Epidemiology, antifungal susceptibility and pathogenicity of Candida africana isolates from the ...

27 Pages·2013·0.65 MB·English

JCM Accepts, published online ahead of print on 9 January 2013
J. Clin. Microbiol. doi:10.1128/JCM.02816-12
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
Epidemiology, antifungal susceptibility and
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pathogenicity of Candida africana isolates from the
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United Kingdom
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6 Andrew M. Borman*, Adrien Szekely, Chistopher J. Linton, Michael D. Palmer,
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7 Phillipa Brown, and Elizabeth M. Johnson. m

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10 UK National Mycology Reference Laboratory (MRL), Health Protection Agency a
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11 South-West, Bristol, United KINGDOM. .o
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14 Running Title: Candida africana in the UK e
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16 Key words: Pathogenic yeasts / Candida africana / genital / yeast identification / 8
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17 antifungal susceptibility / pathogenicity/ Candida albicans complex 0
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20 *Corresponding author footnote: t
21 Mycology Reference Laboratory
22 Myrtle Road
23 Kingsdown
24 Bristol BS2 8EL
25 Telephone : [phone]
26 Fax : [phone]
27 Email : [email protected]
28
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29 ABSTRACT
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31 Candida africana was previously proposed as a new species within the Candida
32 albicans species complex, together with C. albicans and C. dubliniensis, although
33 further phylogenetic analyses better support its status as an unusual variant within C. D
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34 albicans. Here we show that C. africana can be distinguished from C. albicans and C.
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35 dubliniensis by Pyrosequencing® of a short region of ITS2, and have evaluated its a
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36 occurrence in clinical samples by Pyrosequencing® all presumptive isolates of C.
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37 albicans submitted to the Mycology Reference Laboratory over a 9 month period. The m

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38 C. albicans complex constituted 826/1839 (44.9%) of yeast isolates received over the t
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39 study period and included 783 isolates of C. albicans, 28 isolates of C. dubliniensis jc
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40 and 15 isolates of C. africana. In agreement with previous reports, C. africana was a
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41 isolated exclusively from genital specimens, in women in the 18-35 years age group. .o
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42 Indeed, C. africana constituted 15/251 (6%) of “C. albicans” isolates from female /
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43 genital specimens during the study period. C. africana isolates were germ-tube D
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44 positive, grew significantly more slowly than C. albicans and C. dubliniensis on e
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45 conventional mycological media, could be distinguished from the other members of e
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46 the C. albicans complex by appearance on chromogenic agar, and were incapable of 8
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47 forming chlamydospores. Here we present the detailed evaluation of epidemiological, 0
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48 phenotypic and clinical features and antifungal susceptibility profiles of UK isolates b
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49 of C. africana. Furthermore, we demonstrate that C. africana is significantly less u
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50 pathogenic than C. albicans and C. dubliniensis in the Galleria mellonella insect t
51 systemic infection model.
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53 248 words
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54 INTRODUCTION
55 The incidence of invasive fungal infections caused by unusual Candida spp.
56 continues to rise, in part due to increased populations of immunocompromised
57 patients and those undergoing invasive procedures (19, 23, 25, 26, 32, 33, 45, 46).
58 Nevertheless, Candida albicans remains the most frequently isolated Candida species D
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59 in the clinical setting, is the principal agent of nosocomial yeast infections (19, 26, 32, n
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60 33), and is widely accepted as being the most pathogenic Candida species (reviewed d
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61 in 20). In addition to C. albicans sensu stricto, two closely related species have been f
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62 described and added to the C. albicans species complex. In 1995, Candida

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63 dubliniensis was described, predominantly associated with cases of oral candidiasis in p
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64 human immunodeficiency virus-infected patients (40). Whilst phylogenetically jc
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65 closely related to C. albicans, and sharing the ability to produce germ tubes, true s
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66 hyphae and chlamydospores, epidemiological analyses have revealed that C. o
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67 dubliniensis is far less prevalent than C. albicans (17, 36, 37, 39), is comparatively o
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68 rarely associated with systemic infection (17, 39), and is less pathogenic than C.
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69 albicans in a variety of infection models (21, 30, 37, 39). m
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70 Similarly, Candida africana was initially described as an atypical, e
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71 chlamydospore-negative variant of C. albicans (12, 41), but was proposed as a new
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72 species on the basis of morphological, biochemical and physiological differences (1, 1
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73 41). While subsequent molecular analyses supported a varietal distinction (C. albicans
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74 var. africana) (15), the taxonomic status of C. africana remains controversial. Although u
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75 germ-tube positive like C. albicans and C. dubliniensis, C. africana isolates studied to
76 date have reportedly grown and produced hyphae more slowly than either C. albicans
77 or C. dubliniensis, and could be distinguished from both by an inability to assimilate
78 several sugars or to produce chlamydospores and by appearance on chromogenic agars
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79 (1, 27, 31, 41, 42). Moreover, despite an almost worldwide distribution, the
80 overwhelming majority of C. africana isolates have been recovered from female genital
81 specimens (reviewed in 30). Epidemiological analyses of C. africana have been
82 hampered by the failure of the commonly employed commercially available
83 identification methods to distinguish it from C. albicans (reviewed in 27, 30). However, D
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84 studies employing PCR amplicon length analyses of the Hwp-1 gene (28) revealed that
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85 C. africana constituted 7.2% of C. albicans complex isolates in three different hospitals a
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86 in southern Italy, a prevalence 3-times that of C. dubliniensis in samples from
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87 principally non-sterile sites from the same cohort of hospitalised patients (29). m

