Multilocus enzyme electrophoresis typing of Candida albicans populations isolated from healthy children according to socioeconomic background

Tipagem de populacões de Candida albicans isoladas de crianças saudáveis que apresentam um fundo socioeconômico por eletroforese de enzima multiloco

ORIGINAL ARTICLES

Multilocus enzyme electrophoresis typing of Candida albicans populations isolated from healthy children according to socioeconomic background

Tipagem de populacões de Candida albicans isoladas de crianças saudáveis que apresentam um fundo socioeconômico por eletroforese de enzima multiloco

Marcelo Fabiano Gomes Boriollo^*, I; Edvaldo Antônio Ribeiro Rosa^II; Wagner Luis de Carvalho Bernardo^I; Denise Madalena Palomari Spolidorio^III; Reginaldo Bruno Gonçalves^I; José Francisco Höfling^I

^IMicrobiology and Immunology Laboratory, Dental School of Piracicaba, State University of Campinas, Piracicaba, Brazil

^IIStomatology Laboratory, Center of Biological and Health Sciences, Pontifical Catholic University of Paraná, Curitiba, Brazil

^IIIDepartment of Physiology and Pathology, School of Dentistry, Paulista State University, Araraquara, São Paulo, Brazil

ABSTRACT

The aim of this research was to evaluate the genetic diversity within and between C. albicans populations isolated from the oral cavity of healthy Brazilian children classified into five socioeconomic categories (A to E). Multilocus Enzyme Electrophoresis (MLEE) analysis was the method used to assess genetic diversity. High genetic diversity was observed in all populations that showed predominance of some C. albicans subtypes (Electrophoretic Types – ETs). However, no correlation was observed between a specific ET and a specific population of children. Clustering analysis showed one or more highly related ET clusters, suggesting the existence of indirect and direct propagation routes of C. albicans among healthy children. Microevolutionary changes were observed in some C. albicans populations isolated from children with the same or very similar socioeconomic condition. Furthermore, low transition of C. albicans subtypes can be occurring among certain populations of children coming from high and medium/high, or high and medium/low, or medium/high and medium/low socioeconomic categories, which can also be explained by their own socioeconomic and cultural characteristics.

Key Words:Candida albicans. MLEE. Genetic diversity. Healthy children. Socioeconomic category.

RESUMO

O objetivo desta pesquisa foi avaliar o grau de diversidade genética dentro e entre populações de C. albicans isoladas da cavidade bucal de crianças saudáveis brasileiras classificadas em cinco categorias socioeconômicas (A até E), através da análise de Eletroforese de Enzimas Multilocos (MLEE). Alta diversidade genética foi observada em todas as populações, as quais mostraram predominância de alguns subtipos de C. albicans (Tipos Eletroforéticos – ETs). Contudo, nenhuma correlação foi observada entre ET-específico e população-específica de crianças. A existência de um ou mais grupos de ET altamente relacionados foi mostrada pela análise de agrupamento, o que sugere a existência de rotas de propagação direta e indireta de C. albicans entre crianças saudáveis. Alterações microevolucionárias foram observadas em algumas populações de C. albicans isoladas de crianças que tiveram a mesma, ou muito próxima, condição socioeconômica. Além disso, baixa transição de subtipos de C. albicans podem estar ocorrendo entre certas populações de crianças provenientes de alta e média/alta, ou alta e média/baixa, ou média/alta e média/baixa, categorias socioeconômicas, o que pode ser esclarecido pelas suas próprias características socioeconômica e cultural.

Palavras-chave:Candida albicans. MLEE. Diversidade genética. Crianças saudáveis. Categoria socioeconômica.

