation, P.O. Box 3050, Doha, Qatar e-mail: [email protected]

J. F. Meis Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, The Netherlands

A. Kolecka
R. Boesten
T. Boekhout CBS Fungal Biodiversity Centre, Utrecht, The Netherlands M. Almaslamani Infectious Disease Division, Department of Medicine, Hamad Medical Corporation, Doha, Qatar M. Almaslamani Weill Cornell Medical College, Doha, Qatar P. Chandra Medical Research Centre, Hamad Medical Corporation, Doha, Qatar

T. Boekhout Department of Internal Medicine and Infectious Diseases, University Medical Center, Utrecht, The Netherlands T. Boekhout Department of Dermatology, Shanghai Key Laboratory of Molecular Medical Mycology, Institute of Dermatology and Medical Mycology, Changzheng Hospital, Second Military Medical University, Shanghai, People’s Republic of China T. Boekhout Institute of Microbiology, Chinese Academy of Sciences, Beijing, People’s Republic of China

J. F. Meis Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands

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common species isolated (33.8 %; n = 68), whereas nonalbicans Candida species represented 66.2 % (n = 133) of the episodes. The species distribution and outcome of candidemia showed a difference in the crude mortality between patients infected with C. albicans (n = 30; 45.5 %) and nonalbicans Candida species. For example, C. parapsilosis candidemia was associated with the lowest mortality rate (40.6 %), and patients with other non-albicans species had the highest mortality rate (68–71.4 %). High mortality rates were observed among pediatric (\1 year of age) and elderly patients ([60 years of age). All strains showed low minimum inhibitory concentrations (MICs) (MIC90 of 0.063 lg/ml) to isavuconazole. The overall resistance to voriconazole in vitro antifungal activity was 2.5 %. C. glabrata (n = 38) had an MIC90 of 8 lg/ml for fluconazole. Most yeast isolates were susceptible to anidulafungin ([99.5 %) and 81.1 % to caspofungin. Resistance to anidulafungin was detected in 1/8 (12.5 %) isolates of C. orthopsilosis. According to new Clinical and Laboratory Standards Institute (CLSI) breakpoints, C. glabrata (n = 38) showed 100 % resistance, and 37/68 (54.4 %) C. albicans isolates were susceptible dose dependent (SDD) to caspofungin. Identification by MALDITOF MS was in 100 % concordance with molecular identification. Conclusion The Middle East epidemiology of candidemia has a unique species distribution pattern distinct from other parts of the globe. High mortality rates were observed among pediatric (\1 year of age) and elderly patients ([60 years of age). All strains were susceptible to isavuconazole. All isolates of C.glabrata were resistant to caspofungin based on M27 S4. MALDI-TOF MS is a highly useful method for the routine identification of yeast isolates in clinical setting to achieve successful therapeutic treatment. Keywords Epidemiology
Candidemia
Bloodstream infections
Antifungal susceptibility
Risk factors
Outcome
Middle East
MALDI-TOF MS

oncological malignancies and transplant recipients, and it has also been recommended as a treatment option of Candida bloodstream infections. The broad use of azole prophylaxis and therapy has an impact on the incidence of candidemia and the observed changes in the distribution of the etiologic agents. Recent reports of candidemia in a hospital in Germany identified mortality rates at 30 and 100 days of 65 and 67 %, respectively [5], and this ranged from 44.2 to 61 % in Brazil [1, 6] and 37–40.4 % in six UK institutes [7, 8]. The crude 12-week adult mortality rate ranged from 34 to 40 % in the USA [9, 10]. Although the global mortality rate varies greatly between different populations, these data show that, during the last decade, no significant improvement has been observed and the mortality rate remains high. The epidemiology of candidemia has been extensively studied worldwide, but not in the Middle East. Data on candidemia in this region are scarce, with local studies being conducted in a number of medical institutes in the Gulf region [11, 12]. Important geographical differences occur worldwide in Candida species distribution and their patterns of in vitro susceptibilities to antifungal agents. Thus, performing epidemiological surveillance studies is important in this part of the world to evaluate potential changes in species distribution and to assess the antifungal susceptibility, including that to new agents. The aim of this retrospective study was to evaluate the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of bloodstream Candida isolates and to study the epidemiology, risk factors, demographic features, species distribution, and clinical outcome associated with candidemia in patients admitted to a single tertiary-care hospital, Hamad Medical Corporation (HMC) in Doha, Qatar.

Materials and methods Introduction

Yeast nomenclature

Bloodstream infections due to Candida spp. constitute the predominant group of hospital-based fungal infections worldwide [1, 2]. These infections are an important cause of morbidity and mortality in hospitalized patients with serious underlying diseases, such as hemato-oncological malignancies, especially immunocompromised patients [3–5]. The present trend of candidemia shows that a large proportion of bloodstream infections are due to Candida species other than C. albicans, particularly among hematological, transplant, and intensive care unit patients. Fluconazole is often used as a prophylaxis to prevent invasive yeast infection in patient populations at high risk, such as those with hemato-

The taxonomic names of yeasts of the species isolated throughout the present work were based on the most recently published teleomorphic names (Table 1).

