mycoses
Diagnosis,Therapy and Prophylaxis of Fungal Diseases
Original article
Multilocus sequence typing of Candida africana from patients with vulvovaginal candidiasis in New Delhi, India Cheshta Sharma,1 Sumathi Muralidhar,2 Jianping Xu,3 Jacques F. Meis4,5 and Anuradha Chowdhary1 1 Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India, 2Regional STD Teaching, Training & Research Center, Vardhman Mahavir Medical College & Safdarjang Hospital, New Delhi, India, 3Department of Biology, McMaster University, Hamilton, Ontario, Canada, 4 Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, The Netherlands and 5Department of Medical Microbiology and Infectious Diseases, Canisius-Wilhelmina Hospital, Nijmegen, The Netherlands
Summary
We investigated the prevalence of vulvovaginal candidiasis due to C. africana in an STD clinic in India and analysed the genetic relatedness of these C. africana isolates with those outside India. A total of 283 germ-tube-positive yeasts were identified by VITEK2. Molecular characterisation of all isolates was carried out by hwp1gene-specific PCR. Of 283 germ-tube-positive yeast isolates, four were identified as C. africana using hwp1-gene-specific PCR. All hwp1 PCR positive C. africana were subjected to antifungal susceptibility testing, ITS and D1/D2 region sequencing and were typed by using MLST approach. Similar to C. africana isolates from the United Kingdom and unlike those from Africa, the Indian C. africana grew at 42°C. Sequencing of eight gene fragments in MLST identified all four strains to have different genotypes not reported previously. Furthermore, though the Indian C. africana isolates were susceptible to most of the 14 tested antifungal drugs, differences in susceptibility were observed among the four strains. Our results indicate genetic and phenotypic heterogeneity among C. africana from different geographical regions. Due to lack of data on epidemiology and genetic variability of this underreported yeast, more studies using molecular methods are warranted.
Key words: Candida africana, multilocus sequence typing, vulvovaginal candidiasis, antifungal susceptibility test-
ing, India.
Introduction Candida africana is an opportunistic yeast pathogen that was proposed as a new species within the Candida albicans species complex in 2001.1 Although C. africana is also germ-tube positive, it could be differentiated from C. albicans by its inability to produce Correspondence: Anuradha Chowdhary, Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110 007, India. Tel: +91-11-27667560. Fax: + 91-11-27666549. E-mail: [email protected] Submitted for publication 24 February 2014 Accepted for publication 4 March 2014
© 2014 Blackwell Verlag GmbH
chlamydospores and failure to assimilate trehalose, N-acetylglucosamine, glucosamine and DL-lactate.1,2 However, based on commonly used methods in most clinical microbiology laboratories, this species is likely to be misidentified as the typical C. albicans because of their close phenotypic resemblance.1–4 At present, molecular diagnostic methods such as PCR of the hwp1 gene or sequencing the internal transcribed spacer region 2 can be used for definitive identification of C. africana.5,6 It has been reported from 12 countries (Angola, Madagascar, Germany, Saudi Arabia, Spain, Italy, Nigeria, Senegal, United Kingdom, United States, Japan and Chile) predominantly as an agent of vulvovaginal candidiasis (VVC)1,3,4,7–14 (Table 1). However, C. africana has also been isolated from a
doi:10.1111/myc.12193
C. Sharma et al.
Table 1 Global distribution of reported Candida africana clinical isolates. Country
Source
No. of isolates
Year reported
Reference no.
Madagascar Angola Germany Saudi Arabia Poland Chile Spain Italy Nigeria Senegal United Kingdom United States United States India
Vaginal swab
21 9 4 131 1 1 1 27 2 3 15 6 1 4
1995–2001
[1]
2002 2004 2007 2008 2009 2012 2012 2013 2013 2013 2013
[7] [30]2 [8] [9] [4] [11] [12] [6] [13] [14] This study
Urine, high vaginal swab, Foley’s catheter tip Vaginal swab Blood Vaginal swab Vaginal swab Vaginal swab Vaginal swab Vaginal swab Vaginal swab Blood culture with renal involvement Vaginal swab
1
Molecular identification of the isolates not done.
2
The case is reported from Germany, however, the patient was from Poland.
blood culture in Chile and more recently implicated in invasive infection with renal involvement in a preterm newborn from the United States.8,14 In spite of the global distribution of C. africana and its association with a wide clinical spectrum, actual epidemiological data about its prevalence are still lacking because of inefficiency of the commonly employed commercial methods for its identification. Indeed, molecular markers are required for accurate identification. Sequence analyses at two of the commonly used fungal phylogenetic markers, the internal transcribed spacers (ITS) and the large subunit of the ribosomal RNA gene (the D1/D2 domains) for establishing fungal species relationships, have revealed very low interspecies divergence of about 0.5% between C. albicans and C. africana, similar to that among most of the clades within the C. albicans species complex.2,15–17 Thus, there has been debate about whether C. africana is a distinct species or a variety of C. albicans. Molecular phylogenetics of a global collection of C. albicans isolates characterised by multilocus sequence typing (MLST) revealed C. africana isolates as belonging to a highly distinct C. albicans clade with a global distribution.8 However, compared to what we know about the typical C. albicans, relatively little is known about the biology of this related species, despite more than a decade has passed since its first description. The purpose of this study was to explore the prevalence of C. africana in patients with vaginal candidiasis attending a STD centre in Delhi, India. We also assessed the usefulness of the consensus C. albicans MLST markers as well as the hwp1 gene for investigating the genetic relatedness of C. africana isolates among each other and with isolates from outside
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India. In addition, the isolates were subjected to several phenotypic analyses to gain a better understanding of the phenotypic variation among the strains of C. africana.
Materials and methods Fungal isolates and their phenotypic characterisation
The study analysed 283 germ-tube-positive yeast isolates during December 2011 to January 2013. Of 283 yeast isolates, 128 were from patients with diagnosed vaginal candidiasis at a regional sexually transmitted disease (STD) treatment centre within a tertiary care hospital in New Delhi. This STD centre provides services to the patients of Delhi and the adjoining states Uttar Pradesh and Haryana. The remaining 155 isolates (including 96 oropharyngeal swabs, 26 sputum, 11 blood, eight urine, five endotracheal aspirate, four each from stool and bronchoalveolar lavage and one cerebrospinal fluid) were from the culture collection of the Department of Medical Mycology, V. P. Chest Institute, Delhi. All isolates were subcultured on Sabouraud dextrose agar plates and incubated at 37°C before they were subjected to phenotypic and genotypic analyses. Preliminary species identification was based on green colony colour of the isolates on CHROMagar Candida medium (Becton Dickinson & Company, Baltimore, USA), positive germ-tube test and morphology on rice tween 80 agar. Growth at 37°C and 42°C was also tested. The carbohydrate assimilation profiles were determined by VITEK2 compact (bioMerieux, Marcy I’Etoile, France). Reference strains included Candida albicans (ATCC 90028), C. dubliniensis
© 2014 Blackwell Verlag GmbH
MLST of Candida africana
(CD 36), C. stellatoidea (CBS 1905) and C. africana (CBS 8781). PCR amplification targeting the hwp1 gene and sequence analyses of the ITS and D1/D2 gene regions
Following a previously established protocol,5 all 283 yeast isolates were subjected to PCR amplification targeting the hwp1 gene. The hwp1 gene encodes hyphal wall protein 1 and has shown to be able to distinguish C. africana and C. stellatoidea from typical C. albicans.5 The isolates identified as C. africana by PCR were subjected to sequencing of the ITS and D1/D2 regions using previously described primers.17,18 Amplified DNA was sequenced in both strands on an ABI 3130XL Genetic Analyser (Applied Biosystems, Foster City, CA, USA) using the BigDye Terminator Kit v3.1 (Applied Biosystems). Sequences were aligned using the Sequencing Analysis 5.3.1 software (Applied Biosystems). GenBank basic local alignment search tool (BLAST) searches (http://www.ncbi.nlm. nih.gov/BLAST/Blast.cgi) were performed for species identification. Multilocus sequence typing and analysis
