Mycopathologia DOI 10.1007/s11046-015-9924-z
Molecular Characterization of Highly Susceptible Candida africana from Vulvovaginal Candidiasis Seyed Amir Yazdanparast . Sadegh Khodavaisy . Hamed Fakhim . Tahereh Shokohi . Iman Haghani . Mojtaba Nabili . Haniyeh Gholami . Imaneh Ahmadi . Hamid Badali
Received: 20 April 2015 / Accepted: 6 July 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Phylogenetic studies highlight Candida africana as an atypical variant within Candida albicans species complex which is dominantly recovered from vaginal specimens. This study aimed to characterize C. africana isolates from patients with vulvovaginal candidiasis (VVC) by molecular methods and in vitro susceptibilities. One hundred and fifty-six (48.44 %) Candida strains were collected from 322 patients diagnosed with VVC. Of these, 114 (73.07 %) were germ tube positive and presented green color on the chromogenic medium, thus classified as C. albicans species complex. One hundred and nine (95.61 %) out of 114 isolates were identified as C. albicans, while five (4.38 %) isolates were identical with C. africana based on hwp1 PCR. C. africana
appeared to be highly susceptible to the tested antifungals. For all strains of C. africana, fluconazole MIC was 2-log2-dilution steps less active than amphotericin B, which in turn was 2-log2-dilution steps and 3-log2-dilution steps less active than other azoles and echinocandin agents, respectively. In conclusion, among the C. albicans species complex, C. albicans predominantly and C. africana rarely occur in vaginal mucosa. Due to limited information on molecular epidemiology of this novel yeast, more studies using molecular methods are needed to elucidate the interand intraspecific genomic variations of C. africana isolates.
S. A. Yazdanparast Department of Medical Parasitology and Mycology, School of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran
Medicine, Mazandaran University of Medical Sciences, Sari, Iran e-mail: [email protected]
S. Khodavaisy Department of Medical Mycology and Parasitology, Kurdistan University of Medical Sciences, Sanandaj, Iran H. Fakhim Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran T. Shokohi I. Haghani M. Nabili H. Gholami H. Badali (&) Department of Medical Mycology and Parasitology/ Invasive Fungi Research Centre (IFRC), School of
Keywords VVC C. africana hwp1 In vitro susceptibility Iran
M. Nabili Iranian Social Security Organization, Mazandaran, Iran I. Ahmadi Department of Obstetrics and Gynecology, Imam Hospital, Mazandaran University of Medical Sciences, Sari, Iran H. Badali Molecular Cell Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran
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Introduction Vulvovaginal candidiasis (VVC) is one of the most common forms of Candida infections which affect up to 75 % of women at least once in their lifetime [1, 2]. It is mostly caused by Candida albicans, but other species including C. glabrata, C. tropicalis and C. krusei may also be involved [3]. Women with recurrent vulvovaginal candidiasis (RVVC) have higher chance of infections by other non-albicans species, reaching approximately 10–15 % of the affected population [4]. Within last few years, several reports have described atypical isolates of C. albicans, which have often been difficult to identify at the species level, due to their heterogeneousity in morphological, biochemical and genetic characteristics [5–7]. In some cases, Candida africana was initially described as an atypical, chlamydospore-negative variant of C. albicans, but later was proposed as a new species on the basis of morphological, biochemical, and physiologic differences [6]. It also has been reported as a cause of VVC from more than ten different countries (Angola, Madagascar, Germany, Saudi Arabia, Spain, Italy, Nigeria, Cameroon, Senegal, United Kingdom, United States, India, China, Japan and Chile) [7–11]. Despite its worldwide distribution, the overwhelming majority of C. africana isolates have been isolated from VVC. Although molecular analyses supported the varietal distinction of C. albicans var. africana, the taxonomic status of C. africana remains unclear [7, 8]. In addition, due to the high degree of phenotypic and physiologic similarity between closely related species, identification problems are imminent. DNA sequencing of the internal transcribed spacers (ITS rDNA) and the large subunit of the ribosomal RNA gene (the D1/D2 domains) have revealed low interspecies divergence of 0.5 % between C. albicans and C. africana, similar with the most of the clades within the C. albicans species complex [12, 14]. Thus, there has been a debate whether C. africana is a distinct species or a variety of C. albicans. Interestingly, Romeo and Criseo described a molecular approach for differentiating C. albicans species complex using a single pair of primers targeting the hyphal wall protein 1 (hwp1) gene in polymerase chain reaction (PCR)based assay [15]. Therefore, the objective of the current study was characterization of C. africana from either VVC or RVVC patients based on conventional
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and molecular assays to determine the epidemiology of C. africana in Iran. Isolates identified as C. africana subjected to in vitro antifungal susceptibility profiles were evaluated.