Introduction

Candida albicans and related species are found ubiquitously and commensally in the microbiota of human cavities (rectal, oral, vaginal, urethral, nasal, and aural) and skin¹. The reasons of their existence in the microbiota of healthy people remain unknown. However, nutritional factors, interactions with bacterial microbiota, and the presence of salivary antibodies were suggested to influence the incidence of those yeasts². In addition, these species are considered opportunistic pathogens capable of causing infections, varying from harmless mucocutaneous disorders to the individual up to invasive diseases involving almost all organs. The frequency of infections caused by Candida has been increasing worldwide due to a multiplicity of predisposing factors (AIDS, diabetes, leukemia, cancer...)^3,4, which facilitates the conversion of the commensal form to the parasitic existence^5,6. The increase of these infections has been associated with immunological deficiencies according to the observations of various cases of oropharyngeal candidiasis in patients with AIDS⁷. The progression of the colonization for infection in mucous membranes was referred as a process that depends on the host defense mechanism and on the ability of Candida spp. to overcome such mechanism⁸.

There has been strong interest in acquiring better understanding of the pathogenesis, epidemiology, genetics and outcome of infections caused by C. albicans. This has led to the development of extensive research, employing fingerprinting methods such as Multilocus Enzyme Electrophoresis (MLEE)^9-16, Random Amplified Polymorphic DNA (RAPD)^15,17, Restriction Endonucleases Analysis (REA)^18,19, Southern Blot hybridization with the Ca3 probe ^15,20-23, and Electrophoretic Karyotyping (EK)^24,25. Strain delineation by MLEE has permitted evaluating the genetic structure and diversity of populations^26,27, and has provided high discriminatory power and reproducibility^12,15,26-29. Considered neutral markers (invariable when they suffer environment selective pressures), metabolic isoenzymes present great potentiality in the taxonomic, systematic, genetic, evolution and epidemiologic characterization of C. albicans and other yeasts of medical importance^9-16,30-40.

The aim of this research was to evaluate by MLEE and clustering analysis, the genetic diversity in C. albicans populations isolated from the oral cavity of healthy Brazilian children classified into five socioeconomic categories (A, B, C, D, and E). Concisely, the results permitted evaluating (i) the genetic diversity degrees among isolates in each population, (ii) the existence of subtypes and highly related isolate clusters, (iii) the distribution and prevalence of these subtypes and highly related isolates clusters in each population, (iv) non-existing correlation between subtypes or isolate clusters and a population of healthy children (different socioeconomic categories), and (v) microevolution within and between isolate populations.

Material and Methods

Population. The study involved 75 C. albicans samples isolated from the oral cavity of 75 clinically healthy children (randomly isolated), with ages varying between six and nine years, of both genders, classified into 5 socioeconomic categories (A = 19, B = 17, C = 15, D = 12, and E = 12) according to the criteria adopted by the Brazilian Association of Advertisers and by the Brazilian Institute of Market Research (ABA/ABIPEME), from the municipal district of Piracicaba, State of São Paulo, Brazil⁴¹. Isolates were previously identified⁴¹ in our laboratory (tube germ formation, chlamydospore test, growth in chromogenic medium CHROMagar Candida^®, and carbohydrate assimilation and fermentation test), and the prevalence of C. albicans (approximately 47% of the total population studied – approximately 2% of non-C. albicans) did not differ substantially between groups A (central area), B (central area and/or outlying area), C (central area and/or outlying area), D (outlying area), and E (outlying area)⁴¹.

Cellular extract preparation. Yeast cultures were grown in flasks containing 50mL of YEPD medium (yeast extract 1% wt/vol, peptone 2% wt/vol, and D-glucose 2% wt/vol) at 37ºC for 18h, under constant agitation at 150rpm (Shaker Incubator mod. NT 712, Nova Técnica Instrumentos e Equipamentos de Laboratório Ltda.)^42,43. After growth, cells were centrifuged at 3,000 × g for 5 minutes and washed twice in a 0.9% wt/vol NaCl solution, submitting each wash to the same centrifuge force^44,45. Pellets (~500mL) were transferred to 2mL microtubes (Biospec Products, Inc.) containing cold distilled water (approximately 8ºC) and glass beads (1:1:1). These mixtures remained in ice (4ºC) for 5 minutes and, afterwards they were agitated 4 times in a BeadBeater^® machine (Biospec Products, Inc.) at 4,200rpm for 30 seconds, with one-minute intervals. Cell fragments were centrifuged at 5,000 × g, 4ºC for 5 minutes. The upper aqueous phases resultants were applied in Whatman n3 (wicks) filter papers, 12x5mm in size, and maintained at -70 °C until the moment of the application^16,46.

