Population-Based Survey of filamentous fungi and Antifungal Resistance in Spain

25 Pages·2013·0.51 MB·English

AAC Accepts, published online ahead of print on 13 May 2013
Antimicrob. Agents Chemother. doi:10.1128/AAC.00383-13
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
1 Running title: Fungal epidemiology and antifungal resistance in Spain
2
3 Population-Based Survey of filamentous fungi and Antifungal
4 Resistance in Spain (FILPOP STUDY)

5
6 A. Alastruey-Izquierdo*1, E. Mellado1, T. Peláez 2, J. Pemán3, S. Zapico4, M. Alvarez5, J.L.
7 Rodríguez-Tudela1, M. Cuenca-Estrella1*, FILPOP Study Group.
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9 1 National Center for Microbiology. Madrid, Spain
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10 2 Hospital General Universitario Gregorio Marañón, Madrid, Spain www
11 3 Hospital Universitario La Fe, Valencia, Spain nnn
12 4 Hospital Universitario Donostia, Guipuzcoa, Spain loaloaloa
13 5 Hospital Universitario Central de Asturias, Oviedo, Spain ddd
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15 *Corresponding author. Mailing address: Mycology Department. Spanish National Center for f f f
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16 Microbiology. Instituto de Salud Carlos III, Majadahonda, Madrid, Spain. ooo
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17 Phone: + 34-91-8223661. Fax: + 34-91-5097966 E-mail: [email protected]

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20 Abstract
21 A population-based survey was conducted to investigate the epidemiology and the antifungal
22 resistance in Spanish clinical strains of filamentous fungi isolated from deep tissue samples,
23 blood cultures and respiratory samples. The study was conducted in two different periods
24 (October 2010 and May 2011) to analyze seasonal variations. A total of 325 strains were
25 isolated in 29 different hospitals. Average prevalence was 0.0016/1,000 habitants. Strains
26 were identified by sequencing of DNA targets and susceptibility testing by EUCAST reference D
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27 procedure. The most frequent genus was Aspergillus accounting for 86.3% of the total isolates, w
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28 followed by Scedosporium 4.7%; Mucorales 2.5%; Penicillium 2.2%, and Fusarium, 1.2%. The lo
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29 most frequent species was Aspergillus fumigatus (48.5%), followed by A. flavus (8.4%), A. d
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30 terreus (8.1%), A. tubingensis (6.8%) and A. niger (6.5%). Cryptic/sibling species of Aspergillus
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31 accounted for 12% of total cases. Resistance to amphotericin B was found in 10.8% of isolates,
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32 while extended spectrum triazole resistance ranged between 10 to 12.7% depending on the h
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33 azole tested. Antifungal resistance was more common among emerging species such as p
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34 Scedosporium and Mucorales and also among cryptic species of Aspergillus with a 40% of these a
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35 isolates showing resistance to all antifungal compounds tested. Cryptic Aspergillus species .
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36 seems to be underestimated and their correct classification could be clinically relevant. m
37 Performing antifungal susceptibility testing of the strains implicated in deep infections and .o
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38 multicentric studies are recommended to evaluate the incidence of these cryptic species in /

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39 other geographical areas. n

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42 INTRODUCTION
43 The number of fungal pathogenic species has increased significantly in the last years (1, 2). The
44 increasing populations at risk for fungal infections and the advances in diagnostic tools have
45 been pointed out as possible reasons for this increment. As the population at risk is expected
46 to keep growing in the coming years the interest for the taxonomy and epidemiology of fungal
47 infections is also increasing. The use of molecular tools for fungal identification has allowed a
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48 deeper study of pathogenic fungi genetics and as a consequence several species have been
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49 revealed to be species complexes (3-5). They are formed by species that are almost
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50 indistinguishable by morphological methods; hence they have been designated as cryptic
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51 species. Therefore, classical identification methods relaying on the phenotypic characteristics e
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52 are no longer suitable for strains classification and the use of molecular tools is yielding a f
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53 continuous description of new taxa (3, 6, 7). m