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88 Here we have shown that C. africana can be distinguished from C. albicans and t
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89 C. dubliniensis by Pyrosequencing of a short fragment of the internal transcribed spacer jc
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90 region 2 (ITS2), and have subsequently used Pyrosequencing to assess the prevalence a
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91 of C. africana amongst Candida isolates from clinical specimens submitted to the UK .o
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92 National Mycology Reference Laboratory (MRL) over a 9 month period spanning /
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93 2011-2012. Isolates identified as C. africana by Pyrosequencing were subjected to D
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94 phenotypic and biochemical analyses, and their antifungal susceptibility profiles were e
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95 established. Finally, we compared the pathogenicity of C. africana, C. albicans and C. e
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96 dubliniensis clinical isolates in vivo in the Galleria mellonella systemic infection 8
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97 model. 0
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98 b
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99 MATERIALS AND METHODS
100 Antifungal agents. Antifungal drugs were obtained from their respective
101 manufacturers as standard powders. Amphotericin B, nystatin and clotrimazole (all
102 Sigma Chemical Co, St Louis, Mo) were dissolved in dimethyl sulfoxide
103 (amphotericin and nystatin) and PEG400 with heating to 70oC (clotrimazole). D
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104 Itraconazole, miconazole, econazole and ketoconazole (all Janssen Research
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105 Foundation, Beerse, Belgium) were prepared in PEG400 with heating to 70oC a
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106 (itraconazole and miconazole), or DMSO (econazole and ketoconazole). Fluconazole
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107 (Pfizer Central Research, Sandwich, UK) was resuspended in sterile water. Serial 2- m

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108 fold dilutions of the various drugs were prepared in RPMI 1640 medium (with L- t
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109 glutamine, without bicarbonate; Sigma Chemical Co, St Louis, Mo), and buffered to jc
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110 pH 7.0 using a 0.165 M solution of MOPS (Sigma Chemical Co, St Louis, Mo). a
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111 Yeast isolates and conventional phenotypic identification approaches. A .o
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112 total of 1839 clinical isolates of yeast were included in the current study. Isolates had /
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113 been submitted to the UK National Mycology Reference Laboratory (MRL) for D
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114 identification, susceptibility testing, or both. Isolates were from both sterile and none- e
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115 sterile sites (female genital n=381; male genital n=10; blood and line tips n= 600; e
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116 sputum and BAL n=199; faeces n= 16; drain fluids n= 144; tissue n= 136; eyes n=37; 8
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117 CSF n=14; urine n=71; ear n=12 and site not stated n=219). All isolates were sub- 0
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118 cultured on plates of Oxoid Sabouraud dextrose agar containing 0.5% w/v b
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119 chloramphenicol (Unipath Limited, Basingstoke, England). Cultures were incubated u
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120 for 24 h at 35oC prior to testing. Isolates of C. africana have been stored in sterile t
121 water in a metabolically inactive state in the National Collection of Pathogenic Fungi
122 (NCPF).
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123 The clinical isolates included in this study were all subjected to initial germ-
124 tube testing (inoculation into sterile horse serum and incubation at 37oC for 3 h).
125 Isolates were also cultured on Dalmau plates (Oxoid cornmeal agar, supplemented
126 with 1% Tween 80, with a sterile coverslip over a single streak of the organism to
127 establish the additional morphological characteristics required for complete D
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128 AUXACOLOR2 profiles) and MAST-ID CHROMagar® Candida plates to establish
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129 the purity of isolates. Strains formally identified as Candida africana by molecular a
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130 approaches were also subjected to the AUXACOLOR2 identification kit (Bio-Rad
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131 Laboratories Ltd, Hemel Hempstead, UK) exactly as described previously (7). m

