mycoses
Diagnosis,Therapy and Prophylaxis of Fungal Diseases
Original article
Investigation of minor species Candida africana, Candida stellatoidea and Candida dubliniensis in the Candida albicans complex among Yaounde (Cameroon) HIV-infected patients Thierry K. Ngouana,1,2,3 Donika Krasteva,2 Pascal Drakulovski,2 Rufin K. Toghueo,3 Charles Kouanfack,1 Akaba Ambe,1 Jacques Reynes,4 Eric Delaporte,2 Fabrice F. Boyom,3 le Mallie 2 and Se bastien Bertout2 Miche 1 Clinical Biology Laboratory, Yaounde Central Hospital, Yaounde, Cameroon, 2Laboratoire de Parasitologie et Mycologie Medicale UFR Pharmacie, UMI 233 UM1-IRD-UCAD-UY1 «TransVIHMI», Universite de Montpellier 1, Montpellier, France, 3Antimicrobial Agents Unit (AMAU), Laboratory for Phytobiochemistry and Medicinal Plants Studies, Department of Biochemistry, Faculty of Science, University of Yaounde I, Yaounde, Cameroon and 4 Service des Maladies Infectieuses et Tropicales, Ho^pital Gui de Chauliac, UMI 233 UM 1-IRD-UCAD-UY1, Montpellier, France
Summary
Minor species of the Candida albicans complex may cause overestimation of the epidemiology of C. albicans, and misidentifications could mask their implication in human pathology. Authors determined the occurrence of minor species of the C. albicans complex (C. africana, C. dubliniensis and C. stellatoidea) among Yaounde HIV-infected patients, Cameroon. Stool, vaginal discharge, urine and oropharyngeal samples were analysed by mycological diagnosis. Isolates were identified by conventional methods and mass spectrometry (MS; carried out by the matrix-assisted laser desorption–ionisation time-offlight MS protocol). Candida albicans isolates were thereafter submitted to the PCR amplification of the Hwp1 gene. The susceptibility of isolates to antifungal drugs was tested using the Clinical and Laboratory Standards Institute M27-A3 protocol. From 115 C. albicans obtained isolates, neither C. dubliniensis nor C. stellatoidea was observed; two strains of C. africana (422PV and 448PV) were identified by PCR electrophoretic profiles at 700 bp. These two C. africana strains were vaginal isolates. The isolate 448PV was resistant to ketoconazole at the minimal inhibitory concentration of 2 lg ml1, and showed reduced susceptibility to amphotericin B at 1 lg ml1. This first report on C. africana occurrence in Cameroon brings clues for the understanding of the global epidemiology of this yeast as well as that of minor species of the C. albicans complex.
Key words: Candida africana, Cameroon, HIV infection, antifungal, Hwp1 gene, matrix-assisted laser desorption–ionisation time of flight.
Introduction Candida species are among the human most dreadful fungi.1–3 They are responsible for a variety of Correspondence: Sebastien Bertout, Laboratoire de Parasitologie et Mycologie Medicale, UFR de Pharmacie, 15 Avenue Charles Flahault, 34093 Montpellier Cedex 05, France. Tel.: +33 4 67 66 81 31. Fax: +33 (0)4 11759462. E-mail: [email protected] Submitted for publication 13 June 2014 Revised 31 August 2014 Accepted for publication 1 September 2014
© 2014 Blackwell Verlag GmbH Mycoses, 2015, 58, 33–39
infections that can be superficial or deep, benign or deadly.2,3 Immunocompromised persons, especially those with HIV infection are particularly susceptible to Candida species.4–6 The genus contains more than 300 species identified till date, with Candida albicans being the most encountered in human infections, and also the most investigated.4,7 Candida albicans produces germ tubes and chlamydospores; and these phenotypic characters have been used to identify the yeast7,8 until Jones and Martin identified some atypical C. albicans strains that did not assimilate sucrose and produced few chlamydospores.9 These strains were named C. stellatoidea and later subdivided into C. stellatoidea Type
doi:10.1111/myc.12266
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I and Type II.10,11 Sullivan et al. [12] identified C. dubliniensis (another species of the C. albicans complex), having inability to assimilate a-methyl-D-glucoside, lactate or xylose while being germ tube and chlamydospore positive.12–17 From the investigation of vaginal yeast isolates from African women Tietz et al. [18] have identified C. africana that showed to be germ tube positive, N-acetylglucosamine and glucosamine negative and failed to produce chlamydospores. Routine identification techniques, such as germ tube production, use of chromogenic media and the genetic amplification of the Internal Transcribed Spacer (ITS4) region of the ribosomal DNA, do not discriminate C. africana from C. albicans, generating a controversy in the classification of C. africana as a distinct species.19–21 However, the amplification of the Hwp1 gene by specific primers has enabled the differentiation between species of the C. albicans complex.1 To date, C. africana has been isolated in Madagascar, Angola, Nigeria, Senegal, United Kingdom, Italy, Germany, Spain, Saudi Arabia, Japan, USA, Chile and India19,21–26 and its impact on human pathology is under investigation. As C. albicans represents an epidemiological concern worldwide, misidentification of members of the C. albicans complex might lead to overestimation of C. albicans burden. In addition, the scarcity of data about these minor species also adds to the misunderstanding of their impact on human pathology. In Cameroon, the global epidemiology of candidiasis is scarce and useful data on C. africana, C. stellatoidea and C. dubliniensis are unavailable. This study describes the occurrence of these species among HIV-infected patients in Yaounde, Cameroon.