Due to the high level of sequence homology between the majority of C. albicans and C. africana open reading frames, all seven loci previously examined for C. albicans MLST were investigated for their potential use in typing C. africana. In addition, hwp1-gene-specific primers were included for C. africana typing. The C. albicans MLST scheme used was based on a previously published consensus set of seven housekeeping genes: CaAAT1a, CaACC1, CaADP1, CaMPIb, CaSYA1, CaVPS13 and CaZWF1b. These seven genes have shown to provide high discriminatory power for strain discrimination and for both population structure analyses and epidemiological studies of C. albicans.19–21 PCR primers used were as described previously.20,21 Fragment amplification by PCR was carried out in a 50 ll reaction volume containing 100 ng of genomic DNA, 10 mM each of forward and reverse primer, 2.5 U of Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA), 5 ll of 10 9 buffer (Invitrogen) and 200 mM deoxynucleoside triphosphate mix (New England Biolabs, Ipswich, MA, USA). The PCR conditions used for all the primer sets were as follows: denaturation for 2 min at 94°C, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 52°C for 1 min, elongation at 72°C for 1 min and a final extension step of 10 min at 72°C in a C1000 Thermal Cycler (Bio-Rad,
© 2014 Blackwell Verlag GmbH
Hercules, CA, USA). DNA sequencing was performed using the same primers as for PCR at 2.5 mM concentration. All sequencing reactions were carried out in a 10 ll reaction volume using Big Dye Terminator Kit v 3.1 (Applied Biosystems) according to the manufacturer’s recommendations and analysed on an ABI 3130XL Genetic Analyser (Applied Biosystems). For all C. africana strains, the eight target loci including hwp1 gene were sequenced in both directions. Sequence data were inspected manually for positions of homozygotic or heterozygotic polymorphisms. Single nucleotide polymorphisms present in both the forward and reverse sequence were included in further analyses. The one-letter IUPAC nucleotide code was used in sequence analyses. Allele numbers for each locus were assigned based on comparison of sequence data from our isolates to sequences deposited into the public MLST database (http://calbicans.mlst.net).20,21 In the case where an exact match was not present in the database, the allele number in the database with 98–99% identity to the query sequence was given followed by ‘00’. In the cases where two unique sequences from this study showed 99% identity to the same allele number in the database, the distinction was made via sequential numbering in the final twodigit suffix (i.e. 00, 01, 02). However, the two unique sequences showing 100% identity with each other were assigned the same two-digit suffix. The composite profile of all seven allele numbers for an individual isolate defined the isolate’s diploid sequence type (DST). Isolates differing by one or more nucleotides (heterozygous or homozygous) at any of the seven loci were represented by a unique DST. DSTs of two C. africana isolates were assigned by authors of this study and do not correspond to DSTs in the Candida MLST database. Phylogenetic inferences were made from distance tree constructed by using the Neighbour-joining (NJ) algorithm implemented in the PAUP v4 software.22 In vitro antifungal susceptibility testing (AFST)
AFST of C. africana isolates was carried out using the Clinical Laboratory Standards Institute (CLSI) broth microdilution method, following the M27-A3 guidelines.23 The antifungals tested were amphotericin B (Sigma, St. Louis, MO, USA), fluconazole (Pfizer, Groton, CT, USA), itraconazole (Lee Pharma, Hyderabad, India), voriconazole (Pfizer), posaconazole (Merck, Whitehouse Station, NJ, USA), isavuconazole (BasileaPharmaceutica, Basel, Switzerland), flucytosine (Sigma), caspofungin (Merck, Whitehouse Station, NJ, USA),
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micafungin (Astellas, Toyama, Japan), anidulafungin (Pfizer), clotrimazole (Sigma), ketoconazole (Sigma), miconazole (Sigma) and terbinafine (Synergene, Hyderabad, India). Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019 were used as two quality control strains with each test. Patient information
The diagnosis of VVC was based on the presence of signs and symptoms of inflammation, microscopic detection of yeast cells and/or pseudohyphae and isolation of Candida species in culture, which has been described previously.24 The records of patients were reviewed and information regarding patients demographics, medical history, risk factors for candidiasis such as diabetes, intrauterine contraceptive device (IUCD) usage, recent antibiotic therapy, vaginal oestrogen replacement, immunocompromised status, corticosteriod use and treatment outcomes were recorded. The treatment regimen included oral fluconazole 150 mg OD once a week for three consecutive weeks followed by periodic review of the patients.
Results Of the 283 germ-tube-positive yeast isolates, four yielded an amplicon of size ~740 bp identical to that of the C. africana reference isolate, CBS 8781 by hwp1 PCR. The remaining 279 isolates gave an amplicon of size 941 bp suggesting their identification as the typical C. albicans. However, none of the isolates showed amplification products at 569 bp or 800 bp, ruling out C. dubliniensis and C. stellatoidea respectively.2,5 All four C. africana isolates showed distinct turquoise green coloured colonies, in contrast to green colour colonies of C. albicans on CHROMagar Candida medium. No chlamydospores were formed on rice tween 80 agar after 5 days of incubation at 28°C. Furthermore, the biochemical profiles generated by VITEK2 identified two of the C. africana isolates as C. sphaerica with 86% ID, whereas the other two remained unidentified. All four isolates failed to assimilate trehalose glucosamine, DL-lactate and lacked N-acetyl-galactosaminidase activity. However, unlike C. stellatoidea, the isolates assimilated sucrose. Our isolates grew at 37°C and showed restricted growth at 42°C. Of the four C. africana isolates, three have been deposited in the CBS Fungal Biodiversity Centre, Utrecht, the Netherlands, with each representing a distinct multilocus genotype based on the consensus C. albicans MLST genotyping markers. The laboratory and CBS accession numbers
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of C. africana isolates are VPCI 891/P/12 (CBS 12764), VPCI 896/P/12 (CBS 12765), VPCI 84/P/13 and VPCI 301/P/13 (CBS 12826). ITS and D1/D2 sequences of the four C. africana isolates (GenBank accession nos. KF741783 to KF741786 for ITS and KF741787 to KF741790 for D1/D2) showed 99% sequence similarity (query coverage ranging from 98–100%) with the only available C. africana type isolate in GenBank (accession no. AY342214) and with C. albicans isolates (accession nos. JN606311, GQ280317 and AF455524 for ITS; AB828128, GU319992 and HQ876051 for D1/D2). Comparisons of the D1/D2 regions between the four Indian C. africana isolates and 10 C. albicans isolates (five each from the United States and Japan) revealed 99.09–100% nucleotide sequence similarity. Similarly, comparison of our ITS sequences revealed >99% identity with three C. albicans isolates submitted to the GenBank (accession no.s AJ551313, AB032174 and AB032173). DNA sequencing of the 373–491 bp fragments from the coding region of each of the seven genes resulted in a total of 2,883 aligned nucleotides for each isolate. All four C. africana isolates shared the common C. albicans allele types 33, 7, 32, 48 and 61 assigned by MLST database at the following five loci, CaAAT1a, CaACC1, CaADP1, CaVPS13 and CaZWF1b, respectively. However, only 98–99% sequence similarities were observed for two loci, CaSYA1 and CaMPIb. Candida africana reference strain (CBS 8781) also shared common C. albicans allele types assigned by MLST database at CaAAT1a, CaACC1, CaADP1, CaVPS13 and CaSYA1, but the sequence similarity for CaMPIb and CaZWF1b was 98–99%. Although the CBS 8781 previously included in MLST analysis by Odds et al. show it to be diploid sequence type (DST) 182, in our hand the strain revealed differences at two allele types. Compared to the C. albicans MLST scheme, three of the four C. africana isolates revealed a 99% sequence identity with allele 43 at the CaMPI1b gene and they were assigned new allele numbers 134 (for strain VPCI 896/P/12) and 135 (for strains VPCI 84/P/13 and VPCI 301/P/13) by the C. albicans MLST database. However, one C. africana isolate showed a 99% sequence identity with a different allele, allele 26 at the CaMPIb gene and this isolate was also assigned new allele number 133 (VPCI 891/P/12) by the database. The sequences of CaAAT1a, CaACC1, CaADP1, CaMPIb, CaSYA1, CaVPS13 and CaZWF1b genes, used for MLST, of the 4 C. africana isolates have been submitted to the C. albicans MLST database. All the allele numbers of seven loci and their corresponding DSTs
© 2014 Blackwell Verlag GmbH
MLST of Candida africana
Table 2 Multilocus sequence typing profile of the four Indian C. africana isolates generated using seven loci of the C. albicans MLST
scheme MPIb
SYA1
Isolate no.
AAT1 Allele no.
ACC1 Allele no.
ADP1 Allele no.
Allele no. (% identity)
New allele assigned
Allele no. (% identity)
New allele assigned
ZWF1b Allele no.
VPSP13 Allele no.
Assigned DST
VPCI VPCI VPCI VPCI
33 33 33 33
7 7 7 7
32 32 32 32
26 43 43 43
133 134 1351 1351
2 (99%) 2 (99%) 2 2
2-00 2-01 -
48 48 48 48
61 61 61 61
DST12 DST22 DST 21913,4 DST 21913,4
891/P/12 896/P/12 84/P/13 301/P/13
(98%) (99%) (99%) (99%)
1
Isolates at MPIb locus were 100% identical. DSTs assigned by the authors.
2 3
DSTs assigned by C. albicans MLST database.