Electrophoresis and specific enzyme staining. Enzymes were separated in starch gel (Penetrose 30^® – Refinações de Milho Brasil Ltda) at 13% wt/vol, with the dimension of 200x120x10mm. Wicks were then immediately soaked in 5 µL (0.02% wt/vol) of bromophenol-blue solution and, afterwards, they were perpendicularly applied on a gel longitudinal cut (20mm). Electrophoresis was performed in a horizontal and continuous system, under a 130-volt tension at 4ºC overnight (bromophenol-blue migration equivalent to 80mm). To assure result reproducibility, the C. albicans^CBS-562 type-strain (Centralbureau voor Schimmelcultures, Delft, The Netherlands) was systematically placed in the ends of each gel. After the electrophoretic run, the gel was put on an acrylic base, and it was sliced (1.5mm layers) with the aid of rulers and a n15 nylon thread. The layers were carefully put inside white porcelain containers and submitted to a staining process by methods previously described for 11 systems (15 enzyme loci)^15,47,48. The enzymatic activities analyzed were: alcohol dehydrogenase, sorbitol dehydrogenase, manitol-1-phosphate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, glucose dehydrogenase, glucose-6-phosphate dehydrogenase, aspartate dehydrogenase, catalase, peroxidase, and leucine aminopeptidase (Table 1). Enzymatic expressions of malate dehydrogenase, isocitrate dehydrogenase, and sorbitol dehydrogenase showed two and three genetically interpretative loci (Mdh-1, Mdh-2, and Mdh-3; Idh-1 and Idh-2; Sdh-1 and Sdh-2).

Genetic interpretation of MLEE patterns. MLEE patterns were interpreted following a commonly accepted rule, which allows the deduction of the allelic composition of a diploid organism. The electromorphs (bands) of each enzyme were numbered and/or alphabetically sorted in descending disposition regarding the anodal enzymatic mobility, and were compared with the alleles of the corresponding structural genic locus. C. albicans populations were characterized by the allelic combinations of 15 enzyme loci, so that different allelic combinations of polymorphic loci designated electrophoretic types (ETs). Thus, the percentile index of polymorphic loci (frequency of the most common allele < 0.99), the average number of alleles per locus, the average number of alleles in each polymorphic locus, and the number of alleles between heterozygotes and homozygotes, were also established^27,49. The lack of enzymatic activity was interpreted as two null alleles of the corresponding genic locus^{12,14,32,47,48,50,51}.

Clustering analysis. The genetic diversity of ea ch C. albicans population was determined by the Nei' coefficient of genetic

distance, , which

accepts the use of data from allelic and genic frequencies⁵². Thus, genetic distance matrices (trellis diagrams) were prepared and treated by the SAHN grouping method (Sequential, Agglomerative, Hierarchic, Non-overlapping Clustering Methods) UPGMA algorithm (Unweighted Pair-Group Method Using an Arithmetic Average), in order to generate trees with two-dimensional classifications, denominated dendrograms⁵³. The Pearson product-moment correlation coefficient

was used as a measure of the agreement between the genetic distance values implied by the UPGMA dendrograms and those of the original genetic distance matrices (d_ij)⁵³. Such agreements were interpreted as follows: 0.9 < r – very good fit; 0.8 < r < 0.9 – good fit; 0.7 < r <0.8 – poor fit; r < 0.7 very poor fit. These analyses were done with the aid of the NTSYS pc version 1.70 software. The C. albicans^CBS-562 type-strain (Centralbureau voor Schimmelcultures, Delft, The Netherlands) was included in this experiment in order to establish the cophenetic correlation among isolates, and to determine method reproducibility⁵⁴.