Materials and methods Ethical considerations and recruitment of participants
The survey was carried out at the Yaounde Central Hospital, and involved HIV-infected patients presenting or not clinical signs of any mucosal candidiasis. This study was approved by the Cameroonian National Ethical Committee. Patients enrolled for this study were HIV-infected individuals of both genders, between 18 and 65 years old, who did not receive any antifungal treatment during the last 3 months. The purpose of the study and potential benefits were explained to patients, and those willing to participate were required to sign a written inform consent form prior to their enrolment as participant.
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Determination of clinical status of patients and collection of study samples
The patient’s age, clinical signs of any mucosal candidiasis and HIV status (CD4+ count, type of HIV, stage of the HIV infection and antiretroviral therapy) were registered before the sample collection. Vaginal discharge, oropharyngeal swab, stools and urine were collected from each patient. Mycological diagnosis
Samples were submitted to direct macroscopic and microscopic analyses using routine laboratory protocols prior to culture on Sabouraud chloramphenicol medium for 24 h at 37 °C. Isolates were thereafter seeded on ChromID CAN2 medium (Biomerieux, Marcy l’Etoile, France) and incubated at 37 °C for 48 h, and colony aspect and colour were observed. Germ tube test was achieved by inoculating 0.5 ml of sheep sterile serum with a loop (5 ll) of yeast from the initial culture. After 3 h of incubation at 37 °C, a drop of the suspension was observed under light microscope. Evidence of chlamydospores production was assessed by culturing yeast on Potato-Carrot-Bile agar medium at 25 °C for 72 h followed by microscopic observation. Growth ability of these isolates at 42 °C was additionally tested using the Sabouraud chloramphenicol agar medium. Biochemical characters were studied with the ID32C kit (Biomerieux, Marcy l’Etoile, France). Reference C. albicans ATCC90028 strain was used throughout the mycological diagnosis as quality control. Identification by Mass Spectrometry
The identification of yeast strains by the matrixassisted laser desorption–ionisation time of flight (MALDI-TOF) was achieved as described by the manufacturer of the Vitek mass spectrometry (MS; Biomerieux). Briefly, yeast cells were grown on Sabouraud dextrose agar medium plates for 24 h at 37 °C. A loopful (1 ll) of yeast cells was directly transferred from the culture medium onto each position of the 48-well flex target plate, and 0.5 ll of 25% formic acid was immediately mixed with the yeast. After evaporation, 0.5 ll matrix solution (75 mg ml1 2,5-dihydroxybenzoic acid in ethanol/water/acetonitrile [1 : 1 : 1] with 0.03% trifluoroacetic acid) was added and gently mixed. All sample mixtures were airdried at room temperature. Each isolate was spotted in duplicate. Analyses were performed on a Vitek MS (Biomerieux) equipped with a nitrogen laser
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Candida africana in Yaounde HIV patients
(337 nm). The mass range from 2000 to 20 000 Da was recorded using the linear mode. An Escherichia coli ATCC 8739 strain was used for external calibration of the spectra. C. albicans ATCC 90028, C. krusei ATCC 6258, C. parapsilosis ATCC 22019, C. africana CBS 8781 and C. dubliniensis CBS 7987 were used as quality control strains. Spectra were exported to the SARAMISTM software package where the final identifications were achieved. Cluster analysis of the MALDI-TOF Intact Cell Mass Spectrometry (ICMS) mass spectral data was performed using the SARAMISTM (Spectral Archiving and Microbial Identification System, AnagnosTec, Postdam-Golm, Germany, http://www.anagnostec.eu) database by comparing database peak lists of individual samples with Super Spectra and/or reference spectra. Molecular identification of isolates
Twenty-four-hour fresh cultures from germ tube-positive isolates and reference strains (C. albicans ATCC 90028, C. africana CBS 8781, C. dubliniensis CBS 7987) were submitted to high-molecular-weight DNA extraction, using the NucleoSpin quickPure kit (Macherey-Nagel, Duren, Germany). Cells were disrupted (by glass bead beading at 6037 g for 40 s repeated three times) by a Magna lyser (Roche Diagnosis, Penzberg, Germany) in tubes containing 0.45 mm glass beads and 350 ll of the lysis buffer (2% Triton X-100, 1% SDS, 100 mmol l1 NaCl, 10 mmol l1 Tris–HCl, pH 8.0, 1 mmol l1 EDTA; Promega Co, Madison, WI, USA). Genomic DNA was extracted by incubating disrupted cells for 15 min at 70 °C in the presence of 25 ll of proteinase K and 200 ll of additional lysis buffer. DNA was thereafter suspended by adding 200 ll of absolute ethanol, centrifuged for 10 s in a short spin until 12 100 g (MiniSpin plus eppendorf). The supernatant was transferred in a silica column and centrifuged for 1 min at 11 000 g. Adsorbed DNA was washed with a buffer containing 80% ethanol and sodium chloride and dried by centrifugation (3 min, 11 000 g). DNA was finally eluted by centrifugation (1 min, 11 000 g) with 50 ll of Tris–HCl, 5 mmol l1, pH 8.5 heated at 70 °C. Extracted DNA ready to use was then titrated with a spectrophotometer and diluted at 50 ng ll1. PCR amplifications were carried out using a Techne TC-5000 thermal cycler (Global Medical Instrumentation, Minnesota, USA). The hyphal wall protein 1 (Hwp1) gene was amplified with specific primers (CR-f 50 -GCTACCACTTCAGAATCATCATC-30 /CR-r 50 GCAC CTTCAGTCGTAGAGACG-30 ) as described by Romeo