4
DST 2191 isolates were distinguishable based on hwp1 gene analysis.
for four Indian C. africana isolates are summarised in Table 2. The second locus showing distinct C. africana polymorphisms was CaSYA1. At this locus, two strains (VPCI 84/P/13 and VPCI 301/P/13) were identical to allele type 2, whereas the other two isolates (VPCI 891/P/12 and VPCI 896/P/12) showed a close relationship with allele 2, but revealed a 1% sequence difference with allele number 2 and they were assigned alleles 2-00 and 2-01 respectively. Combining the allele types at all seven loci resulted in three unique DSTs (Table 2) among the four Indian C. africana isolates viz. DST 1 (VPCI 891/P/12), DST 2 (VPCI 896/P/12) and DST 2191 (VPCI 84/P/13 and VPCI 301/P/13). Consistent with previous reports, all four Indian C. africana strains clustered into Clade 13 of the C. albicans species complex. In addition, the strains in this clade comprised entirely of C. africana isolates and including all known C. africana sequences deposited so far (from Chile, Angola, Germany, Japan, Madagascar and United Kingdom). A Neighbour-Joining (NJ) phylogenetic tree based on concatenated sequences of the seven housekeeping genes was generated that included the four Indian C. africana isolates as well as 14 C. africana isolates available in the MLST database representing Madagascar (n = 5), Angola (n = 3), United Kingdom (n = 2), Germany (n = 2), Chile (n = 1) and Japan (n = 1). Additional 14 sequences comprising 12 C. albicans isolates belonging to Clades 1–12 along with a CBS reference strain of C. albicans (CBS 562) and C. stellatoidea (CBS 1905) were also retrieved for comparison (Fig. 1). All the C. africana isolates, irrespective of their geographical origins, formed a distinct cluster different from other C. albicans strains (Fig. 1). Furthermore, the NJ phylogenetic tree of the most variable marker CaMPIb
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gene revealed extensive intraspecies variation within C. africana (Fig. 2). Interestingly, strains VPCI 891/P/ 12 and VPCI 84/P/13 shared the same allele at the hwp1 locus, whereas strains VPCI 896/P/12 and VPCI 301/P/13 had a different allele. Thus, the inclusion of sequence information from the hwp1 locus separated the two strains of DST 2191 (Table 2) into different genotypes. Of the azole antifungals tested, clotrimazole, isavuconazole, ketoconazole, miconazole and posaconazole exhibited the most potent activity with MICs of 0.015 lg ml 1 followed by itraconazole and voriconazole each showing MICs of 0.03 lg ml 1. Amphotericin B also showed an excellent activity with MIC range of 0.03–0.125 lg ml 1. The isolates were also susceptible to fluconazole (MIC range, 0.5–4 lg ml 1), flucytosine (MIC range, 0.125–0.5 lg ml 1) and had higher MICs of terbinafine (MIC, 2 lg ml 1). All the echinocandins exhibited low MICs of 0.015 lg ml 1. The mean age of four female patients with VVC due to C. africana was 25 years (range 18–35 years). Their high vaginal swabs yielded confluent monofungal population of C. africana in culture. One of the patients gave history of using IUCD and the other was HIV positive, whereas in the remaining two no risk factor was apparent. The patients in this study were all treated successfully with fluconazole 150 mg once a week for 4 weeks.
Discussion The present survey revealed that C. africana constituted 3.1% of the vaginal isolates of the C. albicans species complex. In Europe, C. africana has been reported to cause 6–16% of vaginal infections/colonisation previously attributed to C. albicans, and a solitary strain of C. africana has been reported from
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NJ
C.albicans-Clade1/DST69/UK C.albicans-Clade7/DST300/UK C.albicans-Clade8/DST766/Japan C.africana-Clade13/DST182/496/Germany VPCI891/P/12, India C.africana-Clade13/DST182/1520/Madagascar C.africana-Clade13/DST182/1512/Angola C.africana-Clade13/DST182/1513/Madagascar C.africana-Clade13/DST182/211/UK C.africana-Clade13/DST182/1521/Madagascar C.africana-Clade13/DST182/1516/Angola C.africana-Clade13/DST182/1519/Angola C.africana-Clade13/DST182/1178/Chile C.africana-Clade13/DST182/1514/Germany C.africana-Clade13/DST182/206/UK C.africana-Clade13/DST782/1077/Japan VPCI 301/P/13, India VPCI 84/P/13, India VPCI 896/P/12, India C.africana-Clade13/DST182/1518/Madagascar C.africana-Clade13/DST182/1515/Madagascar C.albicans-Clade12/DST90/Malaysia
Figure 1 Phylogenetic tree, based on
C.albicans-CBS562/DST1030 C.albicans-Clade2/DST124/UK C.albicans-Clade3/DST155/US C.albicans-Clade5/DST881/Belgium C.albicans-Clade11/DST167/US C.albicans-Clade10/DST840/France C.albicans-Clade4/DST345/Belgium C.albicans-Clade6/DST735/Argentina C.albicans-Clade9/DST173/UK C.stellatoidea-CBS1905/DST1031 0.0001 substitutions/site
Spain.4,6,9 In contrast, C. africana was not identified among 195 and 98 vaginal C. albicans isolates from Turkey and Malaysia, respectively.25,26 Also, a significant geographical variation in prevalence of C. africana has been reported from the African continent. Low rates of 2 and 2.4% from Senegal and Nigeria, respectively, have been reported.11,12 On the other hand, Tietz et al. found a very high prevalence of 23% and 40% from Angola and Madagascar, respectively.3 These variations in prevalence of C. africana emphasise the importance of studying the epidemiology of this pathogen. This study employed a well-established molecular subtyping approach, MLST, to determine the genetic
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concatenated sequences of the seven housekeeping genes for comparing four Indian C. africana isolates with 14 C. africana isolates available in the MLST database, obtained by using neighbourjoining phylogenetic analysis. In addition, sequences of 12 C. albicans isolates belonging to Clade 1–12 along with CBS reference strain of C. albicans (CBS 562) and C. stellatoidea (CBS 1905) were retrieved for comparison. The figure includes the diploid sequence type, clade number, strain number and geographical location of the respective isolates.
relatedness of C. africana isolates based upon variability within the particular housekeeping genes. Previously, molecular epidemiology studies based upon MLST data have revealed that C. albicans in fact contained several distinct phylogenetic groups.8 Among these, C. africana has emerged as an important aetiological agent of vaginal infections. All Indian C. africana isolates originated from diagnosed cases of VVC whose vaginal swabs yielded confluent monofungal growth. The seven gene fragments used for MLST of C. africana yielded three distinct DSTs among the four Indian strains. The inclusion of sequence information from hwp1 gene revealed that all four strains had distinct
© 2014 Blackwell Verlag GmbH
MLST of Candida africana
NJ
Clade13/DST182/Strain206/UK Clade13/DST182/Strain211/UK Clade13/DST182/Strain496/Germany Clade13/DST182/Strain1178/Chile Clade13/DST182/Strain1512/Angola Clade13/DST182/Strain1513/Madagascar Clade13/DST782/Strain1077/Japan Clade13/DST182/Strain1514/Germany Clade13/DST182/Strain1515/Madagascar Clade13/DST182/Strain1516/Angola Clade13/DST182/Strain1518/Madagascar Clade13/DST182/Strain1519/Angola Clade13/DST182/Strain1520/Madagascar Clade13/DST182/Strain1521/Madagascar VPCI 84/P/13, India VPCI 301/P/13, India VPCI 896/P/12, India
Figure 2 The Neighbour-joining phyloge-
netic tree based on CaMPIb gene depicting intraspecies variation between Candida africana isolates.
VPCI 891/P/12, India 0.001 substitutions/site
genotypes. These four Indian strains all belonged to Clade 13 of the C. albicans species complex that included all 14 isolates of C. africana analysed previously using the seven gene fragments for C. albicans MLST.8 Also, a close relation among most of the MPIb allele of Indian C. africana isolates with that of MPIb allele type of a Japanese C. africana (JIMS500002 DST 782) isolate described previously was observed. These results confirm that C. africana is a distinct clade within the C. albicans species complex and some of these loci provide reasonable discriminating power for strains of C. africana. The genetic variation observed at the MPIb locus, as well as at the hwp1 locus, seems to be useful for differentiating C. africana isolates. Thus,
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we suggest that hwp1 be included for future sequence typing of C. africana, not only because it can discriminate C. africana from the typical C. albicans but also it allowed further separation of our strains into unique genotypes. So far about 100 isolates have been reported in the global literature and of these only 51 had been confirmed using molecular methods. Among the 51 isolates, only 14 have been typed by MLST markers, with eight isolates from Africa, four from Europe and one each from Chile and Japan and most of these were published in one study in 2007.8 This study expands the MLST database by describing four additional C. africana isolates from India. Furthermore, most C.