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54 Some of these species have been already found in clinical samples but their prevalence and tp
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55 relevance in the clinical setting is still unknown. Several studies have been published about the /
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56 epidemiology of yeast infections (8-10), but in moulds, the limited available data are mainly c
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57 based on retrospective studies or only dealing with specific groups of moulds (11-14). In s
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58 addition, according to some clinical trials, a total of 50 to 75% of cases enrolled are diagnosed .
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59 by microscopic examination of tissues or by detection of fungal components what means that g
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60 species causing the infection are never known in a relevant number of cases (15, 16). However,
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61 the prevalence of rare, emerging, cryptic and sibling species seems to be rising. Species such as J
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62 Scedosporium and Fusarium could cause 5% to 10% of deep mycosis in some geographical u
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63 areas (17, 18). Recently the frequency of cryptic species of moulds has been analyzed in two ry

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64 studies with transplant patients in USA (19, 20). Up to our knowledge, prevalence of these
,

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65 species has not being studied in Europe so far. In addition, some of these cryptic species, such 0
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66 as Aspergillus lentulus and A. calidoustus, are more resistant to the antifungal drugs available 9

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67 (3, 6) highlighting the importance of a correct identification. Moreover, some studies have y

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68 pointed out the emergence of secondary resistance in Aspergillus spp. in Europe (21, 22) but u
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69 its prevalence in Spain has not been investigated yet. t
70 In this study we aimed to analyze species distribution and prevalence of antifungal drugs
71 resistance in Spain through a multicentre prospective study involving 29 hospitals in different
72 regions of Spain.
73
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74 MATERIAL AND METHODS
75 Strains
76 The study was conducted prospectively in two different periods one in fall (October 2010) and
77 one in spring (May 2011). We included all patients admitted in 29 Spanish hospitals with a
78 positive culture of filamentous fungi from respiratory samples, blood cultures and biopsies.
79 The strains were sent to the Spanish National Centre of Microbiology for identification and D
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80 susceptibility testing. A referral form was filled out by isolate including demographic and w
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81 clinical data. Strains were classified as colonizers or as of clinical relevance (proven, probable lo
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82 and suspected infections) according to site of isolation and clinical report (23). d
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83 The prevalence of fungal infections by filamentous fungi was calculated for each hospital, using f
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84 as the reference, the number of admitted patients for each period divided by the average m

