Simultaneous carriage of Can&da albicans strains from HIV-infected patients with oral candidiasis: multilocus enzyme electrophoresis analysis

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1 ELSEVIER FEMS Microbiology Letters 137 (1996) Simultaneous carriage of Can&da albicans strains from HIV-infected patients with oral candidiasis: multilocus enzyme electrophoresis analysis Jacques Reynes a, Claude Pujol b, Catherine Moreau, Michkle Mall% b, Franqois Renaud, Frangois Janbon a, Jean-Marie Bastide bt * Service de Maladies Infectieuses et Tropicales, HGpifal Gui de Chauliac. Centre Hospitalier Unicersitaire, Montpellier. France Laboratoire d lmmunologie et Parasitologic, Faculte de Pharmacie, Montpellier, France L.aboratoire de Parasitologic Compare e, Unicersite des Sciences et Techniques du Languedoc Montpellier, France Received 4 January 1996; revised 8 February 1996; accepted 8 February 1996 Abstract Genetic diversity of 160 Cnndidu albicuns isolates from the oral cavity of 16 HIV-infected adults prior to antifungal treatment was assessed using multilocus enzyme electrophoresis (10 C. albicuns colonies were randomly chosen from each specimen culture). 20 electrophoretic types were distinguished from the analysis of 21 enzyme loci (10 were polymorphic). Five patients (3 1%) were found to be colonized by 2 or 3 genetically distinct strains. Nevertheless, in these five cases, one strain predominated (from 7 to 9 of the 10 colonies). Some HIV + patients with oral candidiasis appear to be simultaneously infected with several genetically different C. albicuns strains before antifungal treatment. Keywords: Multilocus enzyme electrophoresis; Candida albicans; Oral candidiasis; AIDS 1. Introduction Candida albicans and other Candida species are common components of the human oral flora. During human immunodeficiency virus (HIV) infection, progressive immunodeficiency is associated with an increase of Candida spp. oral carriage and high incidence of oropharyngeal candidiasis. C. albicans is the most frequent species involved in this mycosis [Il. A number of recent studies have applied molecu- * Corresponding author. Tel.: ; Fax: lar techniques to the analysis of oral isolates of C. albicans from HIV-infected subjects [2-71. Among these epidemiological typing methods, multilocus enzyme electrophoresis (MEE) is a reproducible and highly discriminatory technique for the genetic characterization of C. albicans strains [8,9]. The relapses of candidiasis frequently observed during the progression of AIDS may be attributed to strains of C. albicans identical to or differing from those initially present [ 10,l 11. It has been generally assumed that the infecting strain isolated in most cases is unique during a specific oral candidiasis episode [2]. When the strain involved in a previous episode of candidiasis is not encountered again, one 037%1097/96/$ Federation of European Microbiological Societies. All rights reserved PII SO (96)