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africana isolates differed from each other at only one or two loci, suggesting that they diverged very recently from a common ancestor.27 Given the information found here, markers MPI1b and hwp1 would be suitable for future genotyping species and strains of C. africana. Large-scale epidemiologic investigations of C. africana using these and other loci are needed to develop a specific and discriminatory MLST scheme for C. africana. Candida africana has been shown to be less pathogenic than C. albicans in an insect model. A recent study showed a significantly slower rate of killing of Galleria mellonella larvae by C. africana isolates than that of C. albicans or C. dubliniensis.6 In larvae inoculated with C. africana, no true mycelium and pseudohyphae were detected. The lower filamentation rate may contribute to the low virulence of this yeast. Also, compared to C. albicans, C. africana showed genetic differences at both the hwp1 and the rlm1 genes.5,28 The hwp1 gene in C. albicans is reported to be an important adhesin that crosslinks C. albicans to epithelial cells. The distinct hwp1 allele found in C. africana may be responsible for the low hyphal production, reduced adhesion and decreased virulence.5,29 Similarly, the rlm1 gene is required for cell wall integrity under stressed conditions such as the presence of the antifungal caspofungin. The rlm1 gene encodes a transcription factor from the MADS (Mcm1p-Agamous-Deficiens-Serum response factor) box family whose deletion impairs growth of Candida glabrata at higher temperature (41°C). Candida africana isolates from Africa (Angola and Madagascar) were unable to grow at higher temperature (42°C), showed a distinct rlm1 allele (named allele 15), and were hypersensitive to several stress agents. It is possible that the rlm1 gene in C. africana may have a reductive role in adaptability of this yeast to specific conditions such as in human hosts.27 Unlike the African isolates, the four Indian isolates and those from the United Kingdom grew at 42°C, consistent with phenotypic variation among strains in C. africana. In addition, our isolates were genetically distinct from the African isolates as identified based on MLST. Aside from differences in genotypes and growth at 42°C, the strains also showed some difference in susceptibilities to several antifungal drugs. For example, one of the four isolates had a high MIC of fluconazole of 4 lg ml 1, whereas the other three were highly sensitive. The new species-specific clinical break points (CBPs) of Candida albicans classify MIC of 4 lg ml 1 as susceptible dose dependent. Furthermore, resistance of C. africana to voriconazole, flucytosine and terbinafine has been demonstrated.7,14 However, the global
8
patterns of resistance/susceptibility to most antifungal drugs remain largely unknown in C. africana. Finally, although C. africana has been largely associated with superficial infections of the genital tract, it has been associated with deep-seated infections, suggesting its invasive potential.14 Together, these features call greater efforts in studying the epidemiology and pathogenesis of C. africana.
Acknowledgments C.S is supported by University Grants Commission Research Fellowship, India (F.2-15/2003 SA-I). J. Xu’s research on human pathogenic fungi is supported by grants from the Natural Science and Engineering Research Council (NSERC) of Canada. J.F.M. is partly supported by Grant NPRP 5-298-3-086 from the Qatar National Research Fund.
Potential conflict of interest J.F.M received grants from Astellas, Basilea and Merck. He has been a consultant to Basilea, Merck and Pfizer and received speaker’s fees from Merck and Gilead. All other authors: no potential conflicts of interest. The authors alone are responsible for the content and writing of the article.
References 1
2 3
4
5
6
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Tietz HJ, Hopp M, Schmalreck A, Sterry W, Czaika V. Candida africana sp. nov., a new human pathogen or a variant of Candida albicans? Mycoses 2001; 44 : 437–45. Romeo O, Criseo G. Candida africana and its closest relatives. Mycoses 2011; 54: 475–86. Tietz HJ, K€ ussner A, Thanos M, De Andrade MP, Presber W, Sch€ onian G. Phenotypic and genotypic characterization of unusual vaginal isolates of Candida albicans from Africa. J Clin Microbiol 1995; 33: 2462–5. Romeo O, Criseo G. Molecular epidemiology of Candida albicans and its closely related yeasts Candida dubliniensis and Candida africana. J Clin Microbiol 2009; 47: 212–4. Romeo O, Criseo G. First molecular method for discriminating between Candida africana, Candida albicans and Candida dubliniensis by using hwp1 gene. Diagn Microbiol Infect Dis 2008; 62: 230–3. Borman AM, Szekely A, Linton CJ, Palmer MD, Brown P, Johnson EM. Epidemiology, antifungal susceptibility, and pathogenicity of Candida africana isolates from the United Kingdom. J Clin Microbiol 2013; 51: 967–72. Al-Hedaithy SSA, Fotedar R. Recovery and studies on chlamydospores-negative Candida albicans isolated from clinical specimens. Med Mycol 2002; 40: 301–6. Odds FC, Bougnoux ME, Shaw DJ et al. Molecular phylogenetics of Candida albicans. Eukaryot Cell 2007; 6: 1041–52. Alonso-Vargas R, Elorduy L, Eraso E et al. Isolation of Candida africana, probable atypical strains of Candida albicans, from a patient with vaginitis. Med Mycol 2008; 46: 167–70.
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Romeo O, Criseo G. Morphological, biochemical and molecular characterisation of the first Italian Candida africana isolate. Mycoses 2009; 52: 454–7. Nnadi NE, Ayanbimpe GM, Scordino F et al. Isolation and molecular characterization of Candida africana from Jos. Nigeria. Med Mycol 2012; 50: 765–7. Dieng Y, Sow D, Ndiaye M et al. Identification of three Candida africana strains in Senegal. J Mycol Med 2012; 22: 335–40. Hazen K (2013) Novel yeast, originally detected in Africa, now isolated from women in the United States. In: The 53rd Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC); Sep 10-13; Denver, CO, USA Romeo O, Tietz HJ, Criseo G. Candida africana: Is it a fungal pathogen? Curr Fungal Infect Rep 2013; 7: 192–7. Ciardo DE, Sch€ ar G, B€ ottger EC, Altwegg M, Bosshard PP. Internal transcribed spacer sequencing versus biochemical profiling for identification of medically important yeasts. J Clin Microbiol 2006; 44: 77– 84. Forche A, Sch€onian G, Gr€ aser Y, Vilgalys R, Mitchell TG. Genetic structure of typical and atypical populations of Candida albicans from Africa. Fungal Genet Biol 1999; 28: 107–25. Kurtzman CP, Robnett CJ. Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5’ end of the large-subunit (26S) ribosomal DNA gene. J Clin Microbiol 1997; 35: 1216–23. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, (eds), PCR protocols: a guide to methods and applications. San Diego, CA: Academic Press, 1990: 315–22. Bougnoux ME, Tavanti A, Bouchier C et al. Collaborative consensus for optimized multilocus sequence typing of Candida albicans. J Clin Microbiol 2003; 41: 5265–6. Tavanti A, Gow NA, Senesi S, Maiden MC, Odds FC. Optimization and validation of multilocus sequence typing for Candida albicans. J Clin Microbiol 2003; 41: 3765–76.
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Chowdhary A, Lee-Yang W, Lasker BA, Brandt ME, Warnock DW, Arthington-Skaggs BA. Comparison of multilocus sequence typing and Ca3 fingerprinting for molecular subtyping epidemiologicallyrelated clinical isolates of Candida albicans. Med Mycol 2006; 44: 405–17. Swofford DL (2002) PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts. Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard-third edition, M27–A3. Wayne, USA, PA: CLSI, 2008. Sharma C, Wankhede S, Muralidhar S et al. Candida nivariensis as an etiologic agent of vulvovaginal candidiasis in a tertiary care hospital of New Delhi, India. Diagn Microbiol Infect Dis 2012; 76: 46–50. Gumral R, Sancak B, Guzel AB, Saracßlı MA, Ilkit M. Lack of Candida africana and Candida dubliniensis in vaginal Candida albicans isolates in Turkey using HWP1 gene polymorphisms. Mycopathologia 2011; 172: 73–6. Yazdanpanah A, Khaithir TM. Issues in identifying germ tube positive yeasts by conventional methods. J Clin Lab Anal 2014; 28: 1–9. Odds FC, Jacobsen MD. Multilocus sequence typing of pathogenic Candida species. Eukaryot Cell 2008; 7: 1075–84. Sampaio P, Nogueira E, Loureiro AS, Delgado-Silva Y, Correia A, Pais C. Increased number of glutamine repeats in the C-terminal of Candida albicans Rlm1p enhances the resistance to stress agents. Antonie Van Leeuwenhoek 2009; 96: 395–404. Romeo O, De Leo F, Criseo G. Adherence ability of Candida africana: a comparative study with Candida albicans and Candida dubliniensis. Mycoses 2011; 54: e57–61. Mendling W, Krauss C, Fladung B. A clinical multicenter study comparing efficacy and tolerability of topical combination therapy with clotrimazole (Canesten, two formats) with oral single dose fluconazole (Diflucan) in vulvovaginal mycoses. Mycoses 2004; 47: 136–42.