Materials and Methods Fungal Isolates Three hundred and twenty-two vaginal swab samples were obtained from patients (age range 15–56 years) with symptoms including pruritus vulvae; pain and vaginal discharge were admitted to the obstructive and gynecology department, from 2013 to 2014. Specimens initially were examined by 10 % KOH, followed by inoculation on Sabouraud dextrose agar (SDA, Difco) and incubation at 37 °C in dark for 48 h. The grown strains were preliminarily identified based on germ tube tests in serum at 37 °C for 2–3 h in dark, green colony color of the isolates on CHROMagar Candida medium (CHROMagar Company, Paris, France), microscopic morphology on Corn-Meal agar (CMA, Difco, laboratories, Detroit, Mich., USA) with 1 % tween 80 and Carbohydrate assimilation tests using the ID 32 C kit (Biome´rieux, France). All identified strains were suspended in tryptic soy broth medium (TSB, Scharlau, Spain) containing 2 % glucose, 2 % peptone and 20 % glycerol at -70 °C for further investigation and deposited at the reference culture collection of Invasive Fungi Research Center (IFRC), Sari, Iran. This research was approved by the ethics committee (nr. 518) of Mazandaran University of Medical Sciences, Sari, Iran, and written informed consent was obtained from the patient’s next of kin for publication of this report. Molecular Identification Fresh colony on SDA and green color colony on the chromogenic medium with germ tube-positive test were selected for gDNA extraction. Two reference strains Candida albicans (CBS 562) and C. africana (CBS 8781) were included in the study. Briefly, DNA was extracted from 3-day-old SDA cultures with an ultraclean microbial DNA isolation kit (Mobio, Carlsbad, CA, U.S.A.) according to the manufacturer’s instructions. DNA extracts were stored at -20 °C prior to use.
Mycopathologia
PCR Amplification Targeting the hwp1 Gene and Sequencing Based on previously described protocol by Romeo and Criseo [15], the partial amplification of the hwp1 gene was used to distinguish C. africana (750 bp) from typical C. albicans isolates (941 bp). The PCR primers used were as follow (CR-f 5-GCTACCACTTCA GAATCATCATC-3) and (CR-r 5-GCAC CTTCAG TCGTAGAGACG-3) [15]. Strains identified as C. africana and C. albicans were confirmed by sequencing of the ITS rDNA regions using previously described primers [12, 14]. Sequencing was performed on an ABI 3730xl automatic sequencer (Applied Biosystems, Foster City, CA). Sequence data obtained in this study were adjusted using the SeqMan of Lasergene software (DNAStar Inc., Madison, Wisconsin, USA.) and compared with GenBank database and local database at the CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands.
conidial suspensions were measured spectrophotometrically at the 530 nm wavelengths to a percent transmission in the range 75–77. Therefore, the final densities of the stock inoculum suspensions of the isolates tested ranged from 2.5 9 103 to 5 9 103 colony-forming units/ml, as determined by quantitative colony counts on Sabouraud glucose agar (SGA, Difco) and incubated at 35 °C and examined visually after 24 and 48 h to determine MICs value. The MIC endpoints were determined with the aid of a reading mirror and were defined as the lowest concentration of drug that prevents any recognizable growth (i.e., exerts 100 % inhibition for amphotericin B) or significant ([50 %) growth diminution levels (all other agents) compared with the growth of a drugfree control. Candida parapsilosis (ATCC 22019) and Candida krusei (ATCC 6258) strains were chosen as quality controls, and analysis of these strains was performed with every new batch of MIC plates
Antifungal Susceptibility Testing Results Minimum inhibitory concentrations (MICs) were assayed for identified C. albicans species complex, i.e., C. africana (n = 5) and C. albicans (n = 109), according to recommendations stated in the Clinical and Laboratory Standards Institute (CLSI) M27-A3 and M27-S4 documents [16, 17]. Amphotericin B (Sigma, St. Louis, MO, USA), fluconazole (Pfizer, Groton, CT, USA), itraconazole (Janssen Research Foundation, Beerse, Belgium), voriconazole (Pfizer), posaconazole (Schering-Plough, Kenilworth, USA), caspofungin (Merck, Whitehouse Station, NJ, USA), anidulafungin and micafungin (Pfizer) were obtained as reagent-grade powders from the respective manufacturers for preparation of the CLSI microdilution trays. The antifungal agents were diluted in the standard RPMI-1640 medium (Sigma Chemical Co.) buffered to pH 7.0 with 0.165 M-morpholinepropanesulfonic acid (MOPS) (Sigma) with L-glutamine without bicarbonate to yield two times their concentrations and dispensed into 96-well microdilution trays with a final concentration of 0.016–16 lg/ml for amphotericin B, itraconazole, voriconazole and posaconazole; and 0.063–64 lg/ml for fluconazole and 0.008–8 lg/ml for caspofungin, anidulafungin and micafungin. Plates were stored at -70° C until they were used. Briefly, homogeneous
One hundred and fifty-six (48.44 %) strains of Candida were obtained from specimens collected from 322 patients with VVC/RVVC. Of these isolates, 114 (73.07 %) were germ tube positive and presented a green colony color on the chromogenic medium, that were classified as C. albicans complex species. One hundred and nine (95.61 %) out of 114 isolates identified as C. albicans yielded a PCR product of 941 bp length which was identical to that of the typical C. albicans strain (ATCC 90028), while five (4.38 %) yielded an amplicon of *740 bp length identical to that of the C. africana reference isolate (CBS 8781) by amplification of hwp1 gene (Fig. 1). Candida albicans was the dominant Candida species (109 strains; 95.61 %) in VVC. Further investigation showed that all five proved C. africana isolates by hwp1 failed to grow at 42 °C, in contrast to green color colonies of C. albicans on CHROMagar Candida medium which grew at both 37 and 42 °C. Chlamydospores were negative after 5 days of incubation at 28 °C, and the carbohydrate assimilation pattern of our C. africana isolates was identical to that (ID 32C code: 7046340011) described for the first time by Tietz et al. [18]. ITS rDNA sequences of five C. africana isolates showed [99 % sequence similarity with the
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Cured VVC vulvovaginal candidiasis, RVVC recurrent vulvovaginal candidiasis, IFRC invasive fungi research center
Cured Clotrimazole
Clotrimazole 7
3 Immunocompetent
Immunocompetent Burning, vaginal discharge
Pruritus, whitish gray discharge Married
Married 38
32 VVC C. africana (IFRC 708)
C. africana (IFRC 720)
4
5
RVVC
Cured
Cured Clotrimazole
Clotrimazole 4
30 Immunocompetent
Immunocompetent Vaginal discharge
Pruritus, vaginal discharge Married
Divorced 26
49 VVC
VVC
C. africana (IFRC 706)
C. africana (IFRC 707)
2
3
Cured Clotrimazole/ metronidazole 30 Immunocompetent Whitish gray discharge Divorced 25 RVVC
Duration of symptoms (day) Host status Symptom and sign Sexual activity Age Source
C. africana (IFRC 705)
Because of high degree of phenotypic similarity between closely related species of Candida, identification problems are imminent. Identification of those species obtained from clinical specimens is usually
1
Discussion
Collection no.
