Candida guilliermondii complex is characterized by high antifungal resistance but low mortality

25 Pages·2017·0.95 MB·English

AAC Accepted Manuscript Posted Online 24 April 2017
Antimicrob. Agents Chemother. doi:10.1128/AAC.00099-17
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
1 Candida guilliermondii complex is characterized by high antifungal
2 resistance but low mortality: a report on 22 cases of candidemia
3
Laura Judith Marcos-Zambrano 1,2
Mireia Puig-Asensio 3
Felipe Pérez-García 1,2
Pilar Escribano 1,2
Carlos Sánchez-Carrillo 1,2,5
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Oscar Zaragoza 4 o
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Belén Padilla 1,2,5 n
Manuel Cuenca-Estrella 4 lo
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Benito Almirante 3 d
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M. Teresa Martin-Gómez 6 d

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Patricia Muñoz 1,2,5,7 ro
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Emilio Bouza 1,2,5,7

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Jesús Guinea # 1,2,5,7 t
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on behalf of the CANDIPOP study :/
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4 1Clinical Microbiology and Infectious Diseases, Hospital General Universitario Gregorio .a
5 Marañón, Madrid, Spain. 2Instituto de Investigación Sanitaria Gregorio Marañón, s
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6 Madrid, Spain. 3Infectious Diseases Department, Hospital Universitari Vall d’Hebron, .
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7 Universitat Autònoma de Barcelona, Barcelona, Spain. 4Mycology Reference r
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8 Laboratory, National Centre for Microbiology, Instituto de Salud Carlos III, /

9 Majadahonda, Madrid, Spain. 5CIBER Enfermedades Respiratorias-CIBERES on
10 (CB06/06/0058), Madrid, Spain. 6Microbiology Department, Hospital Universitari Vall A
11 d´Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain. 7Medicine p
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12 Department, School of Medicine, Universidad Complutense de Madrid, Madrid, Spain. il
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14 Running title: Candidemia caused by Candida guilliermondii 2
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16 Key words: Candida guilliermondii, Candida fermentati, Galleria mellonella, non-albicans

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17 candidemia, biofilm formation.
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19 Abstract word count: 245 u
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20 Text word count: 2569 s
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22 Correspondence:
23 Jesús Guinea
24 Servicio de Microbiología Clínica y Enfermedades Infecciosas
25 Hospital General Universitario Gregorio Marañón
26 C/Dr. Esquerdo, 46
27 28007 Madrid, Spain
28 Phone: [phone]
29 Fax: [phone]
30 Email: [email protected]
1
31 ABSTRACT
32 Objectives: The objectives of our study were to describe the characteristics of
33 patients infected with C. guilliermondii candidemia and to perform an in-depth
34 microbiological characterization of isolates and compare them with those of
35 patients with C. albicans candidemia.
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36 Methods: We described the risk factors and outcome of 22 patients with o
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37 candidemia caused by C. guilliermondii complex. Incident isolates were n
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38 identified using molecular techniques, and susceptibility to fluconazole, d
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39 anidulafungin and micafungin was studied. Biofilm formation was measured fr
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40 using the crystal violet assay (biomass production) and the XTT reduction assay
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41 (metabolic activity), and virulence was studied using the Galleria mellonella p
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42 model. Biofilm formation was compared with that observed for C. albicans. a
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43 Results: The main conditions predisposing to infection were malignancy (68%), s
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44 immunosuppressive therapy (59%), and neutropenia (18%). Clinical o
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45 presentation of candidemia was less severe in patients infected by C. o
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46 guilliermondii complex than in patients infected by C. albicans, and 30-day p
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47 mortality was lower in C. guilliermondii patients (13.6% vs 33.9%, P=0.049). il 2
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48 Isolates were identified as C. guilliermondii sensu stricto (n=17) and Candida 0
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49 fermentati (n=5). The isolates produced biofilms with low metabolic activity and b
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50 moderate biomass. The G. mellonella model showed that C. guilliermondii was u
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51 less virulent than C. albicans (mean of 6 vs 1 days of survival, respectively; t
52 P<0.001).
53 Conclusions: Patients with candidemia caused by C. guilliermondii complex
54 had severe and debilitating underlying conditions. Overall, the isolates showed
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55 diminished susceptibility to fluconazole and echinocandins, although poor
56 biofilm formation and the low virulence were associated with favorable outcome.
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57 INTRODUCTION
58 Candida albicans is the main cause of candidemia, although a shift
59 towards other non-albicans Candida species, such as C. parapsilosis, C.
60 tropicalis, C. glabrata, and C. krusei, has been reported (1-4). The remaining
61 infrequent Candida species account for 8% of episodes (5), however, their
62 epidemiological characteristics remain largely unknown. D
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63 C. guilliermondii is a complex comprising several species, of which 3 n
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64 have been reported as human pathogens (C. guilliermondii sensu stricto d
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65 [Meyerozyma guilliermondii as the teleomorph form], C. fermentati [M. caribbica f
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66 as the teleomorph form], and C. carpophila). The proportion of candidemia