9
Diagnosis,Therapy and Prophylaxis of Fungal Diseases
Original article
Multilocus sequence typing of Candida africana from patients with vulvovaginal candidiasis in New Delhi, India Cheshta Sharma,1 Sumathi Muralidhar,2 Jianping Xu,3 Jacques F. Meis4,5 and Anuradha Chowdhary1 1 Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India, 2Regional STD Teaching, Training & Research Center, Vardhman Mahavir Medical College & Safdarjang Hospital, New Delhi, India, 3Department of Biology, McMaster University, Hamilton, Ontario, Canada, 4 Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, The Netherlands and 5Department of Medical Microbiology and Infectious Diseases, Canisius-Wilhelmina Hospital, Nijmegen, The Netherlands
Summary
We investigated the prevalence of vulvovaginal candidiasis due to C. africana in an STD clinic in India and analysed the genetic relatedness of these C. africana isolates with those outside India. A total of 283 germ-tube-positive yeasts were identified by VITEK2. Molecular characterisation of all isolates was carried out by hwp1gene-specific PCR. Of 283 germ-tube-positive yeast isolates, four were identified as C. africana using hwp1-gene-specific PCR. All hwp1 PCR positive C. africana were subjected to antifungal susceptibility testing, ITS and D1/D2 region sequencing and were typed by using MLST approach. Similar to C. africana isolates from the United Kingdom and unlike those from Africa, the Indian C. africana grew at 42°C. Sequencing of eight gene fragments in MLST identified all four strains to have different genotypes not reported previously. Furthermore, though the Indian C. africana isolates were susceptible to most of the 14 tested antifungal drugs, differences in susceptibility were observed among the four strains. Our results indicate genetic and phenotypic heterogeneity among C. africana from different geographical regions. Due to lack of data on epidemiology and genetic variability of this underreported yeast, more studies using molecular methods are warranted.
Key words: Candida africana, multilocus sequence typing, vulvovaginal candidiasis, antifungal susceptibility test-
ing, India.
Introduction Candida africana is an opportunistic yeast pathogen that was proposed as a new species within the Candida albicans species complex in 2001.1 Although C. africana is also germ-tube positive, it could be differentiated from C. albicans by its inability to produce Correspondence: Anuradha Chowdhary, Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110 007, India. Tel: +91-11-27667560. Fax: + 91-11-27666549. E-mail: [email protected] Submitted for publication 24 February 2014 Accepted for publication 4 March 2014
© 2014 Blackwell Verlag GmbH
chlamydospores and failure to assimilate trehalose, N-acetylglucosamine, glucosamine and DL-lactate.1,2 However, based on commonly used methods in most clinical microbiology laboratories, this species is likely to be misidentified as the typical C. albicans because of their close phenotypic resemblance.1–4 At present, molecular diagnostic methods such as PCR of the hwp1 gene or sequencing the internal transcribed spacer region 2 can be used for definitive identification of C. africana.5,6 It has been reported from 12 countries (Angola, Madagascar, Germany, Saudi Arabia, Spain, Italy, Nigeria, Senegal, United Kingdom, United States, Japan and Chile) predominantly as an agent of vulvovaginal candidiasis (VVC)1,3,4,7–14 (Table 1). However, C. africana has also been isolated from a
doi:10.1111/myc.12193
C. Sharma et al.
Table 1 Global distribution of reported Candida africana clinical isolates. Country
Source
No. of isolates
Year reported
Reference no.
Madagascar Angola Germany Saudi Arabia Poland Chile Spain Italy Nigeria Senegal United Kingdom United States United States India
Vaginal swab
21 9 4 131 1 1 1 27 2 3 15 6 1 4
1995–2001
[1]
2002 2004 2007 2008 2009 2012 2012 2013 2013 2013 2013
[7] [30]2 [8] [9] [4] [11] [12] [6] [13] [14] This study
Urine, high vaginal swab, Foley’s catheter tip Vaginal swab Blood Vaginal swab Vaginal swab Vaginal swab Vaginal swab Vaginal swab Vaginal swab Blood culture with renal involvement Vaginal swab
1
Molecular identification of the isolates not done.
2
The case is reported from Germany, however, the patient was from Poland.
blood culture in Chile and more recently implicated in invasive infection with renal involvement in a preterm newborn from the United States.8,14 In spite of the global distribution of C. africana and its association with a wide clinical spectrum, actual epidemiological data about its prevalence are still lacking because of inefficiency of the commonly employed commercial methods for its identification. Indeed, molecular markers are required for accurate identification. Sequence analyses at two of the commonly used fungal phylogenetic markers, the internal transcribed spacers (ITS) and the large subunit of the ribosomal RNA gene (the D1/D2 domains) for establishing fungal species relationships, have revealed very low interspecies divergence of about 0.5% between C. albicans and C. africana, similar to that among most of the clades within the C. albicans species complex.2,15–17 Thus, there has been debate about whether C. africana is a distinct species or a variety of C. albicans. Molecular phylogenetics of a global collection of C. albicans isolates characterised by multilocus sequence typing (MLST) revealed C. africana isolates as belonging to a highly distinct C. albicans clade with a global distribution.8 However, compared to what we know about the typical C. albicans, relatively little is known about the biology of this related species, despite more than a decade has passed since its first description. The purpose of this study was to explore the prevalence of C. africana in patients with vaginal candidiasis attending a STD centre in Delhi, India. We also assessed the usefulness of the consensus C. albicans MLST markers as well as the hwp1 gene for investigating the genetic relatedness of C. africana isolates among each other and with isolates from outside
2
India. In addition, the isolates were subjected to several phenotypic analyses to gain a better understanding of the phenotypic variation among the strains of C. africana.
Materials and methods Fungal isolates and their phenotypic characterisation
The study analysed 283 germ-tube-positive yeast isolates during December 2011 to January 2013. Of 283 yeast isolates, 128 were from patients with diagnosed vaginal candidiasis at a regional sexually transmitted disease (STD) treatment centre within a tertiary care hospital in New Delhi. This STD centre provides services to the patients of Delhi and the adjoining states Uttar Pradesh and Haryana. The remaining 155 isolates (including 96 oropharyngeal swabs, 26 sputum, 11 blood, eight urine, five endotracheal aspirate, four each from stool and bronchoalveolar lavage and one cerebrospinal fluid) were from the culture collection of the Department of Medical Mycology, V. P. Chest Institute, Delhi. All isolates were subcultured on Sabouraud dextrose agar plates and incubated at 37°C before they were subjected to phenotypic and genotypic analyses. Preliminary species identification was based on green colony colour of the isolates on CHROMagar Candida medium (Becton Dickinson & Company, Baltimore, USA), positive germ-tube test and morphology on rice tween 80 agar. Growth at 37°C and 42°C was also tested. The carbohydrate assimilation profiles were determined by VITEK2 compact (bioMerieux, Marcy I’Etoile, France). Reference strains included Candida albicans (ATCC 90028), C. dubliniensis
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MLST of Candida africana
(CD 36), C. stellatoidea (CBS 1905) and C. africana (CBS 8781). PCR amplification targeting the hwp1 gene and sequence analyses of the ITS and D1/D2 gene regions
Following a previously established protocol,5 all 283 yeast isolates were subjected to PCR amplification targeting the hwp1 gene. The hwp1 gene encodes hyphal wall protein 1 and has shown to be able to distinguish C. africana and C. stellatoidea from typical C. albicans.5 The isolates identified as C. africana by PCR were subjected to sequencing of the ITS and D1/D2 regions using previously described primers.17,18 Amplified DNA was sequenced in both strands on an ABI 3130XL Genetic Analyser (Applied Biosystems, Foster City, CA, USA) using the BigDye Terminator Kit v3.1 (Applied Biosystems). Sequences were aligned using the Sequencing Analysis 5.3.1 software (Applied Biosystems). GenBank basic local alignment search tool (BLAST) searches (http://www.ncbi.nlm. nih.gov/BLAST/Blast.cgi) were performed for species identification. Multilocus sequence typing and analysis
