# Molecular Characterization ofCandida africana in Genital Specimens in Shanghai, China

Authors: Yang Hu; Aihua Yu; Xiangming Chen; Guojiang Wang; Xiaobo Feng
Journal: BioMed Research International
 (2015)

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## Abstract

Candida africana, an emerging yeast pathogen, is closely related to Candida albicans and most commonly involved in vulvovaginal candidiasis (VVC). However, its prevalence in candidal balanoposthitis is still unclear. In this study, the prevalence of C. africana in both candidal balanoposthitis and VVC in a sexually transmitted diseases (STD) clinic in Shanghai, China, was analyzed, and the molecular characterization and susceptible profiles of C. africana isolates were investigated. As results, C. africana was only isolated in 5 out of 79 (6.3%) cases of candidal balanoposthitis rather than cases with vulvovaginal candidiasis. Among them, 4 out of 5 isolates share the same genotype of DST 782 with an isolate from vaginal swab in Japan published previously. All C. africana isolates were susceptible to amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, posaconazole, caspofungin, and micafungin.


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## Body

## 1. Introduction

EmergingCandida species have been detected in cases of candidiasis by using molecular identification, and these species actually belong to diverse species complexes [1–15].Candida africana is an emerging pathogen that was proposed as a new species or variety within theCandida albicans complex since 2001 [16–20]. Due to phenotypic resemblance and unavailability of commercial tools, this pathogen was readily misidentified asC. albicans in clinical laboratory. AlthoughC. africana is also germ tube-positive, it exhibits differences in producing chlamydospores, morphology on CHROMagarCandida medium, and assimilating results withC. albicans [16, 21]. However, precise identification usually relies on molecular techniques such as HWP1 gene amplification or pyrosequencing of a short fragment of the internal transcribed spacer region 2 (ITS2) [1, 22, 23]. Previous studies showed thatC. africana differed fromC. albicans in clinical distribution, and the former was most commonly involved in vulvovaginal candidiasis (VVC), implicating its tropism for vagina [23, 24]. When only theC. albicans complex isolates from vaginal samples were considered,C. africana was much more prevalent thanC. dubliniensis [23]. Furthermore, differences in pathogenicity, adherence ability, and biofilm formation were observed betweenC. africana andC. albicans in previous studies [21, 23, 24], implicating the necessity to differentiate them in clinical laboratory.Vulvovaginal candidosis is one of the most common infection types and accounts for 40%–50% cases of infectious vulvovaginitis. Candidal balanoposthitis, the most frequent genital infection occurring in man, was revealed to be associated with sexual transmission [25, 26]. In two recent studies, similar genotype distributions betweenC. albicans strains causing balanoposthitis and those causing VVC were confirmed, which imply the sexual transmission of genitalC. albicans infection [25]. Although global distribution ofC. africana in VVC was found, no information on the prevalence of this emerging pathogen in candidal balanoposthitis was available. This study will reveal the distribution, antifungal profiles, and genetic characterization ofC. africana isolates from both candidal balanoposthitis and VVC patients in Shanghai, China.


## 2. Materials and Methods

### 2.1. Strains and Conventional Identification

All genital samples were collected from patients presenting with VVC or candidal balanoposthitis in sexually transmitted diseases (STD) clinic of our hospital between December 2013 and December 2014. A total of 166 independentCandida isolates (79 from cases of balanoposthitis and 87 from cases of vulvovaginitis) were recovered. All isolates were phenotypically characterized using conventional methods. Germ tube test was performed by inoculating 0.5 mL of fetal calf serum with a loopful of yeast. Germ tubes were produced byC. albicans complex within 3 h at 37°C. Chlamydospores production was assessed by culturing yeast on cornmeal agar at 30°C for 5 days. Appearance of the colonies on CHROMagarCandida medium and assimilation profiles with the API 20C AUX system (bioMérieux, Marcy l’Etoile, France) were also recorded according to the manufacturer’s instructions.


### 2.2. Molecular Identification by Use of HWP1 Gene Amplification

Isolates showing green color colony on the CHROMagarCandida medium and germ tube-positive in serum were chosen for molecular analysis. Genomic DNA was extracted from each isolate with the MasterPure Yeast DNA Purification kit (Epicentre Biotechnologies, Madison, WI) according to the manufacturer’s instructions. HWP1 gene amplification was performed to distinguishC. albicans,C. dubliniensis, andC. africana on the basis of the distinct size of the amplicons as previously described [22]. Reference strains used as control in this study wereC. albicans ATCC10231,C. dubliniensis CD36, andC. africana Caf1.