© 2014 Blackwell Verlag GmbH Mycoses, 2015, 58, 33–39
and Criseo.27 PCR reactions were performed in 50 ll mixture of 19 Taq buffer, 1.5 mmol l1 MgCl2, 0.2 mmol l1 dNTPs, 0.2 lmol l1 of each primer, 0.05 U ll1 of Go Taq DNA polymerase (Promega Co) and 1 ng ll1 DNA. Amplification conditions were as follows: Initial denaturation (95 °C, 5 min), 30 cycles of denaturation (94 °C, 45 s), primer annealing (58 °C, 40 s) and extension (72 °C, 55 s), followed by a final extension (72 °C, 10 min).27 PCR products were separated on a 1.5% agarose gel electrophoresis, stained with Ethidium bromide and visualised under UV light. A 100 bp molecular-size marker (Promega Co) was loaded to allow analysis of the PCR products. Antifungal susceptibility testing
Candida albicans complex species were thereafter submitted to antifungal susceptibility testing as described by the Clinical and Laboratory Standards Institute (CLSI) 27-A3 protocol.28 Amphotericin B, ketoconazole, fluconazole and itraconazole provided by SigmaAldrich were serially diluted in a 96-well microtitre plate with Roswell Park Memorial Institute medium (RPMI) 1640 broth medium. Final range concentrations were as follows: amphotericin B (0.0313–16 lg ml1), ketoconazole (0.0313–16 lg ml1), fluconazole (0.125–64 lg ml1) and itraconazole (0.0313–16 lg ml1). A standardised fungal inoculum (spectrophotometrically calibrated) was then added to each well for a final volume of 200 ll and a concentration of 0.5–2.5 9 103 CFU ml1. Plates were incubated at 37 °C for 48 h and Minimal Inhibitory Concentration (MIC) values read after 24 and 48 h. Candida krusei ATCC 6258 and C. parapsilosis ATCC 22019 were used as quality control strains. MIC breakpoints are defined for each antifungal; itraconazole and ketoconazole (susceptible, MIC ≤ 0.125 lg ml1; susceptible dosedependent, 0.25 ≤ MIC ≤ 0.5 lg ml1; resistant, fluconazole (susceptible, MIC ≥ 1 lg ml1), 1 susceptible dose-dependent, MIC ≤ 8 lg ml ; 16 ≤ MIC ≤ 32 lg ml1; resistant, MIC ≥ 64 lg ml1) and amphotericin B (susceptible, MIC ≤ 1 lg ml1; resistant, MIC ≥ 2 lg ml1).28
Results Phenotypic and biochemical identification of isolates
For the general survey, 402 patients were included in the study from which 1354 biological samples (296 vaginal swabs, 342 stools, 360 urines and 356 mouth swabs) were obtained and analysed. A total of 283
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(a)
albicans isolates provided PCR profiles with two bands at 800 and 941 bp and at 941 bp and 1080 bp. These strains possessed the common 941 bp band of C. albicans. They were also sucrose positive, and successfully grew at 42 °C and were chlamydospore positive. Furthermore, MALDI-TOF and ID32C analyses identified them as C. albicans.
(b)
Figure 1 Growth of Candida albicans ATCC 90028 (a) and Candida africana (b) on ChromID CAN2 medium at 37 °C for 48 h. C. africana presents a delayed growth compared to C. albicans.
Patient’s information
The C. africana isolates were isolated from vaginal discharge (422PV and 448PV). The two women, (422 and 448) from whom they came, were infected with HIV 1-M strain and were diagnosed in 2003 and 2004 respectively. Their CD4+ counts were 1146 and 746 mm3, respectively, for 422PV and 448PV. They were at the stage I of the HIV infection as per the previous classification, and were under antiretroviral therapy using tenofovir, lamivudine and efavirenz. Their body mass indexes were 31.02 and 29.75 and they aged 43 and 35 years for 422 and 448 respectively. The patient 422 was suffering from vaginal itching since 2 weeks and no clinical sign of vaginal infection was noticed for the other patient.
isolates were obtained and exploited due to their quantification range in favour of colonisation as described by Bouchara et al. [29] Of these isolates, 115 were germ tube positive and presented a blue colour on the chromogenic medium. They were therefore classified as C. albicans complex species. Further analyses effectively identified 113 strains as C. albicans with the ID32C biochemical test and that successfully grew at 42 °C. Two other strains failed to grow at 42 °C and could not be identified on the basis of their biochemical parameters. Their identification code on ID32C was 7046340011 and they did not assimilate N-acetyl-glucosamine, glucosamine, trehalose and DLlactate. They also presented delayed growth on the chromogenic medium as shown in Fig. 1.
Antifungal susceptibility profile
The MIC ranges at 48 h of antifungals against C. albicans complex species were as follows: Amphotericin B (0.015–2 lg ml1); Ketoconazole (0.03 to >16 lg ml1); Itraconazole (0.03 to >16 lg ml1); Fluconazole (0.125 to >64). The two strains of C. africana appeared to be susceptible to tested antifungals except strain 448PV that was resistant to ketoconazole as observed in Table 1.
MS and molecular identification
The MALDI-TOF analysis of the 115 C. albicans isolates identified 113 strains as being C. albicans and two strains as C. africana (422PV and 448PV). These strains also provided PCR profiles at 700 bp, similar to that of the reference C. africana strain (Fig. 2). Ten C.