only available C. africana type isolate in GenBank (Accession No. AY342214). Table 1 summarizes all the data from patients suffering with vaginal itching due to C. africana. Age, marital status, symptoms and signs, period with symptoms, medical history, risk factors (diabetes, intrauterine contraceptive device (IUCD) usage), broad spectrum antibiotic and corticosteroid therapy and treatment outcomes were recorded. Table 2 summarizes the results of in vitro antifungal susceptibility profiles of several antifungal drugs against isolates of C. africana and C. albicans. MIC results among all the isolates of C. albicans complex showed they were fully susceptible to all tested antifungal drugs. However, echinocandin drugs (caspofungin, anidulafungin and micafungin) were more active than triazole (itraconazole, voriconazole and posaconazole) by \1 log2 dilution step. In addition, the five strains of C. africana appeared to be highly susceptible to tested antifungals (Table 2). For all strains of C. africana, the fluconazole MIC was 2-log2-dilution steps less active than amphotericin B, which in turn was 2-log2-dilution steps and 3-log2dilution steps less active than azoles and echinocandin agents, respectively.
Number of cases
Fig. 1 Species-specific amplification of the hwp1 gene. Lanes of 1 and 4 represent C. albicans; 2, 3, 5 and 6 represent C. africana; Lane N is negative control and Lane M is 100-bp ladder molecular size markers
Table 1 Clinical characteristics of vulvovaginal candidiasis caused isolated C. africana strains from VVC/RVVC patients (n = 5)
Therapy
Out come
Mycopathologia
MIC90: 0.063 MIC90: 0.016 MIC90: 0.016 MIC values expressed in lg/ml
MIC90: 0.25 MIC90: 1
MIC90: 0.063
MIC50: 0.125
AmB amphotericin B, FLU fluconazole, ITR itraconazole, VOR voriconazole, POS posaconazole, CAS caspofungin, ANID anidulafungin, MICA micafungin, IFRC Invasive Fungi Research Center, ND not determined
ND
ND
MIC50: 0.5
MIC50: 0.031
MIC50: 0.008
MIC50: 0.008
MIC50: 0.031
ND
ND 0.008
Range 0.008–0.125 Range 0.008–0.063
0.016 0.016
Range 0.008–0.5 Range 0.016–1 Range 0.063–16 C. albicans (n = 109)
0.063 0.016
Range 0.016–1
C. africana (IFRC 720)
0.031
ND ND 0.008 0.016 0.016 0.063 0.031 C. africana (IFRC 708)
0.031
0.008
0.008 ND 0.008 ND
0.008 0.031
0.031 0.016 0.016 0.016
0.016 0.016
0.016 0.016 0.016 0.063 0.063 0.063 0.031 0.031 C. africana (IFRC 706) C. africana (IFRC 707)
0.063 0.031 C. africana (IFRC 705)
0.016
POS VOR ITR FLU AmB C. africana collection no.
Table 2 MIC ranges, MIC50 and MIC90 of nine antifungal drugs against all obtained C. albicans and C. africana isolates
CAS
ANID
MICA
Mycopathologia
based on phenotypic and physiologic tests, viz. sugar assimilation, growth rate and enzymatic profiles [7, 12]. However, identification of atypical species can be a big challenge, and to overcome this problem, molecular tools are highly required [15]. C. africana is one of the atypical forms of C. albicans, and based on the sequencing of D1–D2 regions, phylogenetic studies show that C. africana and C. albicans isolates are sister species, closely related and support the variety status of C. africana [12, 13]. Although C. africana strains are germ tube positive like C. albicans and C. dubliniensis, they can grow and produce hyphae more slowly than both C. albicans and C. dubliniensis and could be distinguished from latter species of Candida by some physiologic criteria, i.e., sugar assimilation and chlamydospores production which are negative [9, 18–20]. In current study based on molecular tools, C. africana constituted 4.38 % of the vaginal isolates of the C. albicans species complex and was much more prevalent than the related germ tube-positive species (C. dubliniensis and C. stellatoidea) in such samples. More recently, the former species has been reported from Europe, Africa and Asia [7–10], and now, we present five cases of C. africana infection from patients suffering from VVC or RVVC in Iran (Table 1). The prevalence of C. africana in Iran was unknown prior to this study, and it is possible that the etiological agent may often have been misdiagnosed, and most of the time strains were identified as C. albicans without taking to consideration their closely related Candida species. Thus, the actual prevalence and distribution of atypical or novel species remain unclear and may be underestimated. Recently, Gumral et al. [21] reidentified 195 vaginal C. albicans strains isolated from Turkey (west neighboring country) to be C. africana and C. dubliniensis using hwp1 gene polymorphisms and showed that all isolates were confirmed to be C. albicans and none were verified as either C. africana or C. dubliniensis [21]. Also, C. africana was not identified among 98 vaginal C. albicans isolates from Malaysia [22]. In contrast, in India C. africana constituted 3.1 % of the vaginal isolates of the C. albicans species complex [8]. Interestingly, in European countries, high prevalence (6–16 %) of C. africana has been reported as agent of VVC previously misidentified as C. albicans [23], and a solitary strain of C. africana has been reported from Spain [10]. Low prevalence of C. africana has been