Results

Genetic interpretation of MLEE patterns. The enzyme profiles of the C. albicans samples on different gels were reproducible after three repetitions of each electrophoretic run. The genetic interpretation of MLEE patterns showed intrinsic genetic characteristics for each C. albicans population:

Population of socioeconomic class A (19 isolates): 14 (93.3%) out of 15 enzymatic loci were polymorphic to two, three or four alleles (2 alleles: Adh, Cat, Lap, Mdh-1, Mdh-2 and Po; 3 alleles: Asd, G6pdh, Idh-1, Idh-2, M1p, Mdh-3 and Sdh-2; 4 alleles: Gdh). Only 1 (6.7%) enzymatic locus was monomorphic (Sdh-1). The average number of alleles per locus was equal to 2.53, while the average number of alleles per polymorphic locus was equal to 2.69. The combination of the existing alleles in 15 enzymatic loci showed 17 (89.4%) ETs. Heterozygotes revealed two and three enzymatic bands (2 bands: Adh, Asd, G6pdh, Gdh, Idh-1, Idh-2, Lap, M1p, Mdh-2, Mdh-3, Po and Sdh-2; 3 bands: Mdh-2). Among homozygotes, one allele was observed in the Adh, Gdh, Idh-2, Lap, Po, Sdh-1 and Sdh-2 loci, two alleles in the Asd, Cat, Idh-1, M1p, Mdh-1 and Mdh-3 loci, and three alleles in the G6pdh locus (Table 2).

Population of socioeconomic class B (17 isolates): 6 (40%) out of 15 enzymatic loci were polymorphic to two alleles (Adh, G6pdh, Lap, Mdh-1, Mdh-2 and Po). Nine (60%) enzymatic loci were monomorphic (Asd, Cat, Gdh, Idh-1, Idh-2, M1p, Mdh-3, Sdh-1 and Sdh-2). The average number of alleles per locus was equal to 1.40, while the average number of alleles per polymorphic locus was equal to 2. The combination of the existing alleles in 15 enzymatic loci showed 11 (64.7%) ETs. Heterozygotes revealed two and three enzymatic bands (2 bands: Adh, G6pdh, Lap, Mdh-1, Mdh-2 and Po; 3 bands: Mdh-2). Among homozygotes, one allele was observed in the Adh, Asd, Cat, Gdh, Idh-1, Idh-2, Lap, M1p, Mdh-1, Mdh-3, Po, Sdh-1 e Sdh-2 loci, and two alleles in the G6pdh locus (Table 2).

Population of socioeconomic class C (15 isolates): 5 (33.3%) out of 15 enzymatic loci were polymorphic to two or three alleles (2 alleles: Mdh-1, Mdh-2, Po and Sdh-2; 3 alleles: Adh). Ten (66.7%) enzymatic loci were monomorphic (Asd, Cat, G6pdh, Gdh, Idh-1, Idh-2, Lap, M1p, Mdh-3 and Sdh-1). The average number of alleles per locus was equal to 1.40, while the average number of alleles per polymorphic locus was equal to 2.20. The combination of the existing alleles in 15 enzymatic loci showed 11 (73.3%) ETs. Heterozygotes revealed two and three enzymatic bands (2 bands: Adh, Mdh-1, Mdh-2, Po and Sdh-2; 3 bands: Mdh-2). Among homozygotes, one allele was observed in the Adh, Asd, Cat, G6pdh, Gdh, Idh-1, Idh-2, Lap, M1p, Mdh-1, Mdh-2, Mdh-3, Po and Sdh-1 loci, and two alleles in the Sdh- 2 locus (Table 2).