Due to the high level of sequence homology between the majority of C. albicans and C. africana open reading frames, all seven loci previously examined for C. albicans MLST were investigated for their potential use in typing C. africana. In addition, hwp1-gene-specific primers were included for C. africana typing. The C. albicans MLST scheme used was based on a previously published consensus set of seven housekeeping genes: CaAAT1a, CaACC1, CaADP1, CaMPIb, CaSYA1, CaVPS13 and CaZWF1b. These seven genes have shown to provide high discriminatory power for strain discrimination and for both population structure analyses and epidemiological studies of C. albicans.19–21 PCR primers used were as described previously.20,21 Fragment amplification by PCR was carried out in a 50 ll reaction volume containing 100 ng of genomic DNA, 10 mM each of forward and reverse primer, 2.5 U of Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA), 5 ll of 10 9 buffer (Invitrogen) and 200 mM deoxynucleoside triphosphate mix (New England Biolabs, Ipswich, MA, USA). The PCR conditions used for all the primer sets were as follows: denaturation for 2 min at 94°C, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 52°C for 1 min, elongation at 72°C for 1 min and a final extension step of 10 min at 72°C in a C1000 Thermal Cycler (Bio-Rad,
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Hercules, CA, USA). DNA sequencing was performed using the same primers as for PCR at 2.5 mM concentration. All sequencing reactions were carried out in a 10 ll reaction volume using Big Dye Terminator Kit v 3.1 (Applied Biosystems) according to the manufacturer’s recommendations and analysed on an ABI 3130XL Genetic Analyser (Applied Biosystems). For all C. africana strains, the eight target loci including hwp1 gene were sequenced in both directions. Sequence data were inspected manually for positions of homozygotic or heterozygotic polymorphisms. Single nucleotide polymorphisms present in both the forward and reverse sequence were included in further analyses. The one-letter IUPAC nucleotide code was used in sequence analyses. Allele numbers for each locus were assigned based on comparison of sequence data from our isolates to sequences deposited into the public MLST database (http://calbicans.mlst.net).20,21 In the case where an exact match was not present in the database, the allele number in the database with 98–99% identity to the query sequence was given followed by ‘00’. In the cases where two unique sequences from this study showed 99% identity to the same allele number in the database, the distinction was made via sequential numbering in the final twodigit suffix (i.e. 00, 01, 02). However, the two unique sequences showing 100% identity with each other were assigned the same two-digit suffix. The composite profile of all seven allele numbers for an individual isolate defined the isolate’s diploid sequence type (DST). Isolates differing by one or more nucleotides (heterozygous or homozygous) at any of the seven loci were represented by a unique DST. DSTs of two C. africana isolates were assigned by authors of this study and do not correspond to DSTs in the Candida MLST database. Phylogenetic inferences were made from distance tree constructed by using the Neighbour-joining (NJ) algorithm implemented in the PAUP v4 software.22 In vitro antifungal susceptibility testing (AFST)
AFST of C. africana isolates was carried out using the Clinical Laboratory Standards Institute (CLSI) broth microdilution method, following the M27-A3 guidelines.23 The antifungals tested were amphotericin B (Sigma, St. Louis, MO, USA), fluconazole (Pfizer, Groton, CT, USA), itraconazole (Lee Pharma, Hyderabad, India), voriconazole (Pfizer), posaconazole (Merck, Whitehouse Station, NJ, USA), isavuconazole (BasileaPharmaceutica, Basel, Switzerland), flucytosine (Sigma), caspofungin (Merck, Whitehouse Station, NJ, USA),
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micafungin (Astellas, Toyama, Japan), anidulafungin (Pfizer), clotrimazole (Sigma), ketoconazole (Sigma), miconazole (Sigma) and terbinafine (Synergene, Hyderabad, India). Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019 were used as two quality control strains with each test. Patient information
The diagnosis of VVC was based on the presence of signs and symptoms of inflammation, microscopic detection of yeast cells and/or pseudohyphae and isolation of Candida species in culture, which has been described previously.24 The records of patients were reviewed and information regarding patients demographics, medical history, risk factors for candidiasis such as diabetes, intrauterine contraceptive device (IUCD) usage, recent antibiotic therapy, vaginal oestrogen replacement, immunocompromised status, corticosteriod use and treatment outcomes were recorded. The treatment regimen included oral fluconazole 150 mg OD once a week for three consecutive weeks followed by periodic review of the patients.
Results Of the 283 germ-tube-positive yeast isolates, four yielded an amplicon of size ~740 bp identical to that of the C. africana reference isolate, CBS 8781 by hwp1 PCR. The remaining 279 isolates gave an amplicon of size 941 bp suggesting their identification as the typical C. albicans. However, none of the isolates showed amplification products at 569 bp or 800 bp, ruling out C. dubliniensis and C. stellatoidea respectively.2,5 All four C. africana isolates showed distinct turquoise green coloured colonies, in contrast to green colour colonies of C. albicans on CHROMagar Candida medium. No chlamydospores were formed on rice tween 80 agar after 5 days of incubation at 28°C. Furthermore, the biochemical profiles generated by VITEK2 identified two of the C. africana isolates as C. sphaerica with 86% ID, whereas the other two remained unidentified. All four isolates failed to assimilate trehalose glucosamine, DL-lactate and lacked N-acetyl-galactosaminidase activity. However, unlike C. stellatoidea, the isolates assimilated sucrose. Our isolates grew at 37°C and showed restricted growth at 42°C. Of the four C. africana isolates, three have been deposited in the CBS Fungal Biodiversity Centre, Utrecht, the Netherlands, with each representing a distinct multilocus genotype based on the consensus C. albicans MLST genotyping markers. The laboratory and CBS accession numbers
4
of C. africana isolates are VPCI 891/P/12 (CBS 12764), VPCI 896/P/12 (CBS 12765), VPCI 84/P/13 and VPCI 301/P/13 (CBS 12826). ITS and D1/D2 sequences of the four C. africana isolates (GenBank accession nos. KF741783 to KF741786 for ITS and KF741787 to KF741790 for D1/D2) showed 99% sequence similarity (query coverage ranging from 98–100%) with the only available C. africana type isolate in GenBank (accession no. AY342214) and with C. albicans isolates (accession nos. JN606311, GQ280317 and AF455524 for ITS; AB828128, GU319992 and HQ876051 for D1/D2). Comparisons of the D1/D2 regions between the four Indian C. africana isolates and 10 C. albicans isolates (five each from the United States and Japan) revealed 99.09–100% nucleotide sequence similarity. Similarly, comparison of our ITS sequences revealed >99% identity with three C. albicans isolates submitted to the GenBank (accession no.s AJ551313, AB032174 and AB032173). DNA sequencing of the 373–491 bp fragments from the coding region of each of the seven genes resulted in a total of 2,883 aligned nucleotides for each isolate. All four C. africana isolates shared the common C. albicans allele types 33, 7, 32, 48 and 61 assigned by MLST database at the following five loci, CaAAT1a, CaACC1, CaADP1, CaVPS13 and CaZWF1b, respectively. However, only 98–99% sequence similarities were observed for two loci, CaSYA1 and CaMPIb. Candida africana reference strain (CBS 8781) also shared common C. albicans allele types assigned by MLST database at CaAAT1a, CaACC1, CaADP1, CaVPS13 and CaSYA1, but the sequence similarity for CaMPIb and CaZWF1b was 98–99%. Although the CBS 8781 previously included in MLST analysis by Odds et al. show it to be diploid sequence type (DST) 182, in our hand the strain revealed differences at two allele types. Compared to the C. albicans MLST scheme, three of the four C. africana isolates revealed a 99% sequence identity with allele 43 at the CaMPI1b gene and they were assigned new allele numbers 134 (for strain VPCI 896/P/12) and 135 (for strains VPCI 84/P/13 and VPCI 301/P/13) by the C. albicans MLST database. However, one C. africana isolate showed a 99% sequence identity with a different allele, allele 26 at the CaMPIb gene and this isolate was also assigned new allele number 133 (VPCI 891/P/12) by the database. The sequences of CaAAT1a, CaACC1, CaADP1, CaMPIb, CaSYA1, CaVPS13 and CaZWF1b genes, used for MLST, of the 4 C. africana isolates have been submitted to the C. albicans MLST database. All the allele numbers of seven loci and their corresponding DSTs
© 2014 Blackwell Verlag GmbH
MLST of Candida africana
Table 2 Multilocus sequence typing profile of the four Indian C. africana isolates generated using seven loci of the C. albicans MLST
scheme MPIb
SYA1
Isolate no.
AAT1 Allele no.
ACC1 Allele no.
ADP1 Allele no.
Allele no. (% identity)
New allele assigned
Allele no. (% identity)
New allele assigned
ZWF1b Allele no.
VPSP13 Allele no.
Assigned DST
VPCI VPCI VPCI VPCI
33 33 33 33
7 7 7 7
32 32 32 32
26 43 43 43
133 134 1351 1351
2 (99%) 2 (99%) 2 2
2-00 2-01 -
48 48 48 48
61 61 61 61
DST12 DST22 DST 21913,4 DST 21913,4
891/P/12 896/P/12 84/P/13 301/P/13
(98%) (99%) (99%) (99%)
1
Isolates at MPIb locus were 100% identical. DSTs assigned by the authors.
2 3
DSTs assigned by C. albicans MLST database.