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reported in Senegal (2 %) and Nigeria (2.4 %) [24, 25]. Tietz et al. [7] reported high prevalence of C. africana in Angola (23 %) and Madagascar (40 %). We assume that the prevalence of C. africana in Iran is closer to Asia rather than Africa and Europe. Striking variations were seen in the incidence of C. africana as agent of VVC in the world probably due to epidemiology of this pathogen. Sharma et al. [8] have performed molecular sub-typing approach like multilocus sequence typing (MLST) of C. africana isolated from patients with VVC in India, and confirmed that mostly variation in the MPIb and hwp1 locus C. africana is a distinct clade within the C. albicans species being complex. These results were inconcordant with previous MLST analysis by Odds et al. [26] which showed that C. albicans contained several distinct phylogenetic groups. It seems that hwp1 gene as one of the housekeeping genes would be included for MLST analysis to determine the genetic relatedness of C. albicans species complex. Although in the present study, C. albicans were dominantly isolated from VVC, several studies have reported an increasing trend in the occurrence of non-C. albicans species over time [1, 20–22]. Several investigators have reported, with increasing frequency, recovery of atypical isolates of the pathogenic yeast C. albicans from clinical samples [7, 12, 13]. So far 19 out of 100 strains of isolated C. africana had been identified and typed by MLST markers (MPI1b and hwp1). Molecular approach in order to determine the incidence, distribution and genotyping of C. albicans species complex would be a suitable way [8, 26, 27]. By physiologic and molecular tools, we are able to easily and quickly distinguish C. albicans species complex, and results showed uniform susceptibility pattern against conventional and new generation of antifungal agents which are concordant with previously published papers [9, 23, 28] and suggest the C. albicans species complex would be susceptible to all antifungal agents that are appropriate for treating vaginal candidiasis. However, resistance and susceptible dose dependency of C. africana has been reported toward voriconazole, flucytosine and terbinafine and fluconazole. In contrast in our study, all C. albicans species complex were highly susceptible to tested drugs. In fact, species-specific clinical break points (CBPs) of C. africana have not been established yet and are unknown. In conclusion, among the C. albicans species complex, predominantly C. albicans and
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rarely C. africana are isolated from vaginal mucosa, and in contrast absence of C. dubliniensis and C. stellatoidea were highlighted. Further, molecular typing will be performed to elucidate the inter- and intraspecific genomic variations of C. africana isolates. Acknowledgments This study was financially supported by a grant from the School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran, which we gratefully acknowledge. The authors acknowledge the staff of Baghban Clinic for helping with technical assistance of sampling. We are grateful to Ilse Curfs-Breuker for excellent technical assistance and help with antifungal susceptibility testings. Authors would like to thank Dr. Somayeh Dolatabadi for critically reviewing and editing the manuscript. Compliance with Ethical Standards Conflict of interest No potential conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References 1. Sobel JD. Vulvovaginal candidosis. Lancet. 2007;369(9577): 1961–71. 2. Ferrer J. Vaginal candidosis: epidemiological and etiological factors. Int J Gynaecol Obstet. 2000;71:S21–7. 3. Trama JP, Adelson ME, Raphaelli I, Stemmer SM, Mordechai E. Detection of Candida species in vaginal samples in a clinical laboratory setting. Infect Dis Obstet Gynecol. 2005;13:63–7. 4. Sobel JD. Epidemiology and pathogenesis of recurrent vulvovaginal candidiasis. Am J Obstet Gynecol. 1985;152: 924–35. 5. Romeo O, Tietz H-J, Criseo G. Candida africana: is it a fungal pathogen? Curr Fungal Infect Rep. 2013;7:192–7. 6. 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. 7. Tietz H, Ku¨ssner A, Thanos M, De Andrade MP, Presber W, Scho¨nian G. Phenotypic and genotypic characterization of unusual vaginal isolates of Candida albicans from Africa. J Clin Microbiol. 1995;33:2462–5. 8. Sharma C, Muralidhar S, Xu J, Meis JF, Chowdhary A. Multilocus sequence typing of Candida africana from patients with vulvovaginal candidiasis in New Delhi, India. Mycoses. 2014;57:544–52. 9. Shan Y, Fan S, Liu X, Li J. Prevalence of Candida albicansclosely related yeasts, Candida africana and Candida dubliniensis, in vulvovaginal candidiasis. Med Mycol. 2014;52:636–40. 10. Alonso-Vargas R, Elorduy L, Eraso E, FranciscoCano J, Guarro J, Ponto´n J. Isolation of Candida africana, probable atypical strains of Candida albicans, from a patient with vaginitis. Med Mycol. 2008;46:167–70.