Population of socioeconomic class D (12 isolates): 9 (60%) out of 15 enzymatic loci were polymorphic to two alleles (Adh, Asd, G6pdh, Gdh, Idh-1, M1p, Mdh-2, Po and Sdh-2). Six (40%) enzymatic loci were monomorphic (Cat, Idh-2, Lap, Mdh-1, Mdh-3 and Sdh-1). The average number of alleles per locus was equal to 1.60, while the average number of alleles per polymorphic locus was equal to 2. The combination of the existing alleles in 15 enzymatic loci showed 6 (50%) ETs. Heterozygotes revealed two and three enzymatic bands (2 bands: Adh, Asd, Gdh, Idh-1, M1p, Mdh-2, Po and Sdh-2; 3 bands: Mdh-2). Among homozygotes, one allele was observed in the Adh, Cat, Gdh, Idh-1, Idh-2, Lap, Mdh-1, Mdh-3, Po and Sdh-1 loci, and two alleles in the G6pdh, M1p and Sdh-2 loci (Table 2).

Population of socioeconomic class E (12 isolates): 10 (66.7%) out of 15 enzymatic loci were polymorphic to two or three alleles (2 alleles: Asd, G6pdh, Gdh, Idh-1, Lap, M1p, Mdh-2, Po and Sdh-2; 3 alleles: Adh). Five (33.3%) enzymatic loci were monomorphic (Cat, Idh-2, Mdh-1, Mdh-3 and Sdh-1). The average number of alleles per locus was equal to 1.73, while the average number of alleles per polymorphic locus was equal to 2.11. The combination of the existing alleles in 15 enzymatic loci showed 12 (100%) ETs. Heterozygotes revealed two and three enzymatic bands (2 bands: Adh, Asd, Gdh, Idh-1, M1p, Mdh-2, Po and Sdh-2; 3 bands: Mdh-2). Among homozygotes, one allele was observed in the Asd, Cat, Gdh, Idh-1, Idh-2, M1p, Mdh-1, Mdh-3, Sdh-1 and Sdh-2 loci, and two alleles in the Adh, G6pdh, Lap and Po loci (Table 2).

Such results indicated that 31 healthy children (A = 12; B = 4; C = 7; D = 2; E = 6) were carriers of different C. albicans ETs in the oral cavity. However, identical ETs were found in children coming from socioeconomic categories as follows: a) only A (ET1); b) only B (ET10); c) A and B (ET31); d) A, B and C (ET33); e) A, D and E (ET23 and ET24); f) B and C (ET9 and ET37); g) B, C and E (ET32); h) B and E (ET34); and, i) D and E (ET4 and ET28). Identical ETs were not identified in children of socioeconomic classes B and D, C and D, or only E (Table 3, Fig. 1).

Clustering analysis. The genetic diversity among isolates in their respective populations of healthy children was evaluated by UPGMA dendrograms (Fig. 2). Such results showed coexistence of highly related or indistinguishable C. albicans subtypes (0.012 > d_ij > 0) among some healthy children coming from the same socioeconomic category. However, variations of highly related or indistinguishable (0.012 > d_ij > 0), and moderately related or non-related (d_ij > 0.012) isolate numbers were observed in each population of children (Table 4). Thus, the larger percentile index of polymorphism (d_ij >0.012) occurred among isolates from healthy children coming from socioeconomic categories A (47.3% of isolates), followed by E (33.3% of isolates), B (17.6% of isolates), C (13.3% of isolates), and D (8.3% of isolates), whose indexes of genetic distance were of 0.151 ³ d_ij ³ 0, 0.148 > d_ij > 0, 0.123 > d_ij > 0, 0.127 > d_ij > 0, and 0.039 > d_ij > 0, respectively.

The genetic diversity analysis among populations of isolates showed an ancestral convergence in populations B and C, or D and E. However, a low genetic divergence was detected in populations A and BC, A and DE, or BC and DE which, on average, corresponded to >1 and <2.3 allelic substitutions for each 100 loci, from a common ancestral population (Fig. 3).