85 population associated to the hospital (data provided by the hospitals). h
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p
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86 Morphological Identification //
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87 The strains were subcultured in different media to ascertain their macroscopic and .a
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88 microscopic morphology. The media included malt extract agar (MEA, 2% malt extract (Oxoid m
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89 S.A., Madrid, Spain)), potato dextrose agar (PDA, Oxoid S.A.), oatmeal agar (OMA, Oxoid S.A.),
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90 potassium chloride agar (Oxoid S.A.) and Czapek-Dox Agar (Difco, Soria Melgizo S.A., Madrid, /
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91 Spain). Cultures were incubated at 30°C and 37°C. Fungal morphological features were
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92 examined macro and microscopically by conventional methods (24) n
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93 Molecular identification r
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94 Moulds were subcultured in GYEP medium (0.3% yeast extract, 1% peptone, Difco, Soria 2
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95 Melguizo) with 2% glucose (Sigma Aldrich Quimica, Madrid, Spain), for 24 to 48h at 30°C. 1
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96 Genomic DNA was isolated using an extraction procedure previously described (25). Molecular b
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97 identification was performed by sequencing informative targets. DNA segments comprising the g
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98 ITS1 and ITS2 regions, were amplified for all the strains with primer set ITS1 (5’- e
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99 TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) (26). In the case of
100 Aspergillus and Scedosporium isolates, a portion of the beta tubulin gene was sequenced with
101 the following primers: βtub3 (5'-TTCACCTTCAGACCGGT-3') and βtub2 (5′-
102 AGTTGTCGGGACGGAATAG-3′) (3) for Aspergillus and TUB-F (5’-
103 CTGTCCAACCCCTCTTACGGCGACCTGAAC-3’) y TUB-R (5´-
104 ACCCTCACCAGTATACCAATGCAAGAAAGC-3’) (27) for Scedosporium. Also, DNA segments
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105 comprising the elongation factor alpha region were amplified for Fusarium isolates with
106 primers EF1 (5’-ATGGGTAAGARGACAAGAC-3’) and EF2 (5’-GGARGTACCAGTSATCATGTT -3’)
107 (28). All primers were synthesized by Sigma Genosys (Madrid, Spain). The reactions were
108 performed in a GeneAmp PCR System 9700 (Applied Biosystems). The reaction mixtures
109 contained 0.5 μM of each primer, 0.2 μM of each deoxynucleoside triphosphate, 5 μl of PCR
110 10× buffer (Applied Biosystems, Madrid, Spain), 2.5 U Taq DNA polymerase (Amplitaq; Applied
111 Biosystems), and 25 ng of DNA in a final volume of 50 μl. The samples were amplified in a D
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112 GeneAmp PCR System 9700 (Applied Biosystems) by using the following cycling parameters: w
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113 one initial cycle of five min at 94°C, followed by 35 cycles of 30 s at 94°C, 45 s at 56°C (ITS), lo
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114 55°C (β-tubulin) or 47°C (Elongation factor α), and two min at 72°C, with one final cycle of five d
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115 min at 72°C. The reaction products were analyzed in a 0.8% agarose gel. Sequencing reactions d

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116 were done with two μl of a sequencing kit (BigDye Terminator cycle sequencing, ready ro
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117 reaction: Applied Biosystems), one μM of primers [the same as in the PCR except for
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118 Aspergillus β tubulin were βtub1 (5’-AATTGGTGCCGCTTTCTGG-3’) and βtub4 (5’- tp
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119 AGCGTCCATGGTACCAGG-3’) were used] and three μl of the PCR product in a final volume of /a
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120 ten μl. c
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121 Sequences were assembled and edited using the SeqMan II and EditSeq software packages
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122 (Lasergene; DNAstar, Inc., Madison, WI, USA). All sequences were compared with reference r
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123 sequences from GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and Mycobank o
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124 (http://www.mycobank.org/) databases with InfoQuest FP software, version 4.50 (BIORAD J
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125 Laboratories, Madrid, Spain), as well as with the data base belonging to the Department of n
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126 Mycology of the Spanish National Centre for Microbiology which holds 9,000 sequences from a
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127 strains belonging to 270 different fungal species. This database was designed by the Spanish 4
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128 National Centre for Microbiology and has restricted access. 2
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129 Antifungal susceptibility testing.
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130 Microdilution testing was performed following the EUCAST standard methodology (29). g
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131 Aspergillus fumigatus ATCC 2004305 and Aspergillus flavus ATCC 2004304 were used as quality s
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132 control strains. The antifungal agents used in the study were amphotericin B (Sigma-Aldrich
133 Quimica), itraconazole (Janssen Pharmaceutica, Madrid, Spain), voriconazole (Pfizer S.A.,
134 Madrid, Spain), ravuconazole (Bristol-Myers Squibb, Princeton, U.S.A.), posaconazole
135 (Schering-Plough Research Institute, Kenilworth, N.J.), terbinafine (Novartis, Basel,
136 Switzerland), caspofungin (Merck & Co., Inc., Rahway, N.J.), micafungin (Astellas pharma Inc.,
137 Tokio, Japan) and anidulafungin (Pfizer S.A.). The final concentrations tested ranged from 0.03
5
138 to 16 mg/L for amphotericin B, terbinafine, caspofungin, micafungin and anidulafungin, and
139 from 0.015 to 8 mg/L for itraconazole, voriconazole, ravuconazole and posaconazole. The
140 plates were incubated at 35ºC for 48 h in a humid atmosphere. Visual readings were
141 performed at 24 and 48 hours with the help of a mirror. The endpoint for amphotericin B,
142 itraconazole, voriconazole, ravuconazole, posaconazole and terbinafine was the antifungal
143 concentration that produced a complete inhibition of visual growth at 24 and 48 hours. For the
144 echinocandins the endpoint was the antifungal concentration that produced a visible change in D
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145 the morphology of the hyphae compared with the growth control well (minimum effective w
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146 concentration, MEC). The EUCAST have set breakpoints to interpret antifungal susceptibility lo
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147 testing results of amphotericin B (resistant strain MIC value >2 mg/L), itraconazole (MIC >2 d
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148 mg/L), voriconazole (MIC >2 mg/L), and posaconazole (MIC > 0.25 mg/L) d