Mycopathologia 11. Ngouana TK, Krasteva D, Drakulovski P, Toghueo RK, Kouanfack C, Ambe A, et al. Investigation of minor species Candida africana, Candida stellatoidea and Candida dubliniensis in the Candida albicans complex among Yaounde´ (Cameroon) HIV-infected patients. Mycoses. 2015;58:33–9. 12. Romeo O, Criseo G. Candida africana and its closest relatives. Mycoses. 2011;54:475–86. 13. Ciardo DE, Schar G, Bottger 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. 14. Forche A, Schonian G, Graser Y, Vilgalys R, Mitchell TG. Genetic structure of typical and atypical populations of Candida albicans from Africa. Fungal Genet Biol. 1999;28:107–25. 15. 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. 16. Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard-third edition, M27–A3. Wayne: CLSI; 2008. 17. Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of yeasts: fourth informational supplement M27-S4. Wayne: CLSI; 2012. 18. 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. 19. Al-Hedaithy SSA, Fotedar R. Recovery and studies on chlamydospore-negative Candida albicans isolated from clinical specimens. Med Mycol. 2002;40:301–6.
20. Romeo O, Criseo G. Morphological, biochemical and molecular characterization of the first Italian Candida africana isolate. Mycoses. 2009;52:454–7. 21. Gumral R, Sancak B, Guzel AB, Sarac¸lı 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. 22. Yazdanpanah A, Khaithir TM. Issues in identifying germ tube positive yeasts by conventional methods. J Clin Lab Anal. 2014;28:1–9. 23. 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. 24. Dieng Y, Sow D, Ndiaye M, Guichet E, Faye B, Tine R, et al. Identification of three Candida africana strains in Senegal. J Mycol Med. 2012;22:335–40. 25. Nnadi NE, Ayanbimpe GM, Scordino F, Okolo MO, Enweani IB, Criseo G, et al. Isolation and molecular characterization of Candida africana from Jos, Nigeria. Med Mycol. 2012;50:765–7. 26. Odds FC, Bougnoux ME, Shaw DJ. Molecular phylogenetics of Candida albicans. Eukaryot Cell. 2007;6:1041–52. 27. Afsarian MH, Badali H, Boekhout T, Shokohi T, Katiraee F. Multilocus sequence typing of Candida albicans isolates from a burn intensive care unit in Iran. J Med Microbiol. 2015;64:248–53. 28. Fesharaki SH, Haghani I, Mousavi B, Kargar ML, Boroumand M, Anvari MS, et al. Endocarditis due to a coinfection of Candida albicans and Candida tropicalis in a drug abuser. J Med Microbiol. 2013;62(Pt 11):1763–7.
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Molecular Characterization of Highly Susceptible Candida africana from Vulvovaginal Candidiasis Seyed Amir Yazdanparast . Sadegh Khodavaisy . Hamed Fakhim . Tahereh Shokohi . Iman Haghani . Mojtaba Nabili . Haniyeh Gholami . Imaneh Ahmadi . Hamid Badali
Received: 20 April 2015 / Accepted: 6 July 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Phylogenetic studies highlight Candida africana as an atypical variant within Candida albicans species complex which is dominantly recovered from vaginal specimens. This study aimed to characterize C. africana isolates from patients with vulvovaginal candidiasis (VVC) by molecular methods and in vitro susceptibilities. One hundred and fifty-six (48.44 %) Candida strains were collected from 322 patients diagnosed with VVC. Of these, 114 (73.07 %) were germ tube positive and presented green color on the chromogenic medium, thus classified as C. albicans species complex. One hundred and nine (95.61 %) out of 114 isolates were identified as C. albicans, while five (4.38 %) isolates were identical with C. africana based on hwp1 PCR. C. africana
appeared to be highly susceptible to the tested antifungals. For all strains of C. africana, fluconazole MIC was 2-log2-dilution steps less active than amphotericin B, which in turn was 2-log2-dilution steps and 3-log2-dilution steps less active than other azoles and echinocandin agents, respectively. In conclusion, among the C. albicans species complex, C. albicans predominantly and C. africana rarely occur in vaginal mucosa. Due to limited information on molecular epidemiology of this novel yeast, more studies using molecular methods are needed to elucidate the interand intraspecific genomic variations of C. africana isolates.
S. A. Yazdanparast Department of Medical Parasitology and Mycology, School of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran
Medicine, Mazandaran University of Medical Sciences, Sari, Iran e-mail: [email protected]
S. Khodavaisy Department of Medical Mycology and Parasitology, Kurdistan University of Medical Sciences, Sanandaj, Iran H. Fakhim Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran T. Shokohi I. Haghani M. Nabili H. Gholami H. Badali (&) Department of Medical Mycology and Parasitology/ Invasive Fungi Research Centre (IFRC), School of
Keywords VVC C. africana hwp1 In vitro susceptibility Iran
M. Nabili Iranian Social Security Organization, Mazandaran, Iran I. Ahmadi Department of Obstetrics and Gynecology, Imam Hospital, Mazandaran University of Medical Sciences, Sari, Iran H. Badali Molecular Cell Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran
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Introduction Vulvovaginal candidiasis (VVC) is one of the most common forms of Candida infections which affect up to 75 % of women at least once in their lifetime [1, 2]. It is mostly caused by Candida albicans, but other species including C. glabrata, C. tropicalis and C. krusei may also be involved [3]. Women with recurrent vulvovaginal candidiasis (RVVC) have higher chance of infections by other non-albicans species, reaching approximately 10–15 % of the affected population [4]. Within last few years, several reports have described atypical isolates of C. albicans, which have often been difficult to identify at the species level, due to their heterogeneousity in morphological, biochemical and genetic characteristics [5–7]. In some cases, Candida africana was initially described as an atypical, chlamydospore-negative variant of C. albicans, but later was proposed as a new species on the basis of morphological, biochemical, and physiologic differences [6]. It also has been reported as a cause of VVC from more than ten different countries (Angola, Madagascar, Germany, Saudi Arabia, Spain, Italy, Nigeria, Cameroon, Senegal, United Kingdom, United States, India, China, Japan and Chile) [7–11]. Despite its worldwide distribution, the overwhelming majority of C. africana isolates have been isolated from VVC. Although molecular analyses supported the varietal distinction of C. albicans var. africana, the taxonomic status of C. africana remains unclear [7, 8]. In addition, due to the high degree of phenotypic and physiologic similarity between closely related species, identification problems are imminent. DNA sequencing of the internal transcribed spacers (ITS rDNA) and the large subunit of the ribosomal RNA gene (the D1/D2 domains) have revealed low interspecies divergence of 0.5 % between C. albicans and C. africana, similar with the most of the clades within the C. albicans species complex [12, 14]. Thus, there has been a debate whether C. africana is a distinct species or a variety of C. albicans. Interestingly, Romeo and Criseo described a molecular approach for differentiating C. albicans species complex using a single pair of primers targeting the hyphal wall protein 1 (hwp1) gene in polymerase chain reaction (PCR)based assay [15]. Therefore, the objective of the current study was characterization of C. africana from either VVC or RVVC patients based on conventional
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and molecular assays to determine the epidemiology of C. africana in Iran. Isolates identified as C. africana subjected to in vitro antifungal susceptibility profiles were evaluated.