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132 Molecular identification. All 1839 test isolates were subjected to Pyrosequencing t
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133 analysis of a portion of ITS2 exactly as described previously (4,5,6). For isolates jc
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134 identified by Pyrosequencing as C. africana, identity was confirmed by PCR a
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135 amplification and sequencing of a region of the large subunit gene (LSU) and the .o
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136 Internal Transcribed Spacer 1 (ITS1) region using the primers described previously /
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137 (2, 18) and direct inoculation of PCR products with yeast suspensions as described in D
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138 (3). Organisms were identified using BLAST searches against fungal sequences in e
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139 existing public DNA databases (EMBL) and multiple sequence alignments using e
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140 ClustalW and a database of formally identified organisms compiled by the MRL. 8
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141 Broth microdilution determination of yeast minimum inhibitory concentrations 0
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142 (MICs). MICs were determined according to CLSI methodologies (CLSI M27-A3; b
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143 [8]) in round-bottomed 96 well plates with yeast blastospore suspensions prepared in u
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144 saline and then diluted into RPMI 1640 and adjusted to final concentrations of 2.5 x t
145 103 CFU/ml. Inoculated plates were incubated for 24-48 h at 35 oC. MICs were read at
146 24 and 48 h as the concentration of drug that elicited 100% inhibition of growth
147 (amphotericin B, nystatin) or significant (approx. 50 %) inhibition of growth
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148 compared with a drug-free control (itraconazole, fluconazole, miconazole,
149 ketoconazole, econazole and clotrimazole).
150 Galleria mellonella virulence assays. Killing assays were performed in Galleria
151 mellonella essentially as described previously (9, 10, 13, 14). Briefly, final (sixth)
152 instar larvae weighing approximately 300 mg each (Livefood UK Ltd, Rooks Bridge, D
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153 Somerset, UK) that were absent of grey markings were maintained at room
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154 temperature in the dark, and inoculated within one week of receipt. Suspensions of a
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155 individual Candida isolates that had been grown on Sabouraud’s agar for 24 h at 37oC d
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156 were harvested by gentle scraping of colony surfaces with sterile plastic loops, m

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157 washed twice in sterile PBS, counted in hemocytometers and adjusted to 105 cells per tt
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158 microlitre in sterile PBS. Individual larvae were inoculated in the left rear pro-leg jc
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159 with 1 x 106 yeast cells in PBS (final inoculum volume 10 μl) using a 10 μl Hamilton a
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160 syringe fitted with a 26 gauge blunt needle. Fifteen larvae were inoculated per isolate .o
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161 per experiment (experiments employed 4 independent isolates of each Candida test /
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162 species). Control groups of larvae received 10 μl of sterile PBS in exactly the same D
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163 manner. Inoculated larvae were incubated at 30 oC, 37 oC or 42 oC and scored for e
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164 viability at 8 h intervals as described previously (9, 10, 13, 14). Differences in e
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165 resulting Kaplan-Meier survival plots were evaluated using the Mann-Whitney (two 8
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166 sample Wilcoxon) test. In experiments designed to assess differences in fungal cell 1
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167 filamentation post-infection, representative larvae from each inoculum group were cut b
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168 open at 24 h post infection and the fat body/solid internal structures and hemolymph u
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169 were collected aseptically as described previously (14) and examined microscopically.
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171 RESULTS
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173 We have previously demonstrated that Pyrosequencing of 35 base pairs of a
174 portion of the internal transcribed spacer region 2 (ITS2) is sufficient to robustly
175 identify the vast majority of pathogenic yeasts (4), including genetically closely related D
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176 isolates within species complexes (5, 6). This is also the case for the key species within
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177 the C. albicans species complex. To date, we have identified 4 C. albicans sequence a
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178 variants within the 35 bp ITS2 signature sequence we employ for yeast identification, 2
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179 C. dubliniensis variants and a single signature sequence shared by all C. africana m