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149 (http://www.eucast.org/clinical_breakpoints/)(29-31). These breakpoint values have been set ro
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150 only for some Aspergillus spp., but were used in this study to analyze rate of resistance in vitro
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151 for all species. Breakpoints of echinocandins have not been set yet, and rate of resistances tp
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152 were not calculated. /
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153 Statistical analysis .a
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154 Descriptive and comparative analyses were done. Differences in proportions of fungal species .
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155 were determined by Fisher’s exact test or by chi-square analysis. The significance of the g
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156 differences between MIC values was determined by the analysis of variance (ANOVA,
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157 Bonferroni post-hoc) or non-parametric tests. P<0.01 was considered statistically significant. J
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158 Statistical analysis was performed with (IBM SPSS Statistics 19.0, SPSS Iberica, Madrid, Spain). u
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160 RESULTS
161 The average prevalence was of 0.002% (isolates divided by admitted patients) or 0.017 per
162 1,000 habitants in October 2010 and 0.0018% or 0.016 per 1,000 habitants in May 2011. A
163 total of 325 isolates coming from 23 hospitals were included in the study. Two hundred and
164 seven isolates were collected in the first period (October 2010) and 118 in the second period
165 (May 2011). Six hospitals reported no isolates matching the conditions of the study. A total of
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166 309 out of 325 (95%) clinical strains were isolated from respiratory samples. Number of
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167 isolates which were regarded as colonizers was 186/309 (60%). The remaining 123 isolates
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168 (40%) were accounted as of clinical relevance, including 32/123 (26%) recovered from
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169 bronchoalveolar lavages. Fungi were cultured from tissue samples or sterile fluids in 5% of e
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170 patients (16/325). Proven infections were reported in 13 cases, including one of those with f
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171 blood-cultures positive. Three cases with isolates recovered from drainages of deep sites were m

172 regarded as colonization. h
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173 Of the 325 isolates included in the study, 322 were identified by means of observation of /
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174 morphology characteristics, ITS sequencing and part of β-tubulin or elongation factor α genes c
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175 when need; three isolates could not be analyzed at the reference center for absence of growth s
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176 or contamination when received. Table 1 shows the number of isolates classified in each .
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177 genus. The most frequent genus was Aspergillus accounting for 86.3% (278 isolates) of the g
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178 total isolates, followed by Scedosporium 4.7% (15 isolates); Mucorales 3.7% (12 isolates);
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179 Penicillium 2.2% (seven isolates); and Fusarium, 1.2% (four isolates). Table 1, also displays rates J
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180 of genus by clinical significance (colonizers vs. of clinical relevance). No statistical differences u
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181 were found although researchers reported Mucorales as of clinical relevance in 8 cases (8/12, ry

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182 66%) and as colonizers in other four patients. This difference was close to the statistical ,

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183 significance (P=0.04). Of the 13 cases of proven infections, most of them were caused by 0
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184 Aspergillus spp. but Mucorales were found in two cases of sino-orbital infections, Fusarium 9

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185 oxysporum was isolated from blood in a cirrhotic patient suffering from fungemia and y