(a)
(b)
Figure 2 Electrophoretic profile of Hwp1 amplification. M is the molecular weight marker; from picture a, CA, Candida albicans is at
941 bp. CD, C. dubliniensis is about 569 bp, CAf, C. africana is about 700 bp; Lanes I and II are isolates identified as C. africana based on their identical profile with the reference C. africana strain. From picture b, Lanes 1 and 2 have a similar profile (800 and 941 bp), different from that of Lane 3 (941 and 1080 bp) and Lanes 5–7 (941 bp). All these strains that have the common 941 bp profile (to C. albicans) were identified as C. albicans by conventional methods and matrix-assisted laser desorption–ionisation time-of-flight analysis. Lane 4 is C. africana. Arrows indicate C. africana PCR profiles.
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Candida africana in Yaounde HIV patients
Table 1 Minimal inhibitory concentrations of the two isolated Candida africana strains. Minimal inhibitory concentration (lg/ml) Ketoconazole (h)
Strain 422PV Strain 448PV
Amphotericin B (h)
Fluconazole (h)
Itraconazole (h)
24
48
24
48
24
48
24
48
0.03 2
0.06 2
0.5 1
1 1
0.25 1
1 2
0.03 0.125
0.06 0.125
Isolates were tested for susceptibility to standard drugs in vitro. Minimal inhibitory concentration is the lowest drug concentration showing no visible growth (for amphotericin B) or 50% growth inhibition (for azoles) of the tested yeast.
Discussion The C. albicans complex is one of the major fungal groups, which is involved in more than 50% of Candida infections,30 therefore pointing out their significant burden among human beings. Species of this group are more often involved in human fungal infections, thereby justifying the interest of studying them. C. dubliniensis and C. stellatoidea Type I were not identified in this study. However, Longdoh et al. [30] recently identified C. dubliniensis in the oral cavity of some HIV-infected patients in Buea, Cameroon by phenotypic and biochemical analyses. The identification of C. dubliniensis by biochemical profiles is now available in some commercial kits, since the yeast has the inability to assimilate a-methyl-D-glucoside, lactate or xylose.12 Moreover, the yeast can be detected by the latex agglutination test, Bichro-Dubli of Fumouze diagnosis.13 C. stellatoidea has not yet been described in Cameroon. However, since C. stellatoidea is a sucrosenegative mutant of C. albicans, only molecular approaches can efficiently separate C. stellatoidea Type I from C. stellatoidea Type II.7,8 The two species can be identified by electrocaryotyping.11 In this study, we report for the first time the occurrence of C. africana among HIV-infected patients in Cameroon. The isolation of the yeast in vaginal fluid is in accordance with what has been found previously,31 supporting the vaginal mucosa as a particular niche for C. africana. The patients’ CD4+ counts were higher enough to not consider the immunosuppression as being involved in the presence of the yeast. In fact, it has already been shown that C. africana might colonise mucosae without a real implication in an infection.20,31 The patient with itching also presented Gardnerella vaginalis, a bacterium that colonises vaginal mucosa, thereby destroying Lactobacillus, providing a free space for the development of opportunistic pathogens such as Candida species.32 Gardnerella vaginalis also creates vaginosis which is associated with clinical signs as itching.32
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C. africana has been demonstrated as being less pathogenic than C. albicans using Galleria mellonella larvae infection model.21,31 Phenotypic characteristics that were observed for these two strains are close to those previously described 23–25,33 and discriminate clearly the yeast from the classic C. albicans. For instance, the ID32C biochemical codes obtained for the two isolates are identical to the codes published previously.34 Since the publication of the Hwp1 as a particular target for differentiation of the C. albicans complex species, the identification of C. africana has been achieved.26 This molecular approach helped to confirm the atypical C. albicans strains as being identical to previously described C. africana.1,20,27 Moreover, by this approach, the identification of C. albicans seems to be disputable since the heterozygotes or aneuploid strains for the amplified gene might present different PCR profiles as we observed in the present work. C. africana has been proposed as a particular species of the C. albicans complex for a number of reasons including biochemical (no assimilation of glucosamine and N-acetylglucosamine), phenotypic (germ tube positive and chlamydospore negative) and some genetic characteristics (differences in Hwp1 PCR profiles) that are clearly different from those of C. albicans.18–20,22 In addition, C. africana has a particular location in the vaginal mucosa, while C. albicans is found almost everywhere. However, Multi Locus Sequence Typing (MLST) studies of the C. albicans complex presented C. africana co-clustered with C. stellatoidea, thereby arguing no separation of C. africana from C. albicans.7 Recently, Borman et al. [21] differentiated C. albicans from C. africana by pyrosequencing a portion of the ITS2, and proposed the latter to be considered as a particular species. C. africana isolates have been described as being susceptible to current antifungal drugs.20,21 However, 448PV strain presented a significantly reduced susceptibility to ketoconazole and was categorised resistant according to the criteria of the CLSI M27-A3 protocol
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that described Candida species to be resistant to this drug when the MIC is greater than 1 lg ml1.28 To a lesser extent, the strain also showed reduced susceptibility to amphotericin B, underlying the need for detailed molecular investigations to elucidate the mechanisms of the observed resistance.
Conclusion This work was designed to search for minor species of the C. albicans complex (C. africana, C. dubliniensis and C. stellatoidea) among HIV-infected patients in Yaounde. From the study sample population, results highlighted the absence of C. dubliniensis and C. stellatoidea Type I, but the presence of C. africana in Yaounde and among HIV-infected patients. The vaginal mucosa was the preferential niche for this yeast. One of the C. africana strains (448PV) with reduced susceptibility to ketoconazole and amphotericin B is of particular interest since no case of resistance has been described for C. africana to date. Further detailed molecular studies will be performed to elucidate the mechanisms of resistance adopted by this particular strain.
Acknowledgements The authors are thankful to Dominic Castel for its technical help. We also thank the staff of the Yaounde Central Hospital for their logistic support.
Conflict of interest No conflict of interest to be mentioned.