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Study design and population The study was a single-center, retrospective analysis covering the period from January 1st, 2004 to December 31st, 2010. We analyzed the information pertaining to Candida bloodstream infections of patients hospitalized in all departments of HMC, such as hematology/oncology, pediatric and adult intensive care units, and other medical

Epidemiology of bloodstream candidiasis in Qatar Table 1 Relationship between genus and species names of anamorphs and teleomorphs of yeasts detected in this study, based on [40] Teleomorpha

Anamorpha

Commonly known synonymsa

Unknown

Candida albicans (1923)b

Candida stellatoidea (1939), Candida albicans var. stellatoidea (1942)

Unknown

Candida dubliniensis (1995)b

Unknown

Candida glabrata (1978)b

Unknown

Candida intermedia (1938)b

Unknown

Candida orthopsilosis (2005)b

Unknown

Candida parapsilosis (1932)b

Unknown Unknown

Candida pararugosa (1978)b Candida tropicalis (1923)b

Clavispora lusitaniae (1979)b

Candida lusitaniae (1959)

Kluyveromyces marxianus (1971)b

Candida kefyr (1970)

Cyberlindnera fabianii (2009)b

Candida fabianii (1964)b

Lodderomyces elongisporus (1971)b

Unknown

Saccharomyces elongisporus (1952)

Meyerozyma guilliermondii (2010)b

Candida guilliermondii (1938)

Pichia guilliermondii (1966)

Pichia kudriavzevii (1965)b

Candida krusei (1923)

Wickerhamomyces anomalus (2008)b

Candida pelliculosa (1925)

Candida castellanii (1953), Issatchenkia orientalis (1960), Pichia orientalis (1964) Hansenula anomala (1919), Pichia anomala (1984)

Yarrowia lipolytica (1980)b

Candida lipolytica (1942)

a

Years of publication are indicated in parentheses

b

Current correct names

wards. The study subject population was composed of all adult and pediatric hospitalized patients of both genders who developed candidemia. Candida bloodstream infections were defined as one or more blood cultures positive for Candida species in patients with relevant clinical signs and symptoms [13]. All patients selected for further analyses had at least one positive blood culture of Candida spp. as identified by the HMC Microbiology Laboratory database. Only the isolate from the first blood culture from each patient at the time of onset of candidemia was included. This study was reviewed and approved by the local ethics committee, Medical Research Center (MRC) at Hamad Medical Corporation (#11308/11); given the use of retrospective laboratory data and preserved yeast species, the requirement for written informed consent was waived because of the retrospective and observational nature of this study. Biochemical identification Blood cultures were performed using the Bactec automated culturing system (BD Diagnostic Systems, Franklin Lakes, NJ, USA). A small inoculum from a single colony of each isolate was inoculated onto Sabouraud dextrose agar (SDA) plates, incubated at 35 °C for 48 h, and used to prepare inoculum for substrate assimilation profiles employing the Vitek 2 Compact yeast identification system (bioMe´rieux, Marcy-l’E´toile, France), as recommended by the

Torulopsis glabrata (1938)

Lindnera fabianii (2008), Hansenula fabianii (1965), Pichia fabianii (1984)

manufacturer, API ID 32C (bioMe´rieux), and partly on CHROMagar Candida plates. Cultures of Candida isolates were preserved at -70 °C using cryo-tubes (Mast Diagnostics, Bootle, Merseyside, UK) until further analysis. DNA isolation and sequencing Sequence analysis of the D1/D2 domains of the large subunit ribosomal DNA (LSU rDNA) and the ITS1 and ITS2 regions of the rDNA was performed according to the method of Okoli et al. [14]. The sequences generated were compared to data available in the NCBI database using the Basic Local Alignment Search Tool (BLASTn) (http:// blast.ncbi.nlm.nih.gov/). MALDI-TOF MS MALDI-TOF MS-based identification of all yeast isolates was performed according to Bruker Daltonics (Bremen, Germany) with the ethanol (EtOH)/formic acid (FA) extraction protocol. For extraction, two loops of yeast biomass (1-ll volume, sterile inoculation loop) not older than 24 h growing on SDA were suspended in 300 ll of molecular graded ionized water and further processed with 900 ll of 95 % EtOH. The volume of FA used for optimization ranged between 20 and 40 ll. However, a volume of 25 ll of FA was found to be optimal and an equal volume of acetonitrile (ACN) was added later. From the

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crude protein extract of each tested strain, 1 ll was spotted in duplicate onto a 96-spot polished steel target plate (Bruker Daltonics) and allowed to dry at room temperature. Bacterial test standard (Bruker Daltonics) was also spotted twice and used as a positive control. Before measurements, all tested spots were overlaid with 1 ll of alpha-cyano-4hydroxycinnamic acid (Bruker Daltonics) saturated matrix solution. The yeast identification was operated by the MALDI Biotyper 3.0 system based on mass spectra generated with the Microflex LT software and compared with two databases simultaneously. The first was the commercially available database (BDAL) that, at the time of conducting the analyses, contained 3,995 main mass spectra (MSPs) (Bruker Daltonics), and the second was an CBSKNAW in-house library of 510 MSPs of different yeasts that was established according to the Bruker internal database creation standard operating procedures. Identification scores were interpreted according to the manufacturer’s recommended criteria: a log score value [2.0 indicated correct identification to the species level, a log score 1.999 [ value [ 1.7 correct genus recognition, and no reliable identification with a score \1.7. Each isolate was considered correctly identified if at least one of the duplicates gained scores[2. Strains with results\2.0 or no peaks found were retested from a fresh culture. The identification was also considered correct if at least one spot from the duplicate gave a reliable identification with score [1.7 that was concordant with the sequencing results.