4
DST 2191 isolates were distinguishable based on hwp1 gene analysis.
for four Indian C. africana isolates are summarised in Table 2. The second locus showing distinct C. africana polymorphisms was CaSYA1. At this locus, two strains (VPCI 84/P/13 and VPCI 301/P/13) were identical to allele type 2, whereas the other two isolates (VPCI 891/P/12 and VPCI 896/P/12) showed a close relationship with allele 2, but revealed a 1% sequence difference with allele number 2 and they were assigned alleles 2-00 and 2-01 respectively. Combining the allele types at all seven loci resulted in three unique DSTs (Table 2) among the four Indian C. africana isolates viz. DST 1 (VPCI 891/P/12), DST 2 (VPCI 896/P/12) and DST 2191 (VPCI 84/P/13 and VPCI 301/P/13). Consistent with previous reports, all four Indian C. africana strains clustered into Clade 13 of the C. albicans species complex. In addition, the strains in this clade comprised entirely of C. africana isolates and including all known C. africana sequences deposited so far (from Chile, Angola, Germany, Japan, Madagascar and United Kingdom). A Neighbour-Joining (NJ) phylogenetic tree based on concatenated sequences of the seven housekeeping genes was generated that included the four Indian C. africana isolates as well as 14 C. africana isolates available in the MLST database representing Madagascar (n = 5), Angola (n = 3), United Kingdom (n = 2), Germany (n = 2), Chile (n = 1) and Japan (n = 1). Additional 14 sequences comprising 12 C. albicans isolates belonging to Clades 1–12 along with a CBS reference strain of C. albicans (CBS 562) and C. stellatoidea (CBS 1905) were also retrieved for comparison (Fig. 1). All the C. africana isolates, irrespective of their geographical origins, formed a distinct cluster different from other C. albicans strains (Fig. 1). Furthermore, the NJ phylogenetic tree of the most variable marker CaMPIb
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gene revealed extensive intraspecies variation within C. africana (Fig. 2). Interestingly, strains VPCI 891/P/ 12 and VPCI 84/P/13 shared the same allele at the hwp1 locus, whereas strains VPCI 896/P/12 and VPCI 301/P/13 had a different allele. Thus, the inclusion of sequence information from the hwp1 locus separated the two strains of DST 2191 (Table 2) into different genotypes. Of the azole antifungals tested, clotrimazole, isavuconazole, ketoconazole, miconazole and posaconazole exhibited the most potent activity with MICs of 0.015 lg ml 1 followed by itraconazole and voriconazole each showing MICs of 0.03 lg ml 1. Amphotericin B also showed an excellent activity with MIC range of 0.03–0.125 lg ml 1. The isolates were also susceptible to fluconazole (MIC range, 0.5–4 lg ml 1), flucytosine (MIC range, 0.125–0.5 lg ml 1) and had higher MICs of terbinafine (MIC, 2 lg ml 1). All the echinocandins exhibited low MICs of 0.015 lg ml 1. The mean age of four female patients with VVC due to C. africana was 25 years (range 18–35 years). Their high vaginal swabs yielded confluent monofungal population of C. africana in culture. One of the patients gave history of using IUCD and the other was HIV positive, whereas in the remaining two no risk factor was apparent. The patients in this study were all treated successfully with fluconazole 150 mg once a week for 4 weeks.
Discussion The present survey revealed that C. africana constituted 3.1% of the vaginal isolates of the C. albicans species complex. In Europe, C. africana has been reported to cause 6–16% of vaginal infections/colonisation previously attributed to C. albicans, and a solitary strain of C. africana has been reported from
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NJ
C.albicans-Clade1/DST69/UK C.albicans-Clade7/DST300/UK C.albicans-Clade8/DST766/Japan C.africana-Clade13/DST182/496/Germany VPCI891/P/12, India C.africana-Clade13/DST182/1520/Madagascar C.africana-Clade13/DST182/1512/Angola C.africana-Clade13/DST182/1513/Madagascar C.africana-Clade13/DST182/211/UK C.africana-Clade13/DST182/1521/Madagascar C.africana-Clade13/DST182/1516/Angola C.africana-Clade13/DST182/1519/Angola C.africana-Clade13/DST182/1178/Chile C.africana-Clade13/DST182/1514/Germany C.africana-Clade13/DST182/206/UK C.africana-Clade13/DST782/1077/Japan VPCI 301/P/13, India VPCI 84/P/13, India VPCI 896/P/12, India C.africana-Clade13/DST182/1518/Madagascar C.africana-Clade13/DST182/1515/Madagascar C.albicans-Clade12/DST90/Malaysia
Figure 1 Phylogenetic tree, based on
C.albicans-CBS562/DST1030 C.albicans-Clade2/DST124/UK C.albicans-Clade3/DST155/US C.albicans-Clade5/DST881/Belgium C.albicans-Clade11/DST167/US C.albicans-Clade10/DST840/France C.albicans-Clade4/DST345/Belgium C.albicans-Clade6/DST735/Argentina C.albicans-Clade9/DST173/UK C.stellatoidea-CBS1905/DST1031 0.0001 substitutions/site
Spain.4,6,9 In contrast, C. africana was not identified among 195 and 98 vaginal C. albicans isolates from Turkey and Malaysia, respectively.25,26 Also, a significant geographical variation in prevalence of C. africana has been reported from the African continent. Low rates of 2 and 2.4% from Senegal and Nigeria, respectively, have been reported.11,12 On the other hand, Tietz et al. found a very high prevalence of 23% and 40% from Angola and Madagascar, respectively.3 These variations in prevalence of C. africana emphasise the importance of studying the epidemiology of this pathogen. This study employed a well-established molecular subtyping approach, MLST, to determine the genetic
6
concatenated sequences of the seven housekeeping genes for comparing four Indian C. africana isolates with 14 C. africana isolates available in the MLST database, obtained by using neighbourjoining phylogenetic analysis. In addition, sequences of 12 C. albicans isolates belonging to Clade 1–12 along with CBS reference strain of C. albicans (CBS 562) and C. stellatoidea (CBS 1905) were retrieved for comparison. The figure includes the diploid sequence type, clade number, strain number and geographical location of the respective isolates.
relatedness of C. africana isolates based upon variability within the particular housekeeping genes. Previously, molecular epidemiology studies based upon MLST data have revealed that C. albicans in fact contained several distinct phylogenetic groups.8 Among these, C. africana has emerged as an important aetiological agent of vaginal infections. All Indian C. africana isolates originated from diagnosed cases of VVC whose vaginal swabs yielded confluent monofungal growth. The seven gene fragments used for MLST of C. africana yielded three distinct DSTs among the four Indian strains. The inclusion of sequence information from hwp1 gene revealed that all four strains had distinct
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MLST of Candida africana
NJ
Clade13/DST182/Strain206/UK Clade13/DST182/Strain211/UK Clade13/DST182/Strain496/Germany Clade13/DST182/Strain1178/Chile Clade13/DST182/Strain1512/Angola Clade13/DST182/Strain1513/Madagascar Clade13/DST782/Strain1077/Japan Clade13/DST182/Strain1514/Germany Clade13/DST182/Strain1515/Madagascar Clade13/DST182/Strain1516/Angola Clade13/DST182/Strain1518/Madagascar Clade13/DST182/Strain1519/Angola Clade13/DST182/Strain1520/Madagascar Clade13/DST182/Strain1521/Madagascar VPCI 84/P/13, India VPCI 301/P/13, India VPCI 896/P/12, India
Figure 2 The Neighbour-joining phyloge-
netic tree based on CaMPIb gene depicting intraspecies variation between Candida africana isolates.
VPCI 891/P/12, India 0.001 substitutions/site
genotypes. These four Indian strains all belonged to Clade 13 of the C. albicans species complex that included all 14 isolates of C. africana analysed previously using the seven gene fragments for C. albicans MLST.8 Also, a close relation among most of the MPIb allele of Indian C. africana isolates with that of MPIb allele type of a Japanese C. africana (JIMS500002 DST 782) isolate described previously was observed. These results confirm that C. africana is a distinct clade within the C. albicans species complex and some of these loci provide reasonable discriminating power for strains of C. africana. The genetic variation observed at the MPIb locus, as well as at the hwp1 locus, seems to be useful for differentiating C. africana isolates. Thus,
© 2014 Blackwell Verlag GmbH
we suggest that hwp1 be included for future sequence typing of C. africana, not only because it can discriminate C. africana from the typical C. albicans but also it allowed further separation of our strains into unique genotypes. So far about 100 isolates have been reported in the global literature and of these only 51 had been confirmed using molecular methods. Among the 51 isolates, only 14 have been typed by MLST markers, with eight isolates from Africa, four from Europe and one each from Chile and Japan and most of these were published in one study in 2007.8 This study expands the MLST database by describing four additional C. africana isolates from India. Furthermore, most C.