Materials and Methods Fungal Isolates Three hundred and twenty-two vaginal swab samples were obtained from patients (age range 15–56 years) with symptoms including pruritus vulvae; pain and vaginal discharge were admitted to the obstructive and gynecology department, from 2013 to 2014. Specimens initially were examined by 10 % KOH, followed by inoculation on Sabouraud dextrose agar (SDA, Difco) and incubation at 37 °C in dark for 48 h. The grown strains were preliminarily identified based on germ tube tests in serum at 37 °C for 2–3 h in dark, green colony color of the isolates on CHROMagar Candida medium (CHROMagar Company, Paris, France), microscopic morphology on Corn-Meal agar (CMA, Difco, laboratories, Detroit, Mich., USA) with 1 % tween 80 and Carbohydrate assimilation tests using the ID 32 C kit (Biome´rieux, France). All identified strains were suspended in tryptic soy broth medium (TSB, Scharlau, Spain) containing 2 % glucose, 2 % peptone and 20 % glycerol at -70 °C for further investigation and deposited at the reference culture collection of Invasive Fungi Research Center (IFRC), Sari, Iran. This research was approved by the ethics committee (nr. 518) of Mazandaran University of Medical Sciences, Sari, Iran, and written informed consent was obtained from the patient’s next of kin for publication of this report. Molecular Identification Fresh colony on SDA and green color colony on the chromogenic medium with germ tube-positive test were selected for gDNA extraction. Two reference strains Candida albicans (CBS 562) and C. africana (CBS 8781) were included in the study. Briefly, DNA was extracted from 3-day-old SDA cultures with an ultraclean microbial DNA isolation kit (Mobio, Carlsbad, CA, U.S.A.) according to the manufacturer’s instructions. DNA extracts were stored at -20 °C prior to use.
Mycopathologia
PCR Amplification Targeting the hwp1 Gene and Sequencing Based on previously described protocol by Romeo and Criseo [15], the partial amplification of the hwp1 gene was used to distinguish C. africana (750 bp) from typical C. albicans isolates (941 bp). The PCR primers used were as follow (CR-f 5-GCTACCACTTCA GAATCATCATC-3) and (CR-r 5-GCAC CTTCAG TCGTAGAGACG-3) [15]. Strains identified as C. africana and C. albicans were confirmed by sequencing of the ITS rDNA regions using previously described primers [12, 14]. Sequencing was performed on an ABI 3730xl automatic sequencer (Applied Biosystems, Foster City, CA). Sequence data obtained in this study were adjusted using the SeqMan of Lasergene software (DNAStar Inc., Madison, Wisconsin, USA.) and compared with GenBank database and local database at the CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands.