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Diagnosis,Therapy and Prophylaxis of Fungal Diseases
Original article
Investigation of minor species Candida africana, Candida stellatoidea and Candida dubliniensis in the Candida albicans complex among Yaounde (Cameroon) HIV-infected patients Thierry K. Ngouana,1,2,3 Donika Krasteva,2 Pascal Drakulovski,2 Rufin K. Toghueo,3 Charles Kouanfack,1 Akaba Ambe,1 Jacques Reynes,4 Eric Delaporte,2 Fabrice F. Boyom,3 le Mallie 2 and Se bastien Bertout2 Miche 1 Clinical Biology Laboratory, Yaounde Central Hospital, Yaounde, Cameroon, 2Laboratoire de Parasitologie et Mycologie Medicale UFR Pharmacie, UMI 233 UM1-IRD-UCAD-UY1 «TransVIHMI», Universite de Montpellier 1, Montpellier, France, 3Antimicrobial Agents Unit (AMAU), Laboratory for Phytobiochemistry and Medicinal Plants Studies, Department of Biochemistry, Faculty of Science, University of Yaounde I, Yaounde, Cameroon and 4 Service des Maladies Infectieuses et Tropicales, Ho^pital Gui de Chauliac, UMI 233 UM 1-IRD-UCAD-UY1, Montpellier, France
Summary
Minor species of the Candida albicans complex may cause overestimation of the epidemiology of C. albicans, and misidentifications could mask their implication in human pathology. Authors determined the occurrence of minor species of the C. albicans complex (C. africana, C. dubliniensis and C. stellatoidea) among Yaounde HIV-infected patients, Cameroon. Stool, vaginal discharge, urine and oropharyngeal samples were analysed by mycological diagnosis. Isolates were identified by conventional methods and mass spectrometry (MS; carried out by the matrix-assisted laser desorption–ionisation time-offlight MS protocol). Candida albicans isolates were thereafter submitted to the PCR amplification of the Hwp1 gene. The susceptibility of isolates to antifungal drugs was tested using the Clinical and Laboratory Standards Institute M27-A3 protocol. From 115 C. albicans obtained isolates, neither C. dubliniensis nor C. stellatoidea was observed; two strains of C. africana (422PV and 448PV) were identified by PCR electrophoretic profiles at 700 bp. These two C. africana strains were vaginal isolates. The isolate 448PV was resistant to ketoconazole at the minimal inhibitory concentration of 2 lg ml1, and showed reduced susceptibility to amphotericin B at 1 lg ml1. This first report on C. africana occurrence in Cameroon brings clues for the understanding of the global epidemiology of this yeast as well as that of minor species of the C. albicans complex.
Key words: Candida africana, Cameroon, HIV infection, antifungal, Hwp1 gene, matrix-assisted laser desorption–ionisation time of flight.
Introduction Candida species are among the human most dreadful fungi.1–3 They are responsible for a variety of Correspondence: Sebastien Bertout, Laboratoire de Parasitologie et Mycologie Medicale, UFR de Pharmacie, 15 Avenue Charles Flahault, 34093 Montpellier Cedex 05, France. Tel.: +33 4 67 66 81 31. Fax: +33 (0)4 11759462. E-mail: [email protected] Submitted for publication 13 June 2014 Revised 31 August 2014 Accepted for publication 1 September 2014
© 2014 Blackwell Verlag GmbH Mycoses, 2015, 58, 33–39
infections that can be superficial or deep, benign or deadly.2,3 Immunocompromised persons, especially those with HIV infection are particularly susceptible to Candida species.4–6 The genus contains more than 300 species identified till date, with Candida albicans being the most encountered in human infections, and also the most investigated.4,7 Candida albicans produces germ tubes and chlamydospores; and these phenotypic characters have been used to identify the yeast7,8 until Jones and Martin identified some atypical C. albicans strains that did not assimilate sucrose and produced few chlamydospores.9 These strains were named C. stellatoidea and later subdivided into C. stellatoidea Type
doi:10.1111/myc.12266
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I and Type II.10,11 Sullivan et al. [12] identified C. dubliniensis (another species of the C. albicans complex), having inability to assimilate a-methyl-D-glucoside, lactate or xylose while being germ tube and chlamydospore positive.12–17 From the investigation of vaginal yeast isolates from African women Tietz et al. [18] have identified C. africana that showed to be germ tube positive, N-acetylglucosamine and glucosamine negative and failed to produce chlamydospores. Routine identification techniques, such as germ tube production, use of chromogenic media and the genetic amplification of the Internal Transcribed Spacer (ITS4) region of the ribosomal DNA, do not discriminate C. africana from C. albicans, generating a controversy in the classification of C. africana as a distinct species.19–21 However, the amplification of the Hwp1 gene by specific primers has enabled the differentiation between species of the C. albicans complex.1 To date, C. africana has been isolated in Madagascar, Angola, Nigeria, Senegal, United Kingdom, Italy, Germany, Spain, Saudi Arabia, Japan, USA, Chile and India19,21–26 and its impact on human pathology is under investigation. As C. albicans represents an epidemiological concern worldwide, misidentification of members of the C. albicans complex might lead to overestimation of C. albicans burden. In addition, the scarcity of data about these minor species also adds to the misunderstanding of their impact on human pathology. In Cameroon, the global epidemiology of candidiasis is scarce and useful data on C. africana, C. stellatoidea and C. dubliniensis are unavailable. This study describes the occurrence of these species among HIV-infected patients in Yaounde, Cameroon.