### 2.3. ITS Sequencing and Multilocus Sequence Typing

AllC. africana isolates identified by use of HWP1 gene amplification were subjected to ITS sequencing using primers ITS1 and ITS4 as previously depicted [3]. TheC. africana isolates were further identified based on the ITS2 sequence as described previously [23]. The MLST scheme using seven housekeeping genes, namely, AAT1a, ACC1, ADP1, MPIb, SYA1, VPS13, and ZWF1b, was conducted for theC. africana isolates obtained herein as previously described [18, 27]. Sequencing was performed on ABI 3730 automatic sequencer (Applied Biosystems, Foster City, CA). The seven loci were sequenced in both directions, and sequence data were checked manually for positions of homozygotic or heterozygotic polymorphisms. Single nucleotide polymorphisms that occurred in sequence were included in analysis. Allele numbers for each locus were assigned on the basis of comparison of sequences obtained here to those deposited at the public MLST database (http://pubmlst.org/calbicans/). The composite profile of the seven allele numbers for an isolate determined the isolate’s diploid sequence type (DST). A unique DST will be assigned if our isolates differ from isolates published in one or more nucleotides at any of the seven loci.


### 2.4.In Vitro Antifungal Susceptibility Testing

AllC. africana isolates obtained herein were tested forin vitro susceptibility to amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, and posaconazole (Sigma, St. Louis, MO, USA), caspofungin (Merck & Co., Inc.), and micafungin (Astellas Pharma) by using broth microdilution method as described in CLSI document M27-A3 and M27-S4. The MIC values were read following 24 h of incubation and determined visually as the lowest concentration in which prominent decrease in turbidity was observed excepting for amphotericin B which is defined as the lowest concentration in which the absence of turbidity is observed. The recently revised CLSI clinical breakpoints (CBPs) values forC. albicans were used as reference [28]. Quality control was performed as recommended in CLSI documents usingC. parapsilosis ATCC22019 andC. krusei ATCC 6258.


## 2.1. Strains and Conventional Identification

All genital samples were collected from patients presenting with VVC or candidal balanoposthitis in sexually transmitted diseases (STD) clinic of our hospital between December 2013 and December 2014. A total of 166 independentCandida isolates (79 from cases of balanoposthitis and 87 from cases of vulvovaginitis) were recovered. All isolates were phenotypically characterized using conventional methods. Germ tube test was performed by inoculating 0.5 mL of fetal calf serum with a loopful of yeast. Germ tubes were produced byC. albicans complex within 3 h at 37°C. Chlamydospores production was assessed by culturing yeast on cornmeal agar at 30°C for 5 days. Appearance of the colonies on CHROMagarCandida medium and assimilation profiles with the API 20C AUX system (bioMérieux, Marcy l’Etoile, France) were also recorded according to the manufacturer’s instructions.


## 2.2. Molecular Identification by Use of HWP1 Gene Amplification

Isolates showing green color colony on the CHROMagarCandida medium and germ tube-positive in serum were chosen for molecular analysis. Genomic DNA was extracted from each isolate with the MasterPure Yeast DNA Purification kit (Epicentre Biotechnologies, Madison, WI) according to the manufacturer’s instructions. HWP1 gene amplification was performed to distinguishC. albicans,C. dubliniensis, andC. africana on the basis of the distinct size of the amplicons as previously described [22]. Reference strains used as control in this study wereC. albicans ATCC10231,C. dubliniensis CD36, andC. africana Caf1.