Meyerozyma guilliermondii. AND and CSP MIC endpoints B0.12 lg/ml were considered to be S for C. glabrata. C. albicans and C. tropicalis strains for which the echinocandin MIC was C1 lg/ml (C0.5 lg/ml for C. glabrata) are considered to be resistant (R). C. parapsilosis and M. guilliermondii strains for which the echinocandin MIC was C8 lg/ml were considered to be R. FLC MIC endpoints were categorized as B2 (S), four susceptible dose-dependent (SDD), and C8 lg/ml (R) for C. albicans, C. tropicalis, and C. parapsilosis. FLC MIC values B32 and C64 lg/ml were considered to be SDD and R, respectively, for C. glabrata. For voriconazole (VOC), C. albicans, C. tropicalis, and C. parapsilosis were categorized as MIC B0.12 lg/ml (S), MIC 0.25–0.5 lg/ml (SDD), and MIC C1 lg/ml. MIC endpoints were determined for all other Candida species following the M27-S4 CLSI guidelines [16]. The epidemiological cut-off value (ECV) C1 lg/ml was used to detect VOC resistance in C. glabrata [17]. A breakpoint B1 lg/ml was selected to define the isolates as S to AmB [18]. The ranges of MICs and MICs at which 50 and 90 % of the isolates population of Candida spp. were inhibited (MIC50 and MIC90, respectively) were calculated. The MICs of the quality control strains of C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 were all within the reference ranges (data not shown).

Susceptibility testing

Categorical and continuous values were expressed as frequency (percentage) and mean ± standard deviation (SD). Descriptive statistics were used to summarize all demographic and other clinical characteristics of the patients. Associations between two or more qualitative or categorical variables were assessed using the Chi-square test. For small cell frequencies, the Chi-square test with continuity correction factor was used. Quantitative variables means between more than two independent groups were analyzed using one-way analysis of variance (ANOVA). Various diagnostic measures to assess the diagnostic performance and accuracy of MALDI-TOF MS in the identification of Candida in reference to molecular technique as a gold standard, such as sensitivity, specificity, positive, and negative predictive values, were calculated and reported, along with their corresponding 95 % confidence limits. Kappa agreement analysis was performed to assess an agreement in the identification of Candida between the MALDI-TOF MS and molecular tests. Pictorial presentations of the key results were made using appropriate statistical graphs. A two-sided p-value \0.05 was considered to be statistically significant. All statistical analyses were done using the statistical package SPSS 19.0 (SPSS Inc., Chicago, IL, USA).

The susceptibility profiles of the five most commonly isolated species to amphotericin B (AmB) (Bristol-Myers Squibb, Woerden, The Netherlands), itraconazole (ITC) (Janssen Research Foundation, Beerse, Belgium), fluconazole (FLC), voriconazole (VOC), anidulafungin (AND) (Pfizer Central Research, Sandwich, UK), caspofungin (CSP) (Merck Sharp and Dohme BV, Haarlem, The Netherlands), posaconazole (PSC) (Merck Sharp and Dohme), and isavuconazole (ISV) (Basilea Pharmaceutica, Basel, Switzerland, now Astellas) were tested using the standard broth microdilution method (M27-A3) as recommended by the Clinical and Laboratory Standards Institute (CLSI) [15]. The susceptibilities were interpreted taking into account the new species-specific clinical breakpoint, M27-S4 suggested by the CLSI [16]. The concentrations of AmB, ITC, VOC, and PSC ranged from 0.016 to 16 lg/ml, that of FLC from 0.063 to 64 mg/l, those of AND and CSP from 0.008 to 8 lg/ml, and that of ISV from 0.004 to 4 lg/ml. AND and CSP minimal inhibitory concentration (MIC) cut-off values of B0.25 lg/ml were considered to be susceptible (S) for C. albicans and C. tropicalis. MIC values B2 lg/ml were categorized as S for C. parapsilosis and

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Statistical analysis

Epidemiology of bloodstream candidiasis in Qatar Table 2 Demographic clinical characteristics and outcome of candidemia by the most common Candida species Variables

C. albicans, n (%)

C. glabrata, n (%)

C. tropicalis, n (%)

C. orthopsilosis, n (%)

C. parapsilosis, n (%)

Others, n (%)

Total, n (%)

\1 year

37 (54.4)

6 (15.8)

1–18 years

1 (1.5)

2 (5.3)

9 (25.0)

6 (75.0)

14 (41.2)

6 (35.3)

78 (38.8)

2 (5.6)

1 (12.5)

5 (14.7)

4 (23.5)

15 (7.5)

19–40 years

2 (2.9)

4 (10.5)

7 (19.4)

41–60 years

15 (22.1)

10 (26.3)

3 (8.3)

0 (0.0)

3 (8.8)

2 (11.8)

18 (9.0)

0 (0.0)

5 (14.5)

(0.0)

33 (16.4)

[60 years

13 (19.1)

16 (42.1)

15 (41.7)

1 (12.7)