Materials and methods Ethical considerations and recruitment of participants
The survey was carried out at the Yaounde Central Hospital, and involved HIV-infected patients presenting or not clinical signs of any mucosal candidiasis. This study was approved by the Cameroonian National Ethical Committee. Patients enrolled for this study were HIV-infected individuals of both genders, between 18 and 65 years old, who did not receive any antifungal treatment during the last 3 months. The purpose of the study and potential benefits were explained to patients, and those willing to participate were required to sign a written inform consent form prior to their enrolment as participant.
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Determination of clinical status of patients and collection of study samples
The patient’s age, clinical signs of any mucosal candidiasis and HIV status (CD4+ count, type of HIV, stage of the HIV infection and antiretroviral therapy) were registered before the sample collection. Vaginal discharge, oropharyngeal swab, stools and urine were collected from each patient. Mycological diagnosis
Samples were submitted to direct macroscopic and microscopic analyses using routine laboratory protocols prior to culture on Sabouraud chloramphenicol medium for 24 h at 37 °C. Isolates were thereafter seeded on ChromID CAN2 medium (Biomerieux, Marcy l’Etoile, France) and incubated at 37 °C for 48 h, and colony aspect and colour were observed. Germ tube test was achieved by inoculating 0.5 ml of sheep sterile serum with a loop (5 ll) of yeast from the initial culture. After 3 h of incubation at 37 °C, a drop of the suspension was observed under light microscope. Evidence of chlamydospores production was assessed by culturing yeast on Potato-Carrot-Bile agar medium at 25 °C for 72 h followed by microscopic observation. Growth ability of these isolates at 42 °C was additionally tested using the Sabouraud chloramphenicol agar medium. Biochemical characters were studied with the ID32C kit (Biomerieux, Marcy l’Etoile, France). Reference C. albicans ATCC90028 strain was used throughout the mycological diagnosis as quality control. Identification by Mass Spectrometry
The identification of yeast strains by the matrixassisted laser desorption–ionisation time of flight (MALDI-TOF) was achieved as described by the manufacturer of the Vitek mass spectrometry (MS; Biomerieux). Briefly, yeast cells were grown on Sabouraud dextrose agar medium plates for 24 h at 37 °C. A loopful (1 ll) of yeast cells was directly transferred from the culture medium onto each position of the 48-well flex target plate, and 0.5 ll of 25% formic acid was immediately mixed with the yeast. After evaporation, 0.5 ll matrix solution (75 mg ml1 2,5-dihydroxybenzoic acid in ethanol/water/acetonitrile [1 : 1 : 1] with 0.03% trifluoroacetic acid) was added and gently mixed. All sample mixtures were airdried at room temperature. Each isolate was spotted in duplicate. Analyses were performed on a Vitek MS (Biomerieux) equipped with a nitrogen laser
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Candida africana in Yaounde HIV patients
(337 nm). The mass range from 2000 to 20 000 Da was recorded using the linear mode. An Escherichia coli ATCC 8739 strain was used for external calibration of the spectra. C. albicans ATCC 90028, C. krusei ATCC 6258, C. parapsilosis ATCC 22019, C. africana CBS 8781 and C. dubliniensis CBS 7987 were used as quality control strains. Spectra were exported to the SARAMISTM software package where the final identifications were achieved. Cluster analysis of the MALDI-TOF Intact Cell Mass Spectrometry (ICMS) mass spectral data was performed using the SARAMISTM (Spectral Archiving and Microbial Identification System, AnagnosTec, Postdam-Golm, Germany, http://www.anagnostec.eu) database by comparing database peak lists of individual samples with Super Spectra and/or reference spectra. Molecular identification of isolates
Twenty-four-hour fresh cultures from germ tube-positive isolates and reference strains (C. albicans ATCC 90028, C. africana CBS 8781, C. dubliniensis CBS 7987) were submitted to high-molecular-weight DNA extraction, using the NucleoSpin quickPure kit (Macherey-Nagel, Duren, Germany). Cells were disrupted (by glass bead beading at 6037 g for 40 s repeated three times) by a Magna lyser (Roche Diagnosis, Penzberg, Germany) in tubes containing 0.45 mm glass beads and 350 ll of the lysis buffer (2% Triton X-100, 1% SDS, 100 mmol l1 NaCl, 10 mmol l1 Tris–HCl, pH 8.0, 1 mmol l1 EDTA; Promega Co, Madison, WI, USA). Genomic DNA was extracted by incubating disrupted cells for 15 min at 70 °C in the presence of 25 ll of proteinase K and 200 ll of additional lysis buffer. DNA was thereafter suspended by adding 200 ll of absolute ethanol, centrifuged for 10 s in a short spin until 12 100 g (MiniSpin plus eppendorf). The supernatant was transferred in a silica column and centrifuged for 1 min at 11 000 g. Adsorbed DNA was washed with a buffer containing 80% ethanol and sodium chloride and dried by centrifugation (3 min, 11 000 g). DNA was finally eluted by centrifugation (1 min, 11 000 g) with 50 ll of Tris–HCl, 5 mmol l1, pH 8.5 heated at 70 °C. Extracted DNA ready to use was then titrated with a spectrophotometer and diluted at 50 ng ll1. PCR amplifications were carried out using a Techne TC-5000 thermal cycler (Global Medical Instrumentation, Minnesota, USA). The hyphal wall protein 1 (Hwp1) gene was amplified with specific primers (CR-f 50 -GCTACCACTTCAGAATCATCATC-30 /CR-r 50 GCAC CTTCAGTCGTAGAGACG-30 ) as described by Romeo