Discussão

In our research, quantitative and qualitative variations of polymorphic loci, of the average number of alleles per locus, and of the average number of alleles per polymorphic locus were observed in all C. albicans populations coming from healthy children. These variations have been observed in several genetic diversity studies of C. albicans populations isolated from immunocompromised and immunocompetent patients^{11,12,14,15,32,38,39,50}. Like previous results of MLEE studies^11,12,38,39, the heterozygote patterns obtained in the present analysis were also consistent with the diploid nature of C. albicans⁵⁵. Pujol et al. (1993) reported that different allelic frequencies in different populations could be associated with geographical isolation, the same when each separate population remains in panmixia³⁸.

The combination of the existing alleles in 15 enzymatic loci showed a quantitative variation of subtypes (ETs) in the healthy children populations suggesting the existence of high genetic diversity of C. albicans (A = 17 ETs^89,5%, B = 11 ETs^64,7%, C = 11 ETs^73,3%, D = 6 ETs^50%, and E = 12 ETs^100%). The predominance and coexistence of some ETs (ET1, ET4, ET9, ET10, ET23, ET24, ET28, ET31, ET32, ET33, ET34 and ET37) was observed within and between some children populations. These results also suggest the existence of strain groups selected and better adapted than others in the oral cavities of those healthy children. Soll et al. (1991) also demonstrated the existence of Candida spp. strains selected and better adapted in certain human niches⁵⁶. Although certain ETs were identified exclusively in certain children populations, no correlation was observed between a specific ET and a specific population of children. Some researchers have demonstrated the prevalence of C. albicans (60% to 95%) and Candida spp. in approximately 50% of the populations of healthy individuals^2,41,57 regardless of socioeconomic factors⁴¹.

The isoenzymatic typing of C. albicans oral isolates from clinically healthy children (Piracicaba, Brazil) has revealed a way of multiclonal colonization for those yeasts¹⁴. Mehta et al. (1999) have analyzed the distribution of C. albicans genotypes among healthy family members of a same city (United States) by electrophoretic karyotyping, RAPD and REA with HinfI and EcoRI. Their results demonstrated the existence of a genotypic intrafamiliar identity (each member of a family as a carrier of the same genotype). However, different genotypes were also observed inter and intrafamiliarly²⁴. Pujol et al. (1993) identified 41 C. albicans subtypes (74.5% of isolates) in HIV-seropositive patients from a limited geographical area (Montpellier, France) by MLEE and population genetics⁹. Those researchers suggested that the high genetic diversity (11 of 21 enzymatic loci being polymorphics) could be correlated with the existence of some clonal strains that present widespread geographical distribution, as it is the case of some bacteria^58,59 and protozoa^60,61,62. Important biological and medical consequences were pointed out with clonal reproduction, once the correlation between the genetic composition and medical characteristics could facilitate effective method selection for the control of the pathological expression of C. albicans in immunocompromised individuals³⁹.

In contrast with the high genetic diversity of C. albicans observed in healthy children populations, a low genetic diversity has been detected in immunocompromised patients. The epidemiologic analysis of C. albicans isolated from seven patients (Oslo, Norway) submitted to bone marrow transplant showed the existence of 8 ETs (13.1% of isolates) and a low genetic diversity among those yeasts (4 of 10 enzymatic loci being polymorphics)¹². In some patients, the colonization with one or more ETs in different anatomical sites remained during medical follow-up. However, no correlation was observed between those ETs and the sensitivity to some antifungal (amphotericin B – AMB – and flucytosine) or anatomical sites (oral cavity, groin, and feces)¹². Boerlin et al. (1995) identified 3 atypical C. albicans ETs (23% of isolates) colonizing the oral cavity of HIV-seropositive asymptomatic patients (Lausanne, Switzerland). This lower genetic diversity (1 of 16 enzymatic loci being polymorphic) among the isolates was also observed without correlation with clinical parameters, and confirmed by Southern blot hybridization with probe Ca3 analysis. Such results were suggestive of probable colonization by atypical C. albicans subtypes from different origins and without a single limited source of contamination³². C. albicans populations isolated from HIV-seropositive patients (Lausanne, Switzerland) with and without oropharingeal candidiasis symptoms, from patients with invasive candidiasis, and from healthy individuals could not be distinguished by MLEE analysis, given that low genetic diversity was found (10 of 18 enzymatic loci being polymorphic) among isolates. In addition, 52 ETs (27.5% of isolates) were identified without correlation with clinical aspects and reduced in vitro sensitivity to fluconazole (FCZ)¹¹.