7 (20.6)

5 (29.4)

57 (28.4)

Total Genderb

68 (33.8)

38 (18.9)

36 (17.9)

8 (3.9)

34 (16.9)

Male

49 (74.2)

Female

17 (25.8)

22 (66.7)

23 (69.7)

3 (42.9)

17 (53.1)

9 (56.3)

123 (65.8)

11 (33.3)

10 (30.3)

4 (57.1)

15 (46.9)

7 (43.8)

64 (34.2)

Heart/ pulmonary

7 (10.6)

8 (24.2)

9 (27.3)

3 (50.0)

6 (18.8)

5 (31.3)

38 (20.4)

Malignancy

14 (21.2)

9 (27.3)

6 (18.2)

0 (0.0)

3 (9.4)

0 (0.0)

32 (17.2)

6 (9.1)

4 (12.1)

5 (15.2)

0 (0.0)

3 (9.4)

2 (12.5)

20 (10.8) 17 (9.1)

p-Value

Age groupa

17 (8.5)

0.0009*

201 (100) 0.229

Risk categoryb

Renal diseases Preterm

11 (16.7)

2 (6.1)

1 (3.0)

0 (0.0)

2 (6.3)

1 (6.3)

Burn/wound

2 (3.0)

0 (0.0)

0 (0.0)

0 (0.0)

2 (6.3)

3 (18.8)

7 (3.8)

Mental disorders

9 (13.6)

3 (9.1)

3 (9.1)

1 (16.7)

2 (6.3)

2 (12.5)

20 (10.8)

DM

3 (4.5)

3 (9.1)

1 (3.0)

0 (0.0)

0 (0.0)

0 (0.0)

7 (3.8)

GI diseases

2 (3.0)

3 (9.1)

7 (21.2)

1 (16.7)

10 (31.3)

2 (12.5)

25 (13.4)

12 (18.2)

1 (3.0)

1 (3.0)

2 (16.7)

4 (12.5)

1 (6.3)

21 (10.8)

Alive

36 (54.5)

10 (30.3)

10 (30.3)

2 (28.6)

19 (59.4)

5 (31.3)

82 (43.9)

Dead

30 (45.5)

23 (69.7)

23 (69.7)

5 (71.4)

13 (40.6)

11 (68.8)

105 (56.1)

Others

0.153*

Outcomeb 0.025

* Yates’ corrected Chi-square a

14 patients had multiple episodes (n = 201)

b

Based on number of subjects (n = 187)

Results Epidemiology Between the period from January 2004 to December 2010, a total of 201 Candida isolates were reported from bloodstream infections of 187 patients at Hamad hospital, Qatar (14 patients had multiple episodes). The distribution of Candida species isolated from positive blood cultures are presented in Table 2. Among the 201 yeast isolates, C. albicans was the most commonly isolated species (33.8 %; n = 68), whereas other Candida species comprised 66.2 % (n = 133) and consisted of C. glabrata (18.9 %; n = 38), C. tropicalis (17.9 %; n = 36), C. parapsilosis (16.9 %; n = 34), C. orthopsilosis (3.9 %; n = 8), and C. dubliniensis (1.5 %; n = 3). Overall, Pichia kudriavzevii, M. guilliermondii, Clavispora lusitaniae, and C. pararugosa

(n = 2 each) caused 4 % of infections. Uncommon yeast species that caused candidemia, such as C. intermedia, Yarrowia lipolytica, Kluyveromyces lactis, Wickerhamomyces anomalus, Cyberlindnera fabianii, and Lodderomyces elongisporus, were found in single cases each (3 %). The species distribution and outcome of candidemia showed a difference in mortality between those patients infected with C. albicans (n = 30; 45.5 %) and with other Candida species, which showed a significantly higher mortality for all other species (68–71.4 %; p = 0.025), except for C. parapsilosis, which is characterized by a low mortality ratio (40.6 %) (Table 2; Fig. 1). Patients belonging to the pediatric age group\1 year of age and the adult patient age group [60 years of age were most at risk of candidemia, with high crude mortality rates of 32.4 and 43.8 %, respectively (Fig. 2). The overall crude mortality of the total population was 56.1 % (Table 2).

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S. J. Taj-Aldeen et al. Fig. 1 Effect of Candida species on the survival of patients with candidemia

Fig. 2 Effect of age factor on the outcome of candidemia

Underlying diseases In 20 % of the candidemia patients, the underlying disease was heart/pulmonary disorders, followed by malignancies (17 % hematological and solid organ tumors), gastrointestinal (GI) disease including surgery (13 %), and renal diseases including transplant patients (11 %). Candidemia due to other risk factors were between 4 and 11 %. Univariate analysis for risk factors associated with mortality showed that heart/pulmonary diseases (25/105; 24 %), malignancies (hematological and solid organ tumors) (23/ 105; 22.1 %), GI (11/105; 10.5 %), and renal diseases (13/ 105; 12.5 %) were associated with higher mortality. The age group (Table 2) was found to be significantly associated with the isolated Candida species (p = 0.0009). No statistically significant association was observed between gender and different Candida species (p = 0.229). Overall, Candida species were the most frequently isolated from