© 2014 Blackwell Verlag GmbH Mycoses, 2015, 58, 33–39
and Criseo.27 PCR reactions were performed in 50 ll mixture of 19 Taq buffer, 1.5 mmol l1 MgCl2, 0.2 mmol l1 dNTPs, 0.2 lmol l1 of each primer, 0.05 U ll1 of Go Taq DNA polymerase (Promega Co) and 1 ng ll1 DNA. Amplification conditions were as follows: Initial denaturation (95 °C, 5 min), 30 cycles of denaturation (94 °C, 45 s), primer annealing (58 °C, 40 s) and extension (72 °C, 55 s), followed by a final extension (72 °C, 10 min).27 PCR products were separated on a 1.5% agarose gel electrophoresis, stained with Ethidium bromide and visualised under UV light. A 100 bp molecular-size marker (Promega Co) was loaded to allow analysis of the PCR products. Antifungal susceptibility testing
Candida albicans complex species were thereafter submitted to antifungal susceptibility testing as described by the Clinical and Laboratory Standards Institute (CLSI) 27-A3 protocol.28 Amphotericin B, ketoconazole, fluconazole and itraconazole provided by SigmaAldrich were serially diluted in a 96-well microtitre plate with Roswell Park Memorial Institute medium (RPMI) 1640 broth medium. Final range concentrations were as follows: amphotericin B (0.0313–16 lg ml1), ketoconazole (0.0313–16 lg ml1), fluconazole (0.125–64 lg ml1) and itraconazole (0.0313–16 lg ml1). A standardised fungal inoculum (spectrophotometrically calibrated) was then added to each well for a final volume of 200 ll and a concentration of 0.5–2.5 9 103 CFU ml1. Plates were incubated at 37 °C for 48 h and Minimal Inhibitory Concentration (MIC) values read after 24 and 48 h. Candida krusei ATCC 6258 and C. parapsilosis ATCC 22019 were used as quality control strains. MIC breakpoints are defined for each antifungal; itraconazole and ketoconazole (susceptible, MIC ≤ 0.125 lg ml1; susceptible dosedependent, 0.25 ≤ MIC ≤ 0.5 lg ml1; resistant, fluconazole (susceptible, MIC ≥ 1 lg ml1), 1 susceptible dose-dependent, MIC ≤ 8 lg ml ; 16 ≤ MIC ≤ 32 lg ml1; resistant, MIC ≥ 64 lg ml1) and amphotericin B (susceptible, MIC ≤ 1 lg ml1; resistant, MIC ≥ 2 lg ml1).28
Results Phenotypic and biochemical identification of isolates
For the general survey, 402 patients were included in the study from which 1354 biological samples (296 vaginal swabs, 342 stools, 360 urines and 356 mouth swabs) were obtained and analysed. A total of 283
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(a)
albicans isolates provided PCR profiles with two bands at 800 and 941 bp and at 941 bp and 1080 bp. These strains possessed the common 941 bp band of C. albicans. They were also sucrose positive, and successfully grew at 42 °C and were chlamydospore positive. Furthermore, MALDI-TOF and ID32C analyses identified them as C. albicans.
(b)
Figure 1 Growth of Candida albicans ATCC 90028 (a) and Candida africana (b) on ChromID CAN2 medium at 37 °C for 48 h. C. africana presents a delayed growth compared to C. albicans.
Patient’s information
The C. africana isolates were isolated from vaginal discharge (422PV and 448PV). The two women, (422 and 448) from whom they came, were infected with HIV 1-M strain and were diagnosed in 2003 and 2004 respectively. Their CD4+ counts were 1146 and 746 mm3, respectively, for 422PV and 448PV. They were at the stage I of the HIV infection as per the previous classification, and were under antiretroviral therapy using tenofovir, lamivudine and efavirenz. Their body mass indexes were 31.02 and 29.75 and they aged 43 and 35 years for 422 and 448 respectively. The patient 422 was suffering from vaginal itching since 2 weeks and no clinical sign of vaginal infection was noticed for the other patient.
isolates were obtained and exploited due to their quantification range in favour of colonisation as described by Bouchara et al. [29] Of these isolates, 115 were germ tube positive and presented a blue colour on the chromogenic medium. They were therefore classified as C. albicans complex species. Further analyses effectively identified 113 strains as C. albicans with the ID32C biochemical test and that successfully grew at 42 °C. Two other strains failed to grow at 42 °C and could not be identified on the basis of their biochemical parameters. Their identification code on ID32C was 7046340011 and they did not assimilate N-acetyl-glucosamine, glucosamine, trehalose and DLlactate. They also presented delayed growth on the chromogenic medium as shown in Fig. 1.
Antifungal susceptibility profile
The MIC ranges at 48 h of antifungals against C. albicans complex species were as follows: Amphotericin B (0.015–2 lg ml1); Ketoconazole (0.03 to >16 lg ml1); Itraconazole (0.03 to >16 lg ml1); Fluconazole (0.125 to >64). The two strains of C. africana appeared to be susceptible to tested antifungals except strain 448PV that was resistant to ketoconazole as observed in Table 1.
MS and molecular identification
The MALDI-TOF analysis of the 115 C. albicans isolates identified 113 strains as being C. albicans and two strains as C. africana (422PV and 448PV). These strains also provided PCR profiles at 700 bp, similar to that of the reference C. africana strain (Fig. 2). Ten C.