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C. Sharma et al.
africana isolates differed from each other at only one or two loci, suggesting that they diverged very recently from a common ancestor.27 Given the information found here, markers MPI1b and hwp1 would be suitable for future genotyping species and strains of C. africana. Large-scale epidemiologic investigations of C. africana using these and other loci are needed to develop a specific and discriminatory MLST scheme for C. africana. Candida africana has been shown to be less pathogenic than C. albicans in an insect model. A recent study showed a significantly slower rate of killing of Galleria mellonella larvae by C. africana isolates than that of C. albicans or C. dubliniensis.6 In larvae inoculated with C. africana, no true mycelium and pseudohyphae were detected. The lower filamentation rate may contribute to the low virulence of this yeast. Also, compared to C. albicans, C. africana showed genetic differences at both the hwp1 and the rlm1 genes.5,28 The hwp1 gene in C. albicans is reported to be an important adhesin that crosslinks C. albicans to epithelial cells. The distinct hwp1 allele found in C. africana may be responsible for the low hyphal production, reduced adhesion and decreased virulence.5,29 Similarly, the rlm1 gene is required for cell wall integrity under stressed conditions such as the presence of the antifungal caspofungin. The rlm1 gene encodes a transcription factor from the MADS (Mcm1p-Agamous-Deficiens-Serum response factor) box family whose deletion impairs growth of Candida glabrata at higher temperature (41°C). Candida africana isolates from Africa (Angola and Madagascar) were unable to grow at higher temperature (42°C), showed a distinct rlm1 allele (named allele 15), and were hypersensitive to several stress agents. It is possible that the rlm1 gene in C. africana may have a reductive role in adaptability of this yeast to specific conditions such as in human hosts.27 Unlike the African isolates, the four Indian isolates and those from the United Kingdom grew at 42°C, consistent with phenotypic variation among strains in C. africana. In addition, our isolates were genetically distinct from the African isolates as identified based on MLST. Aside from differences in genotypes and growth at 42°C, the strains also showed some difference in susceptibilities to several antifungal drugs. For example, one of the four isolates had a high MIC of fluconazole of 4 lg ml 1, whereas the other three were highly sensitive. The new species-specific clinical break points (CBPs) of Candida albicans classify MIC of 4 lg ml 1 as susceptible dose dependent. Furthermore, resistance of C. africana to voriconazole, flucytosine and terbinafine has been demonstrated.7,14 However, the global
8
patterns of resistance/susceptibility to most antifungal drugs remain largely unknown in C. africana. Finally, although C. africana has been largely associated with superficial infections of the genital tract, it has been associated with deep-seated infections, suggesting its invasive potential.14 Together, these features call greater efforts in studying the epidemiology and pathogenesis of C. africana.
Acknowledgments C.S is supported by University Grants Commission Research Fellowship, India (F.2-15/2003 SA-I). J. Xu’s research on human pathogenic fungi is supported by grants from the Natural Science and Engineering Research Council (NSERC) of Canada. J.F.M. is partly supported by Grant NPRP 5-298-3-086 from the Qatar National Research Fund.
Potential conflict of interest J.F.M received grants from Astellas, Basilea and Merck. He has been a consultant to Basilea, Merck and Pfizer and received speaker’s fees from Merck and Gilead. All other authors: no potential conflicts of interest. The authors alone are responsible for the content and writing of the article.
References 1
2 3
4
5
6
7
8 9
Tietz HJ, Hopp M, Schmalreck A, Sterry W, Czaika V. Candida africana sp. nov., a new human pathogen or a variant of Candida albicans? Mycoses 2001; 44 : 437–45. Romeo O, Criseo G. Candida africana and its closest relatives. Mycoses 2011; 54: 475–86. Tietz HJ, K€ ussner A, Thanos M, De Andrade MP, Presber W, Sch€ onian G. Phenotypic and genotypic characterization of unusual vaginal isolates of Candida albicans from Africa. J Clin Microbiol 1995; 33: 2462–5. Romeo O, Criseo G. Molecular epidemiology of Candida albicans and its closely related yeasts Candida dubliniensis and Candida africana. J Clin Microbiol 2009; 47: 212–4. Romeo O, Criseo G. First molecular method for discriminating between Candida africana, Candida albicans and Candida dubliniensis by using hwp1 gene. Diagn Microbiol Infect Dis 2008; 62: 230–3. Borman AM, Szekely A, Linton CJ, Palmer MD, Brown P, Johnson EM. Epidemiology, antifungal susceptibility, and pathogenicity of Candida africana isolates from the United Kingdom. J Clin Microbiol 2013; 51: 967–72. Al-Hedaithy SSA, Fotedar R. Recovery and studies on chlamydospores-negative Candida albicans isolated from clinical specimens. Med Mycol 2002; 40: 301–6. Odds FC, Bougnoux ME, Shaw DJ et al. Molecular phylogenetics of Candida albicans. Eukaryot Cell 2007; 6: 1041–52. Alonso-Vargas R, Elorduy L, Eraso E et al. Isolation of Candida africana, probable atypical strains of Candida albicans, from a patient with vaginitis. Med Mycol 2008; 46: 167–70.
© 2014 Blackwell Verlag GmbH
MLST of Candida africana
10
11
12 13
14 15
16
17
18
19
20
Romeo O, Criseo G. Morphological, biochemical and molecular characterisation of the first Italian Candida africana isolate. Mycoses 2009; 52: 454–7. Nnadi NE, Ayanbimpe GM, Scordino F et al. Isolation and molecular characterization of Candida africana from Jos. Nigeria. Med Mycol 2012; 50: 765–7. Dieng Y, Sow D, Ndiaye M et al. Identification of three Candida africana strains in Senegal. J Mycol Med 2012; 22: 335–40. Hazen K (2013) Novel yeast, originally detected in Africa, now isolated from women in the United States. In: The 53rd Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC); Sep 10-13; Denver, CO, USA Romeo O, Tietz HJ, Criseo G. Candida africana: Is it a fungal pathogen? Curr Fungal Infect Rep 2013; 7: 192–7. Ciardo DE, Sch€ ar G, B€ ottger EC, Altwegg M, Bosshard PP. Internal transcribed spacer sequencing versus biochemical profiling for identification of medically important yeasts. J Clin Microbiol 2006; 44: 77– 84. Forche A, Sch€onian G, Gr€ aser Y, Vilgalys R, Mitchell TG. Genetic structure of typical and atypical populations of Candida albicans from Africa. Fungal Genet Biol 1999; 28: 107–25. Kurtzman CP, Robnett CJ. Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5’ end of the large-subunit (26S) ribosomal DNA gene. J Clin Microbiol 1997; 35: 1216–23. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, (eds), PCR protocols: a guide to methods and applications. San Diego, CA: Academic Press, 1990: 315–22. Bougnoux ME, Tavanti A, Bouchier C et al. Collaborative consensus for optimized multilocus sequence typing of Candida albicans. J Clin Microbiol 2003; 41: 5265–6. Tavanti A, Gow NA, Senesi S, Maiden MC, Odds FC. Optimization and validation of multilocus sequence typing for Candida albicans. J Clin Microbiol 2003; 41: 3765–76.
© 2014 Blackwell Verlag GmbH
21
22
23
24
25
26 27 28
29
30
Chowdhary A, Lee-Yang W, Lasker BA, Brandt ME, Warnock DW, Arthington-Skaggs BA. Comparison of multilocus sequence typing and Ca3 fingerprinting for molecular subtyping epidemiologicallyrelated clinical isolates of Candida albicans. Med Mycol 2006; 44: 405–17. Swofford DL (2002) PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts. Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard-third edition, M27–A3. Wayne, USA, PA: CLSI, 2008. Sharma C, Wankhede S, Muralidhar S et al. Candida nivariensis as an etiologic agent of vulvovaginal candidiasis in a tertiary care hospital of New Delhi, India. Diagn Microbiol Infect Dis 2012; 76: 46–50. Gumral R, Sancak B, Guzel AB, Saracßlı MA, Ilkit M. Lack of Candida africana and Candida dubliniensis in vaginal Candida albicans isolates in Turkey using HWP1 gene polymorphisms. Mycopathologia 2011; 172: 73–6. Yazdanpanah A, Khaithir TM. Issues in identifying germ tube positive yeasts by conventional methods. J Clin Lab Anal 2014; 28: 1–9. Odds FC, Jacobsen MD. Multilocus sequence typing of pathogenic Candida species. Eukaryot Cell 2008; 7: 1075–84. Sampaio P, Nogueira E, Loureiro AS, Delgado-Silva Y, Correia A, Pais C. Increased number of glutamine repeats in the C-terminal of Candida albicans Rlm1p enhances the resistance to stress agents. Antonie Van Leeuwenhoek 2009; 96: 395–404. Romeo O, De Leo F, Criseo G. Adherence ability of Candida africana: a comparative study with Candida albicans and Candida dubliniensis. Mycoses 2011; 54: e57–61. Mendling W, Krauss C, Fladung B. A clinical multicenter study comparing efficacy and tolerability of topical combination therapy with clotrimazole (Canesten, two formats) with oral single dose fluconazole (Diflucan) in vulvovaginal mycoses. Mycoses 2004; 47: 136–42.
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