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males (male:female ratio = 123:64). C. albicans (n = 37/ 68; 54.4 %), C. parapsilosis (n = 14/34; 41.2 %), and C. orthopsilosis (n = 6/8; 75 %) predominated in the age group \1 year. C. glabrata (n = 16/38; 42.1 %) and C. tropicalis (n = 15/36; 41.7 %) were the predominant yeast species in patients[60 years old (Table 2). Patients that were below 1 year and over 60 years of age comprised the population most vulnerable to candidemia, being n = 78/201 (38.8 %) and n = 57/201 (28.4 %), respectively. Among the total of 93 yeast isolates from pediatric patients (age range 0–18 years), the species distribution was as follows: C. albicans (n = 38/ 68; 55.9 %), C. parapsilosis (n = 19/34; 55.9 %), C. tropicalis (n = 11/36; 30.6 %), C. glabrata (n = 8/38; 21.1 %), C. orthopsilosis (n = 7/8; 87.5 %), and other uncommon species (n = 10/17; 58.8 %). C. glabrata was frequently isolated from adult patients over 41 years of age (Table 2). The following differences (p = 0.153) were found across the Candida species. C. albicans (14/66; 21.2 %) and C. glabrata

Epidemiology of bloodstream candidiasis in Qatar Table 3 Susceptibility of the bloodstream Candida species to the systemic antifungal agents after 48 h Range

MIC50

Geometric mean

Table 3 continued Range

Fluconazole 0.125–2

0.5

0.612

0.5 0.031

0.332 0.018

MIC90

0.5

0.667

1

\0.016–0.125

0.063

0.035

0.125

Voriconazole

\0.016–0.063

0.031

0.020

0.063

Posaconazole

0.063–0.125

0.125

0.091

0.125

1.00

Isavuconazole

\0.016–0.031

\0.016

0.010

0.031

1.0

Fluconazole Itraconazole

0.031–64 \0.016–16

Voriconazole

\0.016–1

\0.016

0.013

0.125

Caspofungin

1–2

1

1.143

2

Posaconazole

\0.016–2

0.031

0.018

0.500

Anidulafungin

1–4

2

1.524

4

Isavuconazole

\0.016–1

Caspofungin Anidulafungin

0.25–2 \0.008–4

4

0.5–1

Geometric mean

Itraconazole

All strains (n = 201) Amphotericin B

MIC50

MIC90

\0.016 \0.016 0.5

0.063

0.498

1

0.031 \0.016

2

C. albicans (n = 68) Amphotericin B

0.25–1

0.5

0.489

Fluconazole

0.031–4

0.25

0.161

Itraconazole

\0.016–0.25

\0.016 \0.016

\0.016

Voriconazole

\0.016–0.125

\0.016 \0.016

\0.016

Posaconazole

\0.016–0.5

\0.016 \0.016

\0.016

Isavuconazole

\0.016–0.063

\0.016 \0.016

\0.016

Caspofungin

0.25–0.5

Anidulafungin \0.008–0.016 C. tropicalis (n = 36)

0.5

0.343

\0.008 \0.008

1 0.25

0.5 \0.008

Amphotericin B

0.5–1

1

0.698

1

Fluconazole

0.25–2

0.5

0.460

1

Itraconazole

\0.016–0.25

0.063

0.026

0.125

Voriconazole

\0.016–0.5

0.031

0.023

0.125

Posaconazole

\0.016–0.5

0.031

0.029

0.25

Isavuconazole

\0.016–0.25

\0.016

0.010

0.031

0.5

0.487

1

0.063

0.023

0.125

Caspofungin Anidulafungin

0.25–1 \0.008–0.125

C. parapsilosis (n = 34) Amphotericin B

1–2

Fluconazole

0.25–4

Itraconazole

\0.016–1

Voriconazole

\0.016–0.125

Posaconazole Isavuconazole Caspofungin

1

1.014

1

1

0.709

2

0.063

0.049

0.25

\0.016

0.011

0.125

\0.016–0.5

0.063

0.037

0.125

\0.016–0.125 0.5–2

0.008 1

0.009 1.108

0.063 2

0.031–4

2

0.365

2

Amphotericin B

0.5–1

1

0.745

1

Fluconazole

0.5–64

4

2.850

8

Itraconazole

0.063 to [16

1

0.438

1

0.125

0.066

0.25 0.5

Anidulafungin C. glabrata (n = 38)