The simultaneous occurrence of genetically different C. albicans strains in HIV-seropositive patients (Montpellier, France) suffering of oropharyngeal candidiasis was also demonstrated by MLEE analysis. Low genetic diversity (10 of 21 enzymatic loci being polymorphics) among the isolates and 20 ETs (12.5% of isolates) were identified in a population of patients. However, there was predominance of a single C. albicans genetic type in the oral cavity of patients. This fact could result from the interspecies competition, which could be altered by the selective pressure of antifungal treatments⁶³. Using MLEE, Nébavi et al. (1998) also demonstrated that most of HIV-seropositive patients (Abdjan, Ivory Coast) suffering from oropharyngeal candidiasis were colonized by identical or variant ETs of C. albicans during antifungal therapy (AMB, KTZ, NYS). These researchers identified 27 ETs (40.3% of isolates) and a low genetic diversity (10 of 21 enzymatic loci being polymorphics) among isolates⁵¹. MLEE analyses were also performed in C. albicans isolates from patients (Montpellier, France) suffering of recurrent oropharyngeal candidiasis that successively developed clinical resistance to the fluconazole (FCZ) and itraconazole (ITZ). These analyses revealed that the infection of the patients occurred for one or more ETs during antifungal therapy, which could be (i) selected from a mixed population or (ii) acquired from an exogenous source. Besides, 14 ETs (14.3% of isolates) and low genetic diversity (12 polymorphic enzymatic loci) were identified in a population of C. albicans isolates, without correlation to antifungal sensitivity tests⁵⁰.

The genetic diversity of isolates in their respective populations was evaluated through UPGMA dendrograms. The coexistence of highly related or indistinguishable C. albicans (0.012 > d_ij > 0) was observed in some healthy children of the same socioeconomic category, probably emerging from a common ancestral strain^55,63. These results suggest (i) the existence of one or more highly related C. albicans oral isolate clusters and usually predominant in healthy children populations with a common socioeconomic background, and (ii) the existence of direct and indirect propagation routes of C. albicans in populations of healthy children, which could be determined by complementary studies as, for instance, the isolation of C. albicans from a shared environment (education and sport schools, and their respective professionals...). Schmid et al. (1999) have showed high genetic similarity among not geographically related C. albicans clusters by Southern blot hybridization with a Ca3 probe. Their results suggested that there was a former small radial propagation of strains among geographically adjacent regions⁶⁴. The frequent and common mechanisms involved in the genetic diversity of Candida species could explain this genetic similarity. These mechanisms comprise chromosomal rearrangements, chromosomal alterations and genic expression control^50,65,66. Besides, repetitive sequences in tandem and subtelomeric and telomeric sequences can be involved in organization and chromosomal rearrangements^67,68. Using MLEE and grouping analyses, other researchers have also showed the existence of highly related C. albicans clusters isolated from healthy and immunocompromised patients without correlations with the clinical aspects, antifungal sensitivity or geographical regions^11,15,50,51. Lupetti et al. (1995) used electrophoretic karyotyping and identified two similar C. albicans clusters displaying prevalence in healthy individuals and HIV-seropositive patients (Pisa, Italy)²⁵. Their observations were suggestive that commensal strains can be probable agents of subsequent oral candidiasis in immunocompromised patients, as also suggested by other researchers^69,70, although strain substitution can also happen²⁵.