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186 Scopulariopsis brevicaulis in a cardiac valve. u
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187 The species distribution is showed in table 2. The most frequent species was Aspergilus
188 fumigatus with 156 isolates (48.5%), followed by Aspergillus flavus with 27 isolates (8.4%),
189 Aspergillus terreus with 26 isolates (8.1%), Aspergillus tubingensis with 22 isolates (6.8%) and
190 Aspergillus niger with 21 isolates (6.5%). The rest of species had less than 10 isolates. The low
191 number of isolates belonging to most of species precludes statistical analysis, but some
192 findings can be noted. Firstly, some species were more frequently considered of clinical
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193 relevance than colonizers but only in the cases of A. terreus, A. nidulans and Rhizopus arrhizus
194 (syn. Rhizopus oryzae), that difference was significant (P<0.01). Table 2 also shows the
195 distribution of fungal species by study period (October vs. May). Some species such as A.
196 tubingensis, A. niger and R. arrhizus were more frequently isolated in October 2010 than in
197 May 2011, unlike Aspergillus calidoustus, Aspergillus alliaceus, Scedosporium boydii and
198 Scedosporium apiospermum that were collected more commonly in May 2011. In order to
199 avoid biases from centers, an analysis of species distribution by participant was done. No D
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200 significant differences were observed and outbreaks due to a specific species were neither w
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201 reported in the study periods. A. fumigatus was the most common fungal species in all lo
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202 participants, followed by other Aspergillus spp., Scedosporium spp. and Mucorales. d
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203 The identification of organisms by PCR amplification and DNA sequencing allowed to detect f
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204 cryptic or sibling fungal species (table 2). Regarding complexes of Aspergillus species, from the m

205 total of 278 Aspergillus strains, 40 (14.5%) isolates were classified as cryptic species. The h
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206 Aspergillus section Fumigati included 162 strains of which 6 (3.7%) were non-A. fumigatus :
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207 sensu stricto, being three Aspergillus lentulus, one Aspergillus viridinutans, one Aspergillus a
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208 fumigatiaffinis and one Neosartorya pseudofischeri. The Aspergillus section Flavi had 30 .a
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209 strains, 27 A. flavus and three A. alliaceus. The Aspergillus section Nigri included 22 A. m
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210 tubingensis and 21 A. niger. The section Terrei 26 A. terreus and one Aspergillus carneus. The
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211 section Nidulantes eight A. nidulans. Other sections such us Usti (four A. calidoustus, one /
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212 Aspergillus insuetus and one Aspergillus keveii), Versicolores (one Aspergillus sydowii) and
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213 Circumdati (one Aspergillus westerdijkiae) were also represented. n
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214 Table 3 shows geometric mean, range, MIC50 (MIC value causing inhibition of 50% of isolates), ry

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215 MIC (MIC value causing inhibition of 90% of isolates) and mode of the species isolated in the
90 ,
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216 study. Only species represented with three or more isolates are displayed. Resistance in vitro 0
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217 was uncommon among the most frequent species. According to EUCAST breakpoint values, 9

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218 the resistance to amphotericin B was found in 35/322 (10.8%) isolates, to itraconazole in y

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219 32/322 (10%), to voriconazole in 36/322 (11.2%) and to posaconazole in 41/322 (12.7%). u
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220 Resistance in vitro was more common among rare and emerging species and multi-resistant t
221 isolates were isolated in some cases.
222 None A. fumigatus isolate was resistant in vitro to amphotericin B, itraconazole and
223 voriconazole. One A. fumigatus strain showed a MIC value of posaconazole of 0.50 mg/L.
224 Other taxa belonging to Aspergillus genus showed some rates of resistance in vitro. A total of
225 4/27 (14.8%) A. flavus and 7/26 (27%) A. terreus were resistant to amphotericin B. Among
226 cryptic/sibling species of Aspergillus complexes, a total of 16 out of 40 (40%) strains were
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227 resistant in vitro to at least one antifungal compound. All A. lentulus were resistant to
228 itraconazole, all A. calidoustus to voriconazole and posaconazole and all A. alliaceus to
229 amphotericin B.
230 Regarding other fungal species, Scedosporium species showed high MICs of most of drugs
231 tested. S. prolificans was clearly a multi-resistant species but some S. boydii and S.
232 apiospermum showed susceptibility in vitro to voriconazole, posaconazole and in some cases
233 to echinocandins (table 3). Fusarium spp. were resistant in vitro to all azole agents and D
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234 echinocandins. Amphotericin B showed some activity in vitro against some Fusarium isolates. w
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235 Regarding Mucorales, five different species were found (R. arrhizus, Lichtheimia ramosa, lo
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236 Lichtheimia corymbifera, Rhizopus microsporus and Rhizomucor pusillus), only amphotericin B d
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237 showed good activity to all species. Echinocandins had no activity to these fungal species. d