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67 episodes caused by C. guilliermondii complex has been poorly studied, tp
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68 although it is thought that this species is involved in up to 2% of cases (3, 6-9). a
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69 C. guilliermondii complex is characterized by diminished susceptibility to azoles as
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70 and echinocandins (9, 10), and has been reported mostly as a cause of .o
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71 candidemia in patients with cancer (11, 12). However, the low number of cases o/
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72 reported means that knowledge of the epidemiology of candidemia caused by A
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73 this complex is limited (9, 11). il
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74 We report 22 cases of candidemia caused by C. guilliermondii complex 0
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75 and compare them with cases of candidemia caused by C. albicans in order to b
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76 identify differential characteristics. We also performed in-depth microbiological g
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77 characterization of C. guilliermondii isolates based on molecular identification of t
78 species, antifungal susceptibility, biofilm formation, and virulence.
79 (This study was presented in part at the 26th European Congress on
80 Clinical Microbiology and Infectious Diseases in Amsterdam [Abstr. P-0350]
81 (13)).
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82 RESULTS
83 Patient demographics, risk factors, and outcome. Episodes caused by
84 C. albicans in the CANDIPOP study were compared with the overall cohort of C.
85 guilliermondii episodes in order to identify differential characteristics. To
86 preserve the assumption of independence of observations, only the first episode
87 of candidemia recorded for an individual patient was included in this analysis, D
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88 and episodes caused simultaneously by different Candida species were n
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89 excluded. Therefore, 22 C. guilliermondii episodes were finally compared with d
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90 337 C. albicans episodes (Table 1). Univariate analysis showed that patients f
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91 infected by C. guilliermondii were more likely to have underlying malignancy or

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92 neutropenia or to have undergone solid organ transplantation. Overall, 59% of tp
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93 these patients had received immunosuppressive therapy within the previous a
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94 month. Previous exposure to antifungals and long-term central venous as
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95 catheters (CVCs) were also more common in this group. Conversely, patients .o
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96 with C. albicans infection were older, had more comorbidities (e.g., diabetes), o/
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97 and were more likely to receive total parenteral nutrition. A
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98 Clinical data, therapeutic measures, and outcomes. The clinical il
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99 presentation of candidemia was less severe in patients infected by C. 0
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100 guilliermondii than in those infected by C. albicans. None of the patients b
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101 infected by C. guilliermondii had septic shock or severe sepsis at onset of g
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102 candidemia. Moreover, the C. guillermondii infections were less likely to occur t
103 during hospitalization in the intensive care unit, and 27.3% of cases were
104 outpatient-acquired, with all but one being health care–related (Table 1).
105 Therapeutic measures are shown in Table 2. Briefly, early removal of the CVC
106 was less frequent in patients infected by C. guilliermondii than in those infected
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107 by C. albicans (15.8% vs 50.2%, P=0.004). However, no differences were seen
108 between patients infected by C. guilliermondii and those infected by C. albicans
109 with respect to the frequency of catheter-related candidemia (27.3% vs 30.3%).
110 Early appropriate antifungal treatment was less common in patients infected by
111 C. guilliermondii than in those infected by C. albicans (36.4% vs 60.5%,
112 P=0.026), mainly because therapy was started more than 48 hours after D
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113 diagnosis (8/14, 57.1%) or because of empirical treatment with fluconazole n
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114 (5/14, 35.7%). d
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115 As for prognosis, persistent candidemia (blood cultures that were f
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116 persistently positive for ≥3 days after the incident blood sample) tended to be