Voriconazole

\0.016–1

Posaconazole

0.063–2

Isavuconazole

\0.016–1

Caspofungin Anidulafungin

0.5–1 \0.008–0.063

0.25

0.279

0.063

0.039

0.25

0.5

0.556

1

0.063

0.034

0.063

1

0.800

1

C. orthopsilosis (n = 8) Amphotericin B

(9/33; 27.3 %) were the most frequently recovered Candida species associated with malignancies. C. tropicalis was predominant in patients with heart/pulmonary disorders (9/33; 27.3 %), and C. parapsilosis was the most common cause of candidemia in patients with GI diseases (10/32; 31.3 %). Resistance The in vitro susceptibility of 201 isolates to eight antifungal agents is presented in Table 3. The MIC ranges, geometric mean, MIC50, and MIC90 were determined for C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, and C. orthopsilosis. Overall, the number of isolates resistant to the tested antifungals was low. Resistance to ISV was not detected in any of the Candida strains; this agent exhibited low MIC values (0.016–1 lg/ml). Resistance to AmB (the MIC cut-off value is B1 lg/ml) was detected in 1/36 (2.7 %) isolates C. parapsilosis. CSP exhibited in vitro activity against Candida species with MIC 0.25–2 versus \0.008–4 lg/ml for AND. 99.5 % of the isolates tested were susceptible to AND. Resistance to AND was detected in 1/8 (12.5 %) isolates of C. orthopsilosis. The activity of CSP was lower (81.1 % of isolates were susceptible). C. glabrata (n = 38) showed 100 % resistance to CSP, and 37/68 (54.4 %) isolates of C. albicans were SDD to CSP. All triazoles demonstrated potent activity against C. albicans, C. parapsilosis, and C. orthopsilosis (susceptible rate 100 %). All isolates of C. glabrata (n = 38; 100 %) were SDD with FLC, and 3/36 (8.3 %) isolates of C. tropicalis were SDD with VOC, whereas resistance to VOC was detected in 2/38 (5.2 %) isolates of C. glabrata using the previously reported ECV values [17]. Resistance to itraconazole was detected in 22/201 (10.9 %) of the strains, the majority of which were C. glabrata. Yeast identification Candida isolates routinely tested by biochemical methods could not be completely recognized to the species level. Twenty-one isolates (10.4 %) were identified to the genus

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S. J. Taj-Aldeen et al. Table 4 Species identifications of Candida and related yeasts by different diagnostic methods

Species

Diagnostic methods Biochemical, n (%)

Molecular, n (%)

MALDI-TOF MSa, n (%)

C. albicans

68 (33.8)

68 (33.8)

68 (33.8)

C. glabrata

26 (12.9)

38 (18.9)

38 (18.9)

C. tropicalis

33 (16.4)

36 (17.9)

36 (17.9)

8 (4.0)

8 (4.0)

C. orthopsilosis

a

Candida with species-level identification (score [2) = 200; one C. dubliniensis isolate had identification score (\2) = 1.9

b

Identified as C. pararugosa by the molecular and MALDITOF MS methods c

Were identified to the species level by the molecular and MALDI-TOF MS methods (C. tropicalis, C. glabrata, M. guilliermondii, C. pararugosa, K. lactis, Cy. fabianii, and L. elongisporus)

Number of isolates (n = 201)

–

C. parapsilosis

42 (20.9)

34 (16.9)

34 (16.9)

C. dubliniensis

2 (1.0)

3 (1.5)

3 (1.5)

M. guilliermondii P. kudriavzevii

– 3 (1.5)

2 (1.0) 2 (1.0)

2 (1.0) 2 (1.0)

C. lusitaniae

2 (1.0)

2 (1.0)

2 (1.0)

1 (0.5)

1 (0.5)

C. pararugosa

–

C. rugosab

1 (0.5)

1 (0.5) C. pararugosa

1 (0.5) C. pararugosa

C. intermedia

1 (0.5)

1 (0.5)

1 (0.5)

W. anomalus

1 (0.5)

1 (0.5)

1 (0.5)

Y. lipolytica

1 (0.5)

1 (0.5)

1 (0.5)

K. lactis

–

1 (0.5)

1 (0.5)

Cy. fabianii

–

1 (0.5)

1 (0.5)

L. elongisporus

–

1 (0.5)

Candida sp.c

21 (10.4)

level only. Other species (total 4.9 %), such as C. orthopsilosis, M. guilliermondii, C. pararugosa, W. anomalus, Y. lipolytica, K. lactis, Cy. fabianii, and L. elongisporus, were not identified (Table 4). Two hundred and one isolates were tested by MALDI-TOF MS and yielded scores that allowed correct species identification. Candida isolates yielded a high percentage of log score values of C2 (99.5 %). Amongst the Candida species, only one isolate of C. dubliniensis was correctly identified with a score value of 1.7 \ value \ 2.0. Considering the sequence analysis of the ITS regions and D1/D2 domains of the rDNA as the gold standard for the identification of yeasts, the MALDI-TOF MS method yields values of diagnostic accuracy measures such as sensitivity, specificity, positive, and negative predictive values of 100 %. Kappa agreement analysis between MALDI-TOF MS and molecular tests revealed a kappa agreement value for Candida identification of 1.00 (i.e., perfect agreement) compared to 0.72 for the biochemical identification methods.

Discussion This retrospective candidemia study represents the largest investigation conducted on Candida-related bloodstream infections in the Gulf area and reveals, for the first time, the large burden of candidemia in a main Qatar tertiary care hospital. In addition, it provides the most representative

123

–

1 (0.5) –

and consistent data from the Middle East region on the epidemiology of candidemia to date [11, 12, 19]. Although C. albicans remains the most frequently encountered species in the clinical laboratory, there has been a worldwide increase in the frequency of infections caused by nonalbicans Candida species, including C. tropicalis, C. parapsilosis, and the intrinsically fluconazole-resistant species C. glabrata and P. kudriavzevii [9]. The reasons for the emergence of non-albicans Candida species are not clear, but some medical conditions may consistently impact the risk of developing candidemia due to rarely occurring species, other than C. albicans. For instance C. parapsilosis candidemia has been associated with vascular catheters and parenteral nutrition [20]. Such an increase may also be attributed to the improvement at the diagnostics level, allowing non-albicans Candida species to be distinguished with more sensitive methods. In this study, the medical conditions that consistently impacted a risk of developing candidemia due to nonalbicans Candida species were as follows: C. glabrata emerged prominently among patients with malignancies, C. tropicalis was associated with heart/pulmonary diseases, and C. parapsilosis was dominant in patients \1 year old and those with GI diseases. Although C. glabrata is particularly common in the northern hemisphere [3], we report a relatively high proportion of C. glabrata (18.9 %) as the second cause of candidemia in this surveillance analysis. European studies