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180 isolates encountered to date (Table 1). All C. dubliniensis and C. africana isolates differ t
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181 from C. albicans at 10-11 and 1-2 positions, respectively, within the 35 base jc
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182 Pyrosequencing region (Table 1). Conversely, the ITS2 sequence for C. stellatoidea a
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183 (based on published ITS2 sequences for the Type strain, isolate CBS 1905; EMBL .o
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184 AJ853768) are indistinguishable from C. albicans variant 1 (Table1), which is the most /
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185 common ITS2 sequence variant encountered in UK isolates. D
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186 In order to evaluate the prevalence and potential clinical significance of C. e
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187 africana in clinical samples in the UK, between September 2011 and July 2012, the UK e
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188 National Mycology Reference Laboratory identified 1839 yeast isolates to species level 8
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189 using Pyrosequencing. C. albicans species complex constituted 44.9% (826/1839) of 0
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190 all isolates identified, and comprised 783 isolates of C. albicans (42.6% of all isolates), b
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191 28 isolates of C. dubliniensis (1.5%) and 15 isolates of C. africana (0.8%). Thus, when u
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192 considering isolates from all clinical sites, C. dubliniensis and C. africana represented t
193 3.4% and 1.8%, respectively, of all C. albicans complex isolates (Table 2). In
194 agreement with the published reports concerning the occurrence of C. africana (31), all
195 15 isolates identified in this study had been isolated from genital specimens (Table 2) in
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196 young female adults (age range 18-31 years; Mean age 24.7 years). Indeed, when only
197 the C. albicans complex isolates from female genital specimens were considered (251
198 isolates in total), C. africana (15 isolates) was much more prevalent than C.
199 dubliniensis (3 isolates) and represented 6% of C. albicans complex isolates from such
200 specimens. D
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201 Candida africana isolates had been submitted to the MRL from hospitals across
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202 central and Southern England and Wales. In agreement with one published evaluation a
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203 (24), all isolates that were tested were found to be susceptible (with very low MICs) to
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204 all antifungal agents that would be appropriate for treating vaginal candidiasis (Table m

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205 2). Examination of clinical details submitted with the isolates did not reveal a t
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206 significant or unusual proportion of recalcitrant or recurrent infections associated with jc
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207 C. africana, as compared to those associated with isolates of C. albicans sensu stricto, a
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208 although generally the majority of yeast isolates from genital sites received by the MRL .o
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209 are from recurrent or recalcitrant infections. In agreement with previous reports (31, 41, /
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210 42), all 15 C. africana isolates were capable of producing germ tubes upon incubation D
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211 in horse serum for 3 h at 37oC (Table 3). Similarly, C. africana isolates formed true e
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212 hyphae in Dalmau cultures, although the quantity of hyphae produced and the speed of e
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213 hyphal formation were much reduced when compared to C. albicans and C. 8
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214 dubliniensis isolates (Table 3; data not shown). All isolates of C. africana failed to 0
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215 produce chlamydospores in culture, even after prolonged incubation, were incapable of b
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216 assimilating trehalose and lacked N-acetyl-galactosaminidase (hexosaminidase) activity u
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217 (31, 41, 42; Table 3). Thus C. africana could be distinguished from C. albicans and C. t
218 dubliniensis by AUXACOLOR2, although the former yielded unique profiles that are
219 not currently in the databases provided with the kit. C. africana could also be
220 distinguished from C. albicans on the basis of its appearance on CHROMagar, with the
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221 former producing smaller and deeper turquoise-green colonies than C. albicans (Figure
222 1). However, in contrast to previously published descriptions (31, 43), all 15 UK
223 isolates of C. africana were capable of growth at 30 oC, 37 oC and 42oC, although
224 growth was restricted at the latter temperature (Table 3). Importantly, CURRENT
225 MALDI-TOF approaches are apparently unable to robustly distinguish C. africana and D
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226 C. albicans (see for example Bruker Daltonik TechNote 1.0, Bruker Daltonik GmbH,
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227 Bremen, Germany). a
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228 Given the reduced formation of hyphae and slower growth of C. africana as
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229 compared to C. albicans, and previous reports demonstrating that C. dubliniensis m

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230 exhibits reduced pathogenicity as compared to C. albicans in a variety of infection t
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231 models (reviewed in 21, 39), we compared the virulence of isolates of C. albicans, C. jc
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232 africana and C. dubliniensis in a systemic infection model using the insect larval a
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233 model, Galleria mellonella (9, 10, 13, 14). Although recent studies have failed to reveal .o
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234 differences in pathogenicity between mucosal as opposed to systemic isolates of key /
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235 pathogenic yeast species in Galleria larvae (16), any such potential bias in the current D
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236 study was avoided by comparing C. africana strains with C. albicans and C. e
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237 dubliniensis isolates that had also been recovered from female genital samples. In e
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238 preliminary experiments, and in agreement with previous reports (9), no significant 8
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239 larval killing was observed with any isolate when larvae were inoculated with 105 0
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240 cfu/larva at any temperature (data not shown). However, when 106 cfu per larva were b
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241 employed, species-specific and temperature-dependent larval killing was observed u
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242 (Figure 2). Candida albicans was the most virulent species studied, and larval killing t
243 increased in a temperature-dependent manner. C. dubliniensis isolates exhibited
244 virulence (in terms of mortality rates and time-to-kill) that was indistinguishable from
245 C. albicans in larvae maintained at 37oC, but were significantly less virulent than C.
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