conidial suspensions were measured spectrophotometrically at the 530 nm wavelengths to a percent transmission in the range 75–77. Therefore, the final densities of the stock inoculum suspensions of the isolates tested ranged from 2.5 9 103 to 5 9 103 colony-forming units/ml, as determined by quantitative colony counts on Sabouraud glucose agar (SGA, Difco) and incubated at 35 °C and examined visually after 24 and 48 h to determine MICs value. The MIC endpoints were determined with the aid of a reading mirror and were defined as the lowest concentration of drug that prevents any recognizable growth (i.e., exerts 100 % inhibition for amphotericin B) or significant ([50 %) growth diminution levels (all other agents) compared with the growth of a drugfree control. Candida parapsilosis (ATCC 22019) and Candida krusei (ATCC 6258) strains were chosen as quality controls, and analysis of these strains was performed with every new batch of MIC plates
Antifungal Susceptibility Testing Results Minimum inhibitory concentrations (MICs) were assayed for identified C. albicans species complex, i.e., C. africana (n = 5) and C. albicans (n = 109), according to recommendations stated in the Clinical and Laboratory Standards Institute (CLSI) M27-A3 and M27-S4 documents [16, 17]. Amphotericin B (Sigma, St. Louis, MO, USA), fluconazole (Pfizer, Groton, CT, USA), itraconazole (Janssen Research Foundation, Beerse, Belgium), voriconazole (Pfizer), posaconazole (Schering-Plough, Kenilworth, USA), caspofungin (Merck, Whitehouse Station, NJ, USA), anidulafungin and micafungin (Pfizer) were obtained as reagent-grade powders from the respective manufacturers for preparation of the CLSI microdilution trays. The antifungal agents were diluted in the standard RPMI-1640 medium (Sigma Chemical Co.) buffered to pH 7.0 with 0.165 M-morpholinepropanesulfonic acid (MOPS) (Sigma) with L-glutamine without bicarbonate to yield two times their concentrations and dispensed into 96-well microdilution trays with a final concentration of 0.016–16 lg/ml for amphotericin B, itraconazole, voriconazole and posaconazole; and 0.063–64 lg/ml for fluconazole and 0.008–8 lg/ml for caspofungin, anidulafungin and micafungin. Plates were stored at -70° C until they were used. Briefly, homogeneous
One hundred and fifty-six (48.44 %) strains of Candida were obtained from specimens collected from 322 patients with VVC/RVVC. Of these isolates, 114 (73.07 %) were germ tube positive and presented a green colony color on the chromogenic medium, that were classified as C. albicans complex species. One hundred and nine (95.61 %) out of 114 isolates identified as C. albicans yielded a PCR product of 941 bp length which was identical to that of the typical C. albicans strain (ATCC 90028), while five (4.38 %) yielded an amplicon of *740 bp length identical to that of the C. africana reference isolate (CBS 8781) by amplification of hwp1 gene (Fig. 1). Candida albicans was the dominant Candida species (109 strains; 95.61 %) in VVC. Further investigation showed that all five proved C. africana isolates by hwp1 failed to grow at 42 °C, in contrast to green color colonies of C. albicans on CHROMagar Candida medium which grew at both 37 and 42 °C. Chlamydospores were negative after 5 days of incubation at 28 °C, and the carbohydrate assimilation pattern of our C. africana isolates was identical to that (ID 32C code: 7046340011) described for the first time by Tietz et al. [18]. ITS rDNA sequences of five C. africana isolates showed [99 % sequence similarity with the
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Cured VVC vulvovaginal candidiasis, RVVC recurrent vulvovaginal candidiasis, IFRC invasive fungi research center
Cured Clotrimazole
Clotrimazole 7
3 Immunocompetent
Immunocompetent Burning, vaginal discharge
Pruritus, whitish gray discharge Married
Married 38
32 VVC C. africana (IFRC 708)
C. africana (IFRC 720)
4
5
RVVC
Cured
Cured Clotrimazole
Clotrimazole 4
30 Immunocompetent
Immunocompetent Vaginal discharge
Pruritus, vaginal discharge Married
Divorced 26
49 VVC
VVC
C. africana (IFRC 706)
C. africana (IFRC 707)
2
3
Cured Clotrimazole/ metronidazole 30 Immunocompetent Whitish gray discharge Divorced 25 RVVC
Duration of symptoms (day) Host status Symptom and sign Sexual activity Age Source
C. africana (IFRC 705)
Because of high degree of phenotypic similarity between closely related species of Candida, identification problems are imminent. Identification of those species obtained from clinical specimens is usually
1
Discussion
Collection no.
only available C. africana type isolate in GenBank (Accession No. AY342214). Table 1 summarizes all the data from patients suffering with vaginal itching due to C. africana. Age, marital status, symptoms and signs, period with symptoms, medical history, risk factors (diabetes, intrauterine contraceptive device (IUCD) usage), broad spectrum antibiotic and corticosteroid therapy and treatment outcomes were recorded. Table 2 summarizes the results of in vitro antifungal susceptibility profiles of several antifungal drugs against isolates of C. africana and C. albicans. MIC results among all the isolates of C. albicans complex showed they were fully susceptible to all tested antifungal drugs. However, echinocandin drugs (caspofungin, anidulafungin and micafungin) were more active than triazole (itraconazole, voriconazole and posaconazole) by \1 log2 dilution step. In addition, the five strains of C. africana appeared to be highly susceptible to tested antifungals (Table 2). For all strains of C. africana, the fluconazole MIC was 2-log2-dilution steps less active than amphotericin B, which in turn was 2-log2-dilution steps and 3-log2dilution steps less active than azoles and echinocandin agents, respectively.
Number of cases
Fig. 1 Species-specific amplification of the hwp1 gene. Lanes of 1 and 4 represent C. albicans; 2, 3, 5 and 6 represent C. africana; Lane N is negative control and Lane M is 100-bp ladder molecular size markers
Table 1 Clinical characteristics of vulvovaginal candidiasis caused isolated C. africana strains from VVC/RVVC patients (n = 5)
Therapy
Out come
Mycopathologia
MIC90: 0.063 MIC90: 0.016 MIC90: 0.016 MIC values expressed in lg/ml
MIC90: 0.25 MIC90: 1
MIC90: 0.063
MIC50: 0.125
AmB amphotericin B, FLU fluconazole, ITR itraconazole, VOR voriconazole, POS posaconazole, CAS caspofungin, ANID anidulafungin, MICA micafungin, IFRC Invasive Fungi Research Center, ND not determined
ND
ND
MIC50: 0.5
MIC50: 0.031
MIC50: 0.008
MIC50: 0.008
MIC50: 0.031
ND
ND 0.008
Range 0.008–0.125 Range 0.008–0.063
0.016 0.016
Range 0.008–0.5 Range 0.016–1 Range 0.063–16 C. albicans (n = 109)
0.063 0.016
Range 0.016–1
C. africana (IFRC 720)
0.031
ND ND 0.008 0.016 0.016 0.063 0.031 C. africana (IFRC 708)
0.031
0.008
0.008 ND 0.008 ND
0.008 0.031
0.031 0.016 0.016 0.016
0.016 0.016
0.016 0.016 0.016 0.063 0.063 0.063 0.031 0.031 C. africana (IFRC 706) C. africana (IFRC 707)
0.063 0.031 C. africana (IFRC 705)
0.016
POS VOR ITR FLU AmB C. africana collection no.