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117 more common in patients infected by C. guilliermondii than in those infected by tp
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118 C. albicans (38.5% vs 26%). However, 30-day mortality was lower in the C. a
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119 guilliermondii group (13.6 % vs 33.9%, P=0.049). as
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120 Microbiological characterization of the isolates. All cases were caused r
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121 by C. guilliermondii sensu stricto (n=17) or C. fermentati (n=5). In terms of o
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122 biofilm formation, the isolates were moderate biofilm-forming (n=13) or low p
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123 biofilm-forming (n=9), with moderate metabolic activity (n=9), low metabolic 2
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124 activity (n=12), or high metabolic activity (n=1). Scanning electron microscopy 0
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125 revealed that C. guilliermondii complex biofilms were formed mainly by a layer b
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126 of clamped blastospores without hyphae or pseudo-hyphae, as seen in Corn- u
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127 meal agar medium (Figure 1). Table 3 shows the antifungal susceptibility of the t
128 isolates both in the planktonic forms and the sessile forms. Up to 9% (2/22) of
129 the isolates had fluconazole MICs above the epidemiological cut-off values
130 (ECOFFs). Differences in susceptibility to all the drugs tested between C.
131 albicans and C. guilliermondii complex isolates were observed in the planktonic
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132 form (P<0.001). According to the susceptibility of the biofilms, no differences
133 reaching statistic significance were observed between anidulafungin and
134 micafungin (SMIC50 geometric mean of 2.46 µg/ml, and 8.28 µg/ml respectively,
135 P=0.096).
136 C. guilliermondii complex isolates were significantly less virulent than C.
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137 albicans isolates (6 vs 1 mean survival days; P<0.001) in the G. mellonella o
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138 model (Figure 2). C. fermentati isolates were more virulent than C. guilliermondii lo
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139 sensu stricto isolates (4 vs 9.5 mean survival days; P<0.001), although the low d
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140 number of isolates of this species limited the analysis. We did not find fr
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141 differences in terms of biofilm formation or in susceptibility between C.
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142 fermentati and C. guilliermondii sensu stricto isolates (data not shown). :
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143 c
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144 DISCUSSION
.
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145 C. guilliermondii complex comprises C. guilliermondii sensu stricto, C. /
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146 fermentati, C. carpophila, and other species (6, 7). In previous studies, C. A
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147 guilliermondii sensu stricto and C. fermentati accounted for around 90%-95% il
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148 and 4.5%-9% of cases, respectively, with the remaining species of the complex 2
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149 playing a marginal role (less than 1%) (9, 14). In contrast, we found a higher 9
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150 frequency of cases caused by C. fermentati (22.7%) and a lower frequency of g
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151 cases caused by C. guilliermondii sensu stricto (77.2%). s
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152 The complex exhibited decreased in vitro susceptibility to all of the
153 antifungal agents studied, with rates of resistance to fluconazole and other
154 azoles of around 10%-15% (9, 12, 14). Up to 9% of the isolates studied here
155 were non–wild-type according to the fluconazole ECOFF. In the absence of
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156 species-specific breakpoints for C. guilliermondii, the rate of true fluconazole
157 resistance remains unknown. The MICs of micafungin and anidulafungin
158 against C. guilliermondii were, respectively, 16-, and 60-fold higher than the
159 MICs against C. albicans reported in the literature (3, 15). We also observed
160 that micafungin and anidulafungin were similarly effective at causing damage to
161 the C. guilliermondii biofilms, although the clinical impact of this observation is D
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162 unknown. n
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163 Candidemia caused by C. guilliermondii commonly originates in the CVC d
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164 (11, 16). We found that the CVC was involved in a high percentage (86.4%) of f
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165 patients, as was the case in patients infected by C. albicans. However, C.