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238 Among azoles, posaconazole showed moderate activity to all species (MICs50 < 0.5 mg/L), ro
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239 voriconazole yield high MICs values and itraconazole was active to Lichtheimia isolates (MICs=
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240 0.5mg/L). Seven strains of Penicillium were isolated, among them, amphotericin B and tp
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241 echinocandins showed activity to all species while azoles showed variable results being /
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242 Penicillium cetrinum and Penicillium minioluteum isolates resistant in vitro to the azoles. c
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243 Finally, terbinafine was active in vitro against Aspergillus spp. apart from A. fumigatus. Other s
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244 fungal species were unsusceptible to this compound. .
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245 g
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247 DISCUSSION
248 The FILPOP study is the first Spanish population-based survey about prevalence of fungal
249 species and antifungal drugs resistance. The design of the study included molecular
250 identification of organisms by sequencing of DNA targets in order to detect cryptic/sibling
251 species and susceptibility testing by the reference methodology of the EUCAST(29).
252 The use of molecular methods in fungal studies has produced several changes in the
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253 taxonomy. New species have been described and others have been discovered to be
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254 complexes of several species. In addition, emergence of resistance seems to increase in fungal
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255 infections (21, 22). These changes in taxonomy and the emergence of resistant strains have
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256 produced a need of strain identification and susceptibility testing and several studies have e
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257 been conducted with the aim of species reclassifications (32-34). f
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258 First among the difficulties to plan a survey about the invasive mould disease is the limitation
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259 to collect cases of proven infections. Mold species are saprophytes of humans and tp
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260 contaminants of laboratory and subsequently their isolation on cultures is not of clinical /
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261 interest for many cases. The results of the FILPOP study show that the prevalence of isolation c
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262 of filamentous fungi on cultures of clinical samples from deep sites is low (0.016 to 0.017 per s
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263 1,000 habitants). That prevalence is very similar regardless the time of the year (spring or fall) .
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264 although some variations were found by participant, as is evidenced by the fact that 6 out of g
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265 29 (20%) centers which take part in the survey did not isolate any organism in the study
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266 periods. Researchers reported more than 60% of isolates as colonizers or without clinical J
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267 relevance and only 13 cases were proven infections. u
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268 The species distribution of the FILPOP study proves that Aspergillus spp. are still the most
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269 common mold isolated of human samples from deep sites (>85%). Emerging pathogens are not 2
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270 as rare as suggested since they were isolated in 14% of samples. Scedosporium spp. were 1
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271 found in 5% of cases, Mucorales in 3.7%, and Penicillium spp. and Fusarium spp. in 2% and b
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272 1.2%, respectively. g
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273 The isolates of A. fumigatus represented less than 50% and other 16 species of this genus were t
274 isolated. Regarding A. flavus, A. terreus, and A. niger, regional differences about the presence
275 of these species in clinical samples have been reported, thus A. flavus has been described as
276 the most common species of Aspergillus isolated in some centers (35) and A. terreus is
277 particularly frequent in Austria (36). Balajee et al (37) analyzing Aspergillus strains from a
278 multicentre study of transplant patients performed in USA found higher rate of A. flavus
10