2 cannot however be certain whether this strain has been eradicated or whether it has only become less representative in the oral cavity. The newly identified strain may represent a new infection subsequent to initial sampling or result from the emergence of a resistant sub-population from an originally mixed infection. At present. little is known about simultaneous multiple C. al&cans-strain infection in HIV-related oral candidiasis [5,6]. This could reflect the logistical problems of sampling and the difficulty of strain discrimination. The purpose of this study was to evaluate the potential diversity in C. ulbicans strains from HIV- I-infected patients with oral candidiasis before antifungal therapy. 2. Materials and methods 2.1. Patients 16 HIV 1 -infected adults were investigated. They were attending the AIDS outpatients Unit at the University Hospital of Montpellier, France, between December 1992 and April 1993 with a first episode of oral candidiasis. Subjects were selected on the basis of all the following criteria: (a) presence of oral signs of erythematous or pseudomembranous candidiasis; (b) when patients were admitted to the AIDS unit, cultured specimens were positive for Cundidu sp.; (c) absence of previous antifungal treatment; (d) informed consent for participation in the study. Among these 16 patients (12 men and 4 women), 13 subjects fulfilled the Centers for Disease Control criteria for AIDS; 7 were male homosexual, 6 were intravenous drug users, 3 were heterosexual. The median CD4 cell count was 88/mm3 (range: ). None of the patients were neutropenic Sampling procedure Specimens were obtained by the oral-rinse technique (each patient rinsed out his/her mouth with 10 ml of sterile water for 60 s). The oral-rinse samples obtained were diluted and plated on Sabouraud chloramphenicol agar in order to separate yeasts colonies. 12 colonies per patient were randomly chosen from each plate and identified with the API 32C yeast identification system. 14 patients were infected with C. albicans only, and 2 patients with 2 or 3 species (patient VIII: C. u/bican.s ( 1 I colonies) + C. purupsilosis (1 colony): patient VII: C. ulbicurzs (IO colonies) + C. tropicalis ( 1 colony) + C..suke (I colony)). Then, 10 C. ulbicans colonies per patient were selected for MEE analysis Multilocus enzyme electrophoresis unulysis Isolation of cytosolic extract, starch gel electrophoresis, and enzymatic assays were performed by following the protocols described elsewhere [ 121. Data were obtained for 19 enzymatic activities: malate dehydrogenase (EC I ) glucose-6- phosphate 1 -dehydrogenase (EC ) sorbitol dehydrogenase (EC ) isocitrate dehydrogenase (NADP+) (EC ), alcohol dehydrogenase (EC l.l.l.l), superoxide dismutase (EC I. 15. I. I), hexokinase (EC l), pyruvate kinase Table I Distribution of electrophoretic types (ET) among 160 C. trlhicrrrz.~ isolates from the 16 patients ET No. of isolates Patients A 39 patient II (7), patient IV (I), patient VI (I ). patient VIII (IO), patient IX (IO), patient X (IO) B 20 patient XI ( IO). patient XII (IO) c 9 patient I- I (9) D I patient I-2 ( I) E 3 patient II-2 (3) F 7 patient III- I (7) G 2 patient III-2 (2) H I patient III-3 (I ) I 9 patient IV-I (9) J 9 patient V-I (9) K I patient V-2 (I) L 8 patient VI- I (8) M I patient VI-3 ( I) N 8 patient VII- I (8) 0 I patient VII-2 (I ) P I patient VII-3 (I) Q 10 patient XIII (IO) R IO patient XIV ( IO) s IO patient XV (IO) T IO patient XVI (IO) Patients I-VII exhibited more than one ET. The number of isolates is indicated in parentheses.

3 J. Re?;nes et al. / FEMS Microbiology Letters 137 (1996) Patient III Patient IV Patient V Patient VI Patient VII Fig. 1. Gel illustrating electrophoretic variation for mannose-6- phosphate isomerase (Mpi) in several C. albicans isolates from oral cavity of 5 patients with simultaneous carriage of different strains. (EC , fumarase (EC , mannose-6- phosphate isomerase (EC ), glucose-6-phosphate isomerase (EC ). Malate dehydrogenase and hexokinase enzymatic activities were each expressed by 2 loci: Mdh-I and Mdh-2, Hk-I and Hk-2. Thus, each isolate was characterized by its combination of alleles at the 21 enzyme loci, and distinctive allele combinations were designated electrophoretic types (ETs). Allelic frequencies were used to compute genetic distances between pairs of taxa, using the index proposed by Thorpe [ 131. (E.C ), aspartate transaminase (EC , phosphoglucomutase (EC ), carboxylesterase (EC ), leucine aminopeptidase (EC >, peptidase A (EC g/substrate: Val-Leu), peptidase B (EC /substrate: Leu-Gly-Gly), peptidase D (EC /substrate: Phe-Pro), aldolase 3. Results For the 160 C. albicans isolates examined, 10 of the 21 loci investigated were polymorphic. 20 different ETs were identified (referred ET-A to ET-T) Table 2 Variability of allelic profiles of C. albicans isolates from patients I-VII Patients ET a Polymorphic Loci b Mdh-l Mdh-2 Hk-I Hk-2 G6Pd Mpi Lap Pep.B Pep.D Pk I C (9) D(1) I 2 l/2 3 I 3/4 3 3/4 /2 2 _ 2 _ II A (7) I 2 I 2/3 I 3/6 2/3 3 2 I E(3) _ 3 III F (7) G (2) H(l) IV I (9) A(l) V J (9) K(1) VI L (8) A(1) M(l) VII N (8) O(1) P(1) 1 2 I 2/3 I 3/ I _ l/2-3 l/2 2/3 3 3/4 l/2 2 l/ /6 3 3/4 2 I I _ I 2/3 I - 2/3 3 - _ 2 l/2 l/2 3 I 3 3 3/4 I I I 2 2/3 - f/2 3/s 2 3/4 2 l/2 I 2 I 3 I 5 l/3 l/ /3-3/6 2/3 3 - I - l/2 /3 l/2 l/ _ I 2 I 2 I 6 2/3 3 2 I l/2 l/3 l/2 l/3 3-2/3 - _ /2 2 3 _ 5 _ 2 I 2 a The number of isolates is indicated in parentheses. s Alleles were numbered in order of increasing anodal mobility. I, homozygote for the allele I. I /2, heterozygote presenting alleles I and 2; -, genotype identical to the one above it. Loci encode the following enzymes: Mdh, malate dehydrogenase; Hk, hexokinase: G6Pd. glucose-6-phosphate I-dehydrogenase; Mpi, mannose-6-phosphate isomerase; Lop, leucine aminopeptidase; Pep, peptidase; Pk. pyruvate kinase.