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166 guilliermondii strains developed biofilms with moderate or low biomass and tp
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167 were significantly different from C. albicans isolates (Figure 1). This apparent a
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168 disagreement between the CVC as the source of infection and the scant ability as
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169 to form biofilms may be due to the fact that many cases of CVC-related .o
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170 candidemia involved long-term CVC (36.8%), unlike those infected by C. o/
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171 albicans (11.6%). Moreover, the method chosen to study the biomass or A
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172 metabolic activity implied shorter biofilm maturity (24 hours). il
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173 C. guilliermondii has been reported to be a cause of infection, mainly in 0
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174 patients with solid tumours and hematological malignancies, patients b
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175 undergoing chemotherapy, patients with neutropenia, patients with intravascular g
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176 and vascular catheters, patients receiving total parental nutrition, and patients t
177 with previous exposure to antibiotics, corticosteroids, and long-term antifungal
178 therapy (mostly fluconazole, amphotericin B, and caspofungin) (11, 12, 17). We
179 found that patients with candidemia caused by C. guilliermondii were more likely
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180 to have solid cancer or hematological malignancies, thus supporting some of
181 the previously reported risk factors associated with this infection.
182 As for prognosis, it seems reasonable to expect mortality to be high,
183 considering the severe underlying conditions of the patients and the low
184 susceptibility of the isolates to the antifungal drugs. However, we found that 7-
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185 day and 30-day mortality was lower and clinical presentation less severe in o
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186 patients infected by C. guilliermondii than in patients infected by C. albicans. lo
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187 This observation was in agreement with the low virulence of C. guilliermondii d
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188 isolates compared with C. albicans, as shown in the G. mellonella model fr
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189 (P<0.001). Although this model has been used to study the virulence of several
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190 Candida species (18), to our knowledge, this is the first time it has been used :
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191 for C. guilliermondii. The low virulence of the isolates can also be explained by a
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192 the limited production of biofilm, as previous studies showed that intense biofilm s
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193 formation is an independent factor of poor prognosis (19). The G. mellonella r
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194 model is relatively easy to handle; the correlation between the favorable o
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195 prognosis of the patients and the high survival of the larvae is encouraging and p
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196 suggests that this model could be used in the clinical microbiology laboratory to 2
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197 screen for the presence of highly virulent isolates. 0
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198 In conclusion, C. guilliermondii complex mainly affected patients with severe b
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199 debilitating conditions. The isolates were characterized by moderate biofilm u
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200 production, low susceptibility to azoles and echinocandins, and low virulence. t
201 However, the prognosis of the patients infected by this species is favorable.
202
203 MATERIALS AND METHODS
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204 Patients and isolates. We described the risk factors and outcome of 22
205 patients with an episode of monomicrobial candidemia caused by C.
206 guilliermondii complex who were admitted to Gregorio Marañon Hospital,
207 Madrid (n=9) or Vall d’Hebron Hospital, Barcelona (n=5) from 2007 to 2014. We
208 also included cases collected in the remaining hospitals participating in the
209 CANDIPOP project (n=8), a multicentre population-based study on candidemia D
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210 conducted from 2010 to 2011 in Spain (3, 20). The incident isolates were n
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211 identified using amplification and sequencing of the ITS1-5.8S-ITS2 region (3, d
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212 21). f
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213 Data collection. Cases were identified from the microbiological database h
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214 of each participating hospital. Data from the CANDIPOP study were collected :/
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215 prospectively, as previously described (3, 20). For cases that were not included c
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216 in the CANDIPOP study, clinical records from Gregorio Marañon Hospital and m
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217 Vall d’Hebron. rg
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218 Definitions. The definitions used have been reported elsewhere (20, 22). A
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219 In summary, episodes detected at or within 2 days of hospital admission were il
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220 considered outpatient-acquired. These episodes could also be classified as 2
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221 health care–related if patients fulfilled any of the following criteria: 9
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222 hospitalization for ≥2 days within the 90 days prior to candidemia, residence in a g
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223 long-term care facility, or chemotherapy or dialysis in the 30 days before s
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224 diagnosis of candidemia (23). The criteria for proven catheter-related
225 candidemia were those set out in the IDSA guidelines (24). Secondary foci
226 required isolation of the same Candida species in the suspected source of
227 infection. Breakthrough episodes were defined as candidemia in patients who
10