## 2.3. ITS Sequencing and Multilocus Sequence Typing

AllC. africana isolates identified by use of HWP1 gene amplification were subjected to ITS sequencing using primers ITS1 and ITS4 as previously depicted [3]. TheC. africana isolates were further identified based on the ITS2 sequence as described previously [23]. The MLST scheme using seven housekeeping genes, namely, AAT1a, ACC1, ADP1, MPIb, SYA1, VPS13, and ZWF1b, was conducted for theC. africana isolates obtained herein as previously described [18, 27]. Sequencing was performed on ABI 3730 automatic sequencer (Applied Biosystems, Foster City, CA). The seven loci were sequenced in both directions, and sequence data were checked manually for positions of homozygotic or heterozygotic polymorphisms. Single nucleotide polymorphisms that occurred in sequence were included in analysis. Allele numbers for each locus were assigned on the basis of comparison of sequences obtained here to those deposited at the public MLST database (http://pubmlst.org/calbicans/). The composite profile of the seven allele numbers for an isolate determined the isolate’s diploid sequence type (DST). A unique DST will be assigned if our isolates differ from isolates published in one or more nucleotides at any of the seven loci.


## 2.4.In Vitro Antifungal Susceptibility Testing

AllC. africana isolates obtained herein were tested forin vitro susceptibility to amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, and posaconazole (Sigma, St. Louis, MO, USA), caspofungin (Merck & Co., Inc.), and micafungin (Astellas Pharma) by using broth microdilution method as described in CLSI document M27-A3 and M27-S4. The MIC values were read following 24 h of incubation and determined visually as the lowest concentration in which prominent decrease in turbidity was observed excepting for amphotericin B which is defined as the lowest concentration in which the absence of turbidity is observed. The recently revised CLSI clinical breakpoints (CBPs) values forC. albicans were used as reference [28]. Quality control was performed as recommended in CLSI documents usingC. parapsilosis ATCC22019 andC. krusei ATCC 6258.


## 3. Results

Among the 166Candida isolates, 129 (77.7%) isolates were germ tube-positive and showed green color on CHROMagarCandida medium. However, 5 out of 129 (3.9%) isolates produced smaller and deeper turquoise-green colonies compared with the other isolates. These five isolates failed to produce chlamydospores on rice tween 80’s agar after 5 days of incubation at 28°C. The five unique strains were all isolated from glans penis or foreskin swab samples, and none was isolated from vaginal samples.Among the 129 isolates presumptively identified asC. albicans, 124 (96.1%) were found to beC. albicans isolates and 5 (3.9%) were found to beC. africana isolates on the basis of HWP1 gene amplification. NoC. dubliniensis isolates were found in this study. All fiveC. africana isolates yielded an amplicon of size ~750 bp identical to that of theC. africana reference strain Caf1 (Figure 1). All fiveC. africana isolates shared the identical ITS sequence which differ fromC. albicans in one position located within the 35 bp ITS2 signature sequence. The result of the molecular identification was consistent with the phenotypic analysis.Figure 1
Agarose gel electrophoresis of PCR products. Species or variety is depicted in brackets. Lanes: M, DL2000 ladder. 1, ATCC10231 (C. albicans). 2, Caf1 (C. africana). 3, CD36 (C. dubliniensis). 4, XH455 (C. africana). 5, XH824 (C. africana). 6, XH520 (C. africana). 7, XH644 (C. africana). 8, XH874 (C. africana). 9, negative control.DNA sequencing of the fragments from the coding region of each of the seven genes resulted in a total of 2,883 nucleotides for eachC. africana isolate. Among them, 4C. africana isolates shared the genotype DST 782 ofC. africana strain JIMS500002 assigned by MLST database at the seven loci, AAT1a, ACC1, ADP1, MPIb, SYA1, VPS13, and ZWF1b. The remaining 1 isolate shared a common genotype DST 182. All the allele numbers of seven loci and their corresponding DSTs for 5 ShanghaiC. africana isolates and referenceC. africana strains were summarized in Table 1.Table 1
Multilocus sequence typing profile of the five ShanghaiC. africana isolates together with three reference strains with distinct genotypes.
Isolate number
ST
AAT1
ACC1
ADP1
MPIb
SYA1
VPS13
ZWF1b
AM2003/0025
182
33
7
32
26
2
61
48
JIMS500002
782
33
7
32
43
2
61
48
VPCI84/P/13
2191
33
7
32
135
2
61
48
XH455∗
782
33
7
32
43
2
61
48
XH824∗
782
33
7
32
43
2
61
48
XH520∗
182
33
7
32
26
2
61
48
XH644∗
782
33
7
32
43
2
61
48
XH874∗
782
33
7
32
43
2
61
48
∗Isolates recovered in this study.All 5C. africana isolates were susceptible to fluconazole (MIC range, 0.12–0.25 μg/mL), itraconazole (MIC range, 0.03–0.12 μg/mL), voriconazole (MIC range, <0.016–0.016 μg/mL), caspofungin (MIC range, 0.06–0.25 μg/mL), and micafungin (MIC range, <0.03–0.06 μg/mL). These isolates also exhibited low MICs to amphotericin B (MIC range, 0.03–0.12 μg/mL), flucytosine (MIC range, <0.12–0.12 μg/mL), and posaconazole (0.016–0.03) (see Table 2).Table 2
In vitro susceptibility of the five Shanghai C. africana isolates.
Isolate number
Patient
Age
Minimum inhibitory concentration (µg/mL)
AMB
5-FC
FLC
ITC
VOR
POS
CAS
MIC
XH455
M
27
0.12
<0.12
0.12
0.12
0.016
0.016
0.06
0.03
XH824
M
42
0.06
<0.12
0.12
0.06
<0.016
0.016
0.06
<0.03
XH520
M
41
0.12
0.12
0.25
0.03
0.016
0.03
0.12
0.06
XH644
M
24
0.03
0.12
0.12
0.12
0.016
0.016
0.25
<0.03
XH874
M
24
0.12
<0.12
0.12
0.03
<0.016
0.016
0.06
<0.03
AMB, amphotericin B; 5-FC, flucytosine; FLC, fluconazole; ITC, itraconazole; VOR, voriconazole; POS, posaconazole; CAS, caspofungin; MIC, micafungin.