Epidemiology of bloodstream candidiasis in Qatar

reported variable proportions of this species in the range from 9.5 to 19.1 % [21, 22]. A recent study in Italy reported a change in the epidemiology from 12.8 to 20.8 % [23], which is close to the proportion reported in the presented study. Even higher proportions were recently reported in the UK (33.31 %) [8] and USA (26 %) [9, 24], suggesting a recent change in the USA epidemiology compared to those previously reported (12–17 %) [25, 26]. Such a trend is probably associated with earlier exposure to prophylactic therapies with fluconazole as a common practice, particularly in patients with malignancies and in intensive care units [25], and this remains a subject of concern when it comes to fluconazole resistance. C. glabrata candidemia is seen more often in older adults and is comparatively less prevalent in neonates and in the pediatric age group [24]. C. tropicalis has the most prominent geographical variation over the globe. Interestingly, the Middle East epidemiology of C. tropicalis is different from Western and Eastern parts of the globe. This species was found to be the third leading cause of candidemia during this study (17.9 %), which is close to the values obtained in other Middle Eastern countries such as Saudi Arabia (19 %) and the United Arab Emirates (15 %) [12, 19]. Recent US studies reported low proportions of C. tropicalis candidemia (8.1 to 8.7 %) [9] and European series have consistently reported the lowest proportions, namely, the UK (\5 %) [7], Italy (8.2 %) [23], and Spain (8.21 %) [27]. In contrast, South East Asian countries reported the highest proportions. In China, the proportion of C. tropicalis candidemia was 21.8 % [28], in Thailand 28 % [29], in Taiwan 23.1 % [30], and in India, C. tropicalis is now the most common cause of hospitalacquired candidemia. Here, epidemiological studies have implicated C. tropicalis in up to 45 % of candidemia cases [31], and such a trend highlights the possibility that this species may be associated with hospital cross-transmission. Similar to C. tropicalis, candidemia due to C. parapsilosis occurred frequently in the present study, with a slightly lower proportion (16.9 %) than C. tropicalis. This high proportion is mainly due to infections in GI patients (n = 10; 31.3 %) and in the pediatric age group of \1year-old patients (n = 14/34; 41.2 %). There is a variation of C. parapsilosis candidemia in different age groups and in some regions; C. parapsilosis counts for the majority of candidemia cases. These findings may be attributed to transmission through the hands of health care workers [32]. Of the total of 201 episodes of candidemia reported in the current survey, the absence of C. metapsilosis is of note and C. orthopsilosis was the fifth most common cause of candidemia (3.9 %), representing 24 % of the proportion of C. parapsilosis. In contrast, in China, C. metapsilosis represented 28.1 % of the C. parapsilosis species complex, with the absence of C. orthopsilosis. This suggests a geographical variation in the distribution of species belonging

to the C. parapsilosis species complex [33]. This is the first report describing C. orthopsilosis as a cause of candidemia in Qatar. The majority of C. orthopsilosis candidemia cases (n = 6/8; 75 %) were in the pediatric patients age group (\1 year of age). C. orthopsilosis has been reported to be involved in a restricted number of infections [27] and candidemia caused by this species may be attributed to their ability to form biofilms [34]. Our study demonstrated that candidemia due to P. kudriavzevii is rare in Qatar (\1 %), and that C. glabrata accounted for the large majority of non-albicans Candida species. The reason that P. kudriavzevii is rare in Qatar is not clear, but the wide geographic variability in species distribution suggests that factors such as health care practice, demographic characteristics, and the use of antibiotics may be important. Although the proportion of P. kudriavzevii infections in Qatar is small, the burden of this species is quite similar to rates recently reported by others [27]. Retrospective cohort studies involving patients with candidemia and varying underlying diseases and age groups have revealed worldwide crude mortality rates of 34–67 % [1, 5–10]. In our study, patients with candidemia had a crude mortality rate of 56.1 %, which is close to values reported from Germany and Brazil [1, 5, 6]. Similar to other reports, patients with C. parapsilosis candidemia had the lowest death rates [20], while C. glabrata and C. tropicalis infections were linked to an unfavorable outcome in more than 40 % of patients [9]. This high crude mortality rate of candidemia caused by these species may be due to their occurrence in elderly patients ([60 years old) and in highrisk category groups, such as patients with cancer. The severity of the underlying medical condition greatly influences the crude mortality rate in these patient populations. In the presented study, the overall susceptibility testing to antifungal agents revealed the presence of isolates that were resistant to caspofungin when applying the new breakpoints (CLSI M27-S4 [16]). All bloodstream isolates of Candida spp. were susceptible to ISV, as all strains had low MIC values (\1 lg/ml) and VOC was the azole showing the best in vitro antifungal activity (97.5 %). Important differences in susceptibility patterns, especially for azoles, were observed. High rates of resistant isolates were only observed for ITC, the majority of which belonged to C. glabrata and, thus, suggesting the possibility of cross-resistance to other azoles, which leads to treatment failure. Based on the M27-S4 guideline, FLC was active against C. albicans, C. tropicalis, and C. parapsilosis, but have intermediate effect on C. glabrata (n = 38 SDD). Susceptibility data reported by others were different based on the M27-A3 document, as FLC resistance rates for European and North American isolates were 8.1 and 6.6 %, respectively [27, 35]. Overall, it was observed during this study that echinocandins exhibited poor activity against C.