(a)
(b)
Figure 2 Electrophoretic profile of Hwp1 amplification. M is the molecular weight marker; from picture a, CA, Candida albicans is at
941 bp. CD, C. dubliniensis is about 569 bp, CAf, C. africana is about 700 bp; Lanes I and II are isolates identified as C. africana based on their identical profile with the reference C. africana strain. From picture b, Lanes 1 and 2 have a similar profile (800 and 941 bp), different from that of Lane 3 (941 and 1080 bp) and Lanes 5–7 (941 bp). All these strains that have the common 941 bp profile (to C. albicans) were identified as C. albicans by conventional methods and matrix-assisted laser desorption–ionisation time-of-flight analysis. Lane 4 is C. africana. Arrows indicate C. africana PCR profiles.
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Table 1 Minimal inhibitory concentrations of the two isolated Candida africana strains. Minimal inhibitory concentration (lg/ml) Ketoconazole (h)
Strain 422PV Strain 448PV
Amphotericin B (h)
Fluconazole (h)
Itraconazole (h)
24
48
24
48
24
48
24
48
0.03 2
0.06 2
0.5 1
1 1
0.25 1
1 2
0.03 0.125
0.06 0.125
Isolates were tested for susceptibility to standard drugs in vitro. Minimal inhibitory concentration is the lowest drug concentration showing no visible growth (for amphotericin B) or 50% growth inhibition (for azoles) of the tested yeast.
Discussion The C. albicans complex is one of the major fungal groups, which is involved in more than 50% of Candida infections,30 therefore pointing out their significant burden among human beings. Species of this group are more often involved in human fungal infections, thereby justifying the interest of studying them. C. dubliniensis and C. stellatoidea Type I were not identified in this study. However, Longdoh et al. [30] recently identified C. dubliniensis in the oral cavity of some HIV-infected patients in Buea, Cameroon by phenotypic and biochemical analyses. The identification of C. dubliniensis by biochemical profiles is now available in some commercial kits, since the yeast has the inability to assimilate a-methyl-D-glucoside, lactate or xylose.12 Moreover, the yeast can be detected by the latex agglutination test, Bichro-Dubli of Fumouze diagnosis.13 C. stellatoidea has not yet been described in Cameroon. However, since C. stellatoidea is a sucrosenegative mutant of C. albicans, only molecular approaches can efficiently separate C. stellatoidea Type I from C. stellatoidea Type II.7,8 The two species can be identified by electrocaryotyping.11 In this study, we report for the first time the occurrence of C. africana among HIV-infected patients in Cameroon. The isolation of the yeast in vaginal fluid is in accordance with what has been found previously,31 supporting the vaginal mucosa as a particular niche for C. africana. The patients’ CD4+ counts were higher enough to not consider the immunosuppression as being involved in the presence of the yeast. In fact, it has already been shown that C. africana might colonise mucosae without a real implication in an infection.20,31 The patient with itching also presented Gardnerella vaginalis, a bacterium that colonises vaginal mucosa, thereby destroying Lactobacillus, providing a free space for the development of opportunistic pathogens such as Candida species.32 Gardnerella vaginalis also creates vaginosis which is associated with clinical signs as itching.32
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C. africana has been demonstrated as being less pathogenic than C. albicans using Galleria mellonella larvae infection model.21,31 Phenotypic characteristics that were observed for these two strains are close to those previously described 23–25,33 and discriminate clearly the yeast from the classic C. albicans. For instance, the ID32C biochemical codes obtained for the two isolates are identical to the codes published previously.34 Since the publication of the Hwp1 as a particular target for differentiation of the C. albicans complex species, the identification of C. africana has been achieved.26 This molecular approach helped to confirm the atypical C. albicans strains as being identical to previously described C. africana.1,20,27 Moreover, by this approach, the identification of C. albicans seems to be disputable since the heterozygotes or aneuploid strains for the amplified gene might present different PCR profiles as we observed in the present work. C. africana has been proposed as a particular species of the C. albicans complex for a number of reasons including biochemical (no assimilation of glucosamine and N-acetylglucosamine), phenotypic (germ tube positive and chlamydospore negative) and some genetic characteristics (differences in Hwp1 PCR profiles) that are clearly different from those of C. albicans.18–20,22 In addition, C. africana has a particular location in the vaginal mucosa, while C. albicans is found almost everywhere. However, Multi Locus Sequence Typing (MLST) studies of the C. albicans complex presented C. africana co-clustered with C. stellatoidea, thereby arguing no separation of C. africana from C. albicans.7 Recently, Borman et al. [21] differentiated C. albicans from C. africana by pyrosequencing a portion of the ITS2, and proposed the latter to be considered as a particular species. C. africana isolates have been described as being susceptible to current antifungal drugs.20,21 However, 448PV strain presented a significantly reduced susceptibility to ketoconazole and was categorised resistant according to the criteria of the CLSI M27-A3 protocol
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that described Candida species to be resistant to this drug when the MIC is greater than 1 lg ml1.28 To a lesser extent, the strain also showed reduced susceptibility to amphotericin B, underlying the need for detailed molecular investigations to elucidate the mechanisms of the observed resistance.
Conclusion This work was designed to search for minor species of the C. albicans complex (C. africana, C. dubliniensis and C. stellatoidea) among HIV-infected patients in Yaounde. From the study sample population, results highlighted the absence of C. dubliniensis and C. stellatoidea Type I, but the presence of C. africana in Yaounde and among HIV-infected patients. The vaginal mucosa was the preferential niche for this yeast. One of the C. africana strains (448PV) with reduced susceptibility to ketoconazole and amphotericin B is of particular interest since no case of resistance has been described for C. africana to date. Further detailed molecular studies will be performed to elucidate the mechanisms of resistance adopted by this particular strain.
Acknowledgements The authors are thankful to Dominic Castel for its technical help. We also thank the staff of the Yaounde Central Hospital for their logistic support.
Conflict of interest No conflict of interest to be mentioned.
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