## 4. Discussion

Simple PCR assay was applied for the identification of cryptic species withinCandida spp. [2, 3, 9, 22]. Here, the result of the HWP1 gene amplification was consistent with the sequencing of the internal transcribed spacer region 2 (ITS2) for identification ofC. africana. Additionally,C. africana could also be distinguished fromC. albicans on the basis of its appearance on CHROMagar, with the former producing smaller and deeper turquoise-green colonies thanC. albicans, which may be used as a preliminary screening method. NoC. africana isolated herein produce chlamydospores, which is consistent with previous reports [16, 21, 27].AlthoughC. africana has been isolated from vaginal specimens worldwide [17], noC. africana was identified in cases with VVC in our study, which was similar to another two studies in whichC. africana was not identified among 195 and 98 vaginalC. albicans complex isolates from Turkey and Malaysia, respectively [29, 30]. We think that this situation may result from regional differences or limited clinical samples. The present study revealed thatC. africana consists of 6.3% of isolates from cases of candidal balanoposthitis. The mean age of the five male patients with candidal balanoposthitis due toC. africana was 31.6 years (range 24–42 years). To our knowledge, this is the first time to study the prevalence of this emerging pathogen in candidal balanoposthitis.We conducted a well-established MLST scheme to determine the genetic relatedness ofC. africana isolates based on seven housekeeping genes. However, noC. africana was isolated from vaginal samples which hampered analyzing genotype distributions betweenC. africana strains causing balanoposthitis and those causing VVC. The seven gene fragments used for MLST ofC. africana yielded two distinct DSTs among the fiveC. africana strains. Among them, 4 out of 5 isolates share the same genotype of DST 782 with an isolate from vaginal swab in Japan published previously, implicating the possibility of sexual transmission of genitalC. africana infection. In another report, a white German patient with balanitis and his African girlfriend with vaginal mycoses caused byC. africana were seen at the institute of mycology in Berlin [31]. Thus, genotyping ofC. africana isolates from patient and sexual partner may indicate the sexual transmission of genitalC. africana infection. The genotype of DST 782 was not found in other countries up to now, and additionally special genotype was identified in a study in India [27]. This means that the distribution ofC. africana genotype may be based partially on geographical variation. MoreC. africana isolates should be included in the MLST analysis in the future to confirm this hypothesis, because only 24C. africana isolates have been typed by the MLST scheme till now. Because only three genotypes (DSTs) were available forC. africana at MLST website, more genetic markers and isolates from worldwide should be included in the molecular typing scheme to elucidate the global epidemiology of this emerging pathogen.In agreement with other reports [23], allC. africana isolates tested were found to be susceptible (with very low MICs) to all antifungal agents, which would be appropriate for treating candidal balanoposthitis due to this emerging pathogen.



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