Table 2 MIC ranges, MIC50 and MIC90 of nine antifungal drugs against all obtained C. albicans and C. africana isolates
CAS
ANID
MICA
Mycopathologia
based on phenotypic and physiologic tests, viz. sugar assimilation, growth rate and enzymatic profiles [7, 12]. However, identification of atypical species can be a big challenge, and to overcome this problem, molecular tools are highly required [15]. C. africana is one of the atypical forms of C. albicans, and based on the sequencing of D1–D2 regions, phylogenetic studies show that C. africana and C. albicans isolates are sister species, closely related and support the variety status of C. africana [12, 13]. Although C. africana strains are germ tube positive like C. albicans and C. dubliniensis, they can grow and produce hyphae more slowly than both C. albicans and C. dubliniensis and could be distinguished from latter species of Candida by some physiologic criteria, i.e., sugar assimilation and chlamydospores production which are negative [9, 18–20]. In current study based on molecular tools, C. africana constituted 4.38 % of the vaginal isolates of the C. albicans species complex and was much more prevalent than the related germ tube-positive species (C. dubliniensis and C. stellatoidea) in such samples. More recently, the former species has been reported from Europe, Africa and Asia [7–10], and now, we present five cases of C. africana infection from patients suffering from VVC or RVVC in Iran (Table 1). The prevalence of C. africana in Iran was unknown prior to this study, and it is possible that the etiological agent may often have been misdiagnosed, and most of the time strains were identified as C. albicans without taking to consideration their closely related Candida species. Thus, the actual prevalence and distribution of atypical or novel species remain unclear and may be underestimated. Recently, Gumral et al. [21] reidentified 195 vaginal C. albicans strains isolated from Turkey (west neighboring country) to be C. africana and C. dubliniensis using hwp1 gene polymorphisms and showed that all isolates were confirmed to be C. albicans and none were verified as either C. africana or C. dubliniensis [21]. Also, C. africana was not identified among 98 vaginal C. albicans isolates from Malaysia [22]. In contrast, in India C. africana constituted 3.1 % of the vaginal isolates of the C. albicans species complex [8]. Interestingly, in European countries, high prevalence (6–16 %) of C. africana has been reported as agent of VVC previously misidentified as C. albicans [23], and a solitary strain of C. africana has been reported from Spain [10]. Low prevalence of C. africana has been
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reported in Senegal (2 %) and Nigeria (2.4 %) [24, 25]. Tietz et al. [7] reported high prevalence of C. africana in Angola (23 %) and Madagascar (40 %). We assume that the prevalence of C. africana in Iran is closer to Asia rather than Africa and Europe. Striking variations were seen in the incidence of C. africana as agent of VVC in the world probably due to epidemiology of this pathogen. Sharma et al. [8] have performed molecular sub-typing approach like multilocus sequence typing (MLST) of C. africana isolated from patients with VVC in India, and confirmed that mostly variation in the MPIb and hwp1 locus C. africana is a distinct clade within the C. albicans species being complex. These results were inconcordant with previous MLST analysis by Odds et al. [26] which showed that C. albicans contained several distinct phylogenetic groups. It seems that hwp1 gene as one of the housekeeping genes would be included for MLST analysis to determine the genetic relatedness of C. albicans species complex. Although in the present study, C. albicans were dominantly isolated from VVC, several studies have reported an increasing trend in the occurrence of non-C. albicans species over time [1, 20–22]. Several investigators have reported, with increasing frequency, recovery of atypical isolates of the pathogenic yeast C. albicans from clinical samples [7, 12, 13]. So far 19 out of 100 strains of isolated C. africana had been identified and typed by MLST markers (MPI1b and hwp1). Molecular approach in order to determine the incidence, distribution and genotyping of C. albicans species complex would be a suitable way [8, 26, 27]. By physiologic and molecular tools, we are able to easily and quickly distinguish C. albicans species complex, and results showed uniform susceptibility pattern against conventional and new generation of antifungal agents which are concordant with previously published papers [9, 23, 28] and suggest the C. albicans species complex would be susceptible to all antifungal agents that are appropriate for treating vaginal candidiasis. However, resistance and susceptible dose dependency of C. africana has been reported toward voriconazole, flucytosine and terbinafine and fluconazole. In contrast in our study, all C. albicans species complex were highly susceptible to tested drugs. In fact, species-specific clinical break points (CBPs) of C. africana have not been established yet and are unknown. In conclusion, among the C. albicans species complex, predominantly C. albicans and
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rarely C. africana are isolated from vaginal mucosa, and in contrast absence of C. dubliniensis and C. stellatoidea were highlighted. Further, molecular typing will be performed to elucidate the inter- and intraspecific genomic variations of C. africana isolates. Acknowledgments This study was financially supported by a grant from the School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran, which we gratefully acknowledge. The authors acknowledge the staff of Baghban Clinic for helping with technical assistance of sampling. We are grateful to Ilse Curfs-Breuker for excellent technical assistance and help with antifungal susceptibility testings. Authors would like to thank Dr. Somayeh Dolatabadi for critically reviewing and editing the manuscript. Compliance with Ethical Standards Conflict of interest No potential conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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