The genetic diversity analysis among populations showed ancestral convergence in C. albicans populations isolated from healthy children of the B and C (mean/high), or D and E (mean/low) socioeconomic categories. Ancestral divergence was observed among C. albicans populations isolated from children of socioeconomic categories A (high) and BC (medium/high), A (high) and DE (medium/low), or BC (medium/high) and DE (medium/low) that, on average, corresponded between >1 and <2.3 allelic substitutions for each 100 loci, from a common ancestral population. These results suggest that microevolutionary changes can occur in some C. albicans populations isolated from healthy children that present the same socioeconomic status. However, microevolution investigation in C. albicans population commensals, comparing other host parameters (nutritional and hygienic habits, hormonal changes, age ) could be explored. Other epidemiologic and microevolutionary studies of C. albicans have been performed using Southern blot hybridization with DNA probe Ca3^{20-23,64,71-73}. Some of these studies have also demonstrated the existence of regional specificity and genetically similar and highly predominant subgroups of C. albicans in various types of infections from various patients living in different geographic areas. Such results were indicative of the existence of a ubiquitous group displaying the predominant etiological agent of candidiasis, which could arise from its high prevalence as a commensal. In addition, strong epidemiologic and microevolutionary agreements were demonstrated by Ca3 fingerprinting, MLEE, and RAPD analyses during the characterization of C. albicans isolated from several anatomical sites of immunocompetent and immunocompromised patients¹⁵.

Using MLEE analysis, the results obtained in the current research showed high genetic diversity of C. albicans oral isolates and predominance and coexistence of some subtypes (ETs) in Brazilian populations of clinically healthy children classified into five socioeconomic categories (A, B, C, D, and E). However, no correlation was observed between a specific ET and a specific population of children. The existence of one or more highly related ET clusters was showed by clustering analysis, suggesting the existence of indirect and direct propagation routes of C. albicans, which could demand certain complementary studies as, for instance, the isolation of C. albicans from shared environments. The genetic diversity analyses among populations showed (i) ancestral convergence in the C. albicans populations isolated from healthy children of socioeconomic categories B and C (medium/high), or D and E (medium/low), and (ii) ancestral divergence among C. albicans populations isolated from children of socioeconomic categories A (high) and BC (medium/high), A (high) and DE (medium/low), or BC (medium/high) and DE (medium/low). These results suggest that microevolutionary changes can occur in some C. albicans populations isolated from healthy children that present a common socioeconomic status. Furthermore, a low transition of C. albicans subtypes can be occurring among certain populations of children (low transition between A and BC, A and DE, or BC and DE), which can also be explained by their own socioeconomic and cultural characteristics. However, microevolution investigations of C. albicans commensal populations, comparing other host parameters, could be explored. Finally, MLEE analysis could be used for current and retrospective analyses of C. albicans isolated from healthy and immunocompromised individuals, in order to detect the existence of a predominant group in candidiasis. Such procedures could lead to the development of strategies for prevention of transmissibility of these yeasts groups in healthy or immunocompromised children, regardless of their socioeconomic and cultural conditions.

Acknowledgment

This research was supported by FAPESP – Fundação de Amparo à Pesquisa do Estado de São Paulo (Proc. 00/03045-5).

recebido em: 06/07/04

versão final reapresentada em: 24/02/05

aprovado em: 28/02/05

Research funding: This research was supported by FAPESP – Fundação de Amparo à Pesquisa do Estado de São Paulo (Processo n. 00/03045-5).

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Publication Dates

• Publication in this collection
  12 June 2007
• Date of issue
  Mar 2005

History

• Received
  04 July 2004
• Reviewed
  05 Feb 2005
• Accepted
  28 Feb 2005