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S. J. Taj-Aldeen et al.

glabrata, C. albicans, and one isolate of C. orthopsilosis. The high resistance among these species for caspofungin can be explained by the recently reported variability of in vitro susceptibility testing [36] and the new CLSI recommended species-specific breakpoints (CLSI M27-S4 [16]). AmB was reported as the agent with the lowest overall resistance rate (0.2 %) [27], and the overall resistance rate to this agent appeared very low in this study as well. Although antifungal resistance patterns vary across geographic regions, the emerging resistance among C. glabrata cases in the Middle East region requires further epidemiological surveillance. The rank order of species varies worldwide and is characteristic for each region; moreover, antifungal susceptibility patterns are species-specific. Consequently, the rapid and accurate identification of the causative organism is critical for successful treatment. When growth in blood culture is detected, yeast cells are usually subcultured on agar plates to obtain colonies that are used for further phenotypic testing aimed at the species-level, which typically takes at least 72 h. When the identification results based on MALDI-TOF MS, biochemical, and morphological features were in concordance, the isolate was identified as the consensus species. When discordant results were obtained, definitive species identification was based on rDNA sequences. Sequence analyses of the D1/D2 domains and the ITS1/ITS2 regions of the rDNA were performed. Comparing the performance of each identification technique, all tested isolates (Table 4) were correctly identified by MALDI-TOF MS. Species like C. orthopsilosis, M. guilliermondii, C. pararugosa, K. lactis, Cy. fabianii, and L. elongisporus were incorrectly identified by phenotypic methods. Other species such as C. glabrata, C. tropicalis, C. parapsilosis, C. dubliniensis, and P. kudriavzevii were correctly identified by routine phenotypic methods in Hamad hospital laboratory (67.7–91.6 %). The overall error in the identification of clinical isolates by phenotypic methods was 10.9 %. Thus, contrary to conventional identification methods, all common yeast isolates were identified by MALDI-TOF MS in a straightforward fashion. In particular, C. orthopsilosis, a newly recognized member of the C. parapsilosis complex that is frequently incorrectly identified by conventional methods as C. parapsilosis sensu stricto, and C. pararugosa, which is also difficult to distinguish by conventional methods, were correctly identified by MALDI-TOF MS. For the routine clinical diagnostics, it will be of further interest to consider the MALDI-TOF MS analyses to identify yeast species and to assess differences in the antifungal susceptibility profiles of Candida species, particularly in their resistance to azoles. MALDI-TOF MS has been reported for the identification of yeasts directly from blood cultures [37], but this

123

procedure has some limitations, such as the limited diversity of yeast genera and species examined, and it not being applicable in routine diagnostic laboratories. Instead, we suggest the culture of yeast isolates onto SDA plates for 24 h before identification by MALDI-TOF MS and when performed will reduce the turnaround time by 48 h compared to the phenotypic methods. By using the CBSKNAW in-house library simultaneously in combination with the commercial Bruker BDAL database, our data showed 100 % correct identifications when compared with molecular analysis as the gold standard (kappa value = 1). Irrespective of the type and number of tested organisms, earlier studies showed 96 % (score C1.7) [38] and 97.9 % [39] correctly identified yeasts by the MALDI-TOF MS method. Our study represents, up to now, the largest candidemia surveillance conducted in the Middle East, Qatar, and provides data on the species distribution, including less commonly occurring yeast species, in vitro susceptibility data of the isolates to eight currently used systemic antifungal agents, and shows the performance of the MALDITOF MS system in the identification of bloodstream-related yeast species. Furthermore, this report shows that candidemia is a significant source of morbidity in Qatar, with a substantial burden of disease, mortality, and, likely, high associated costs. It is important to point out that rapid identification of yeasts to the species level is necessary to achieve successful therapeutic treatment. Resistance to antifungal drugs remains restricted to a few species. However, the Middle East epidemiology of candidemia has a unique species distribution pattern, distinct from that of the Eastern and Western parts of the globe. A prospective epidemiological surveillance to evaluate the change in candidemia patterns for the next 4 years in Qatar is underway. Acknowledgments Supported by Grant NPRP 5-298-3-086 from the Qatar National Research Fund (a member of Qatar Foundation) to Saad J. Taj-Aldeen, Muna Almaslamani, Jacques F. Meis, and Teun Boekhout. The statements herein are solely the responsibility of the authors. Conflict of interest J.F.M. received grants from Astellas, Basilea, and Merck. He has been a consultant to Astellas, Basilea, and Merck and received speaker’s fees from Merck and Gilead. All other authors do not report conflicts of interest. All authors contributed to the content and writing of this manuscript.

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