4 272 J. Rr,vnes et al. / FEMS Microhiolog~ Lettrn 137 f I YY6i (Table 1). Of these ETs, one (ET-A) was found in 6 patients and one (ET-B) was observed in 2 patients (Table 1). With the sampling procedure chosen (10 colonies of C. albicans per patient), 7 patients (patients I-VII> exhibited more than one ET (Table 2). For 3 patients (patients I-III) MEE showed isolates displaying two closely related enzyme profiles, differing only by one enzyme allele (Table 2). Five patients (patients III-VII) exhibited 2-3 ETs with at least one isolate differing in 5-8 alleles (Table 2; Fig. 1); the analysis of genetic distance showed that these isolates could be separated into distinct strains. 4. Discussion We have used the MEE typing method to assess the genetic diversity among oral isolates of C. albicans from HIV-infected patients sampled over a short period of time in the same hospital in Montpellier, with identical sampling procedure. 20 ETs were distinguished on the analysis of 21 enzyme loci (10 were polymorphic). In clinical samples from 6 subjects, C. ulbicuns had an identical ET. These patients appeared to have had no contact outside the care unit. This common electrophoretic pattern has already been identified in a larger group of HIV-infected patients during a longer study period [9]. For 3 patients, some isolates from the same clinical specimen differed only by one allele. They could derive from a unique strain (consequence of the loss of one allele by mitotic recombination or a chromosomal rearrangment sensu late) [14]. Yet, interestingly, 5 patients (patients III-VII) were found to be colonized by 2 or 3 genetically distinct strains. The random sampling of only 10 C. albicuns colonies per patient makes it quite possible that we have not identified the entire genetic diversity of the populations of Cundidu albicuns present in each patient. However, probably only a few strains with low incidence have been missed in our study. The oralrinse sampling technique leads to the collection, not only of pathogenic yeasts from candidiasis lesion sites, but perhaps also of commensal yeasts from non-lesional sites. However, a study by DNA fingerprinting of strains of C. ulbicuns from the oral cavities of HIV-seronegative individuals has shown genetic similarity of commensal and pathogenic strains [ 151. Thus, our results indicate that HIV-infected adults with oropharyngeal candidiasis and free of previous antifungal treatment are frequently infected with more than one strain of C. ulbicuns. Other investigators have reported various results regarding strain variation among isolates of C. ulbicuns from HIV + patients. Our results corroborate the recent study by Pfaller et al. [5]. Using DNA subtyping by pulsedfield gel electrophoresis, these authors found that the majority (62%) of AIDS patients with oropharyngeal candidiasis and undergoing azole therapy were infected with more than one strain of C. ulbicuns. In contrast, Miyasaki et al. [2], using the species-specific DNA probe Ca3 to track C. ulbicuns oral isolates from 30 HIV + subjects reported that no more than one strain of C. ulbicuns was isolated simultaneously from the oral cavity of a single patient. In our study, even though 5 patients were found to be colonized with more than one strain, in all these cases one strain predominated. This fact could result from intraspecies competition. If such a phenomenon exists, this could be modified by antifungal treatments. The use of MEE has allowed the demonstration of the simultaneous occurrence of several genetically different strains of C. ulbicuns in the oral cavities of HIV-infected adults. Susceptibility testing of only a single colony during an initial episode may lead to the non-detection of a second, more resistant strain, which might emerge secondarily under the selective pressure of antifungal treatment. Therefore, choosing one or a few representative colonies could lead to some misinterpretation in the evaluation of oral thrush and has implications for protocols related to the diagnosis and the treatment of C. ulbicuns infections. Considering the high frequency of recurrent oral candidiasis and the emergence of azole resistance, it is important to clarify these events leading to resistance with studies including sampling of multiple colonies [ 161. References [I] Korting, H.C., Ollert M., Georgii A. and Froschl M. (1989) In vitro susceptibilities and biotypes of Candidu ulbicans

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