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 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. Marcelo Fabiano Gomes Boriollo*1 Edvaldo Antônio Ribeiro Rosa2 Wagner Luis de Carvalho Bernardo1 Denise Madalena Palomari Spolidorio3 Reginaldo Bruno Gonçalves1 José Francisco Höfling1 Microbiology and Immunology Laboratory, Dental School of Piracicaba, State University of Campinas, Piracicaba, Brazil 1 Stomatology Laboratory, Center of Biological and Health Sciences, Pontifical Catholic University of Paraná, Curitiba, Brazil 2 Department of Physiology and Pathology, School of Dentistry, Paulista State University, Araraquara, São Paulo, Brazil 3 Research funding: This research was supported by FAPESP – Fundação de Amparo à Pesquisa do Estado de São Paulo (Processo n. 00/03045-5). *Correspondência: Av. Limeira 901, CEP13414-90, Piracicaba, SP, Brasil. E-mail: marcelofgb@yahoo.com.br 51 Rev Bras Epidemiol 2005; 8(1): 51-66 Resumo Introduction 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. 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 existence5,6. 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)916 , 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 populations26,27, and has provided high discriminatory power and reproducibility12,15,26-29. Considered neutral markers (invariable when they suffer environment selective pressures), Palavras-chave: Candida albicans. MLEE. Diversidade genética. Crianças saudáveis. Categoria socioeconômica. Rev Bras Epidemiol 2005; 8(1): 51-66 52 Multilocus enzyme electrophoresis typing of Candida albicans Boriollo, M.F.G. et al. metabolic isoenzymes present great potentiality in the taxonomic, systematic, genetic, evolution and epidemiologic characterization of C. albicans and other yeasts of medical importance9-16,30-40. 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 cul- tures 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 force44,45. Pellets (~500mL) were transferred to 2mL microtubes (Biospec Products, Inc.) containing cold distilled water (approximately 8 oC) 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 application16,46. 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 de- Multilocus enzyme electrophoresis typing of Candida albicans Boriollo, M.F.G. et al. 53 Rev Bras Epidemiol 2005; 8(1): 51-66 hydrogenase, sorbitol dehydrogenase, manitol-1-phosphate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, glucose dehydrogenase, glucose-6phosphate 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 established27,49. The lack of enzymatic activity was interpreted as two null alleles of the corresponding genic locus12,14,32,47,48,50,51. 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, Nonoverlapping Clustering Methods) UPGMA algorithm (Unweighted Pair-Group Method Rev Bras Epidemiol 2005; 8(1): 51-66 54 Multilocus enzyme electrophoresis typing of Candida albicans Boriollo, M.F.G. et al. 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 (dij)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. albicansCBS-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, Tabela 1 – Sistemas e soluções utilizados para análise de MLEE a partir de enzimas metabólicas de C. albicans. Table 1 – Systems and solutions utilized for MLEE analysis from metabolic enzymes of C. albicans. Enzyme Compound for Staining EC number Name Symbol Substrate Buffer 1.1.1.1. Alcohol dehydrogenase Sorbitol dehydrogenase Mannitol-1-phosphate dehydrogenase Malate dehydrogenase Isocitrate dehydrogenase Glucose dehydrogenase Glucose-6phosphate dehydrogenase Aspartate dehydrogenase Catalase 8 Peroxidase ADH Ethanol (3 mL) Isopropanol (2 mL) Sorbitol (250 mg) 200 mM Tris-HCl pH 8.0 (50 mL) 1 Tris-HCl 50 mM pH 8.0 (50 mL) 2 Tris-HCl 100 mM pH 8.5 (50 mL) 3 Tris-HCl 200 mM pH 8.0 (40 mL) 1 Tris-HCl 200 mM pH 8.0 (40 mL) 1 Tris-HCl 200 mM pH 8.0 (50 mL) 1 Tris-HCl 200 mM pH 8.0 (50 mL) 1 1.1.1.14. 1.1.1.17. 1.1.1.37. Multilocus enzyme electrophoresis typing of Candida albicans Boriollo, M.F.G. et al. 1.1.1.42. 1.1.1.47. 1.1.1.49. 1.4.3.x. 1.11.1.6. 1.11.1.7. 3.4.11.1. Leucine aminopeptidase SDH M1P MDH IDH GDH G6PDH Mannitol-1phosphate (5 mg) 2M Malic acid (6 mL) 4 1M Isocitric acid (2 mL) 5 D-glucose (500 mg) ASD Glicose-6phosphate disodium salt (100 mg) Aspartic acid (50 mg) CAT PO H2O2 3% (1 mL) LAP L-leucine bnaphthylamide HCl (30 mg) Salt Coenzyme Dye Catalyser NAD 1% (2mL) PMS 1% (500 µL) MTT 1.25% (1 mL) PMS 1% (500 µL) MTT 1.25% (1 mL) PMS 1% (500 µL) MTT 1.25% (1 mL) PMS 1% (500 µL) MTT 1.25% (1 mL) PMS 1% (500 µL) MTT 1.25% (1 mL) PMS 1% (500 µL) MTT 1.25% (1 mL) PMS 1% (500 µL) MTT 1.25% (1 mL) NAD 1% (2mL) NAD 1% (2mL) NAD 1% (2mL) 100 mM MgCl2 (1 mL) 6 NADP 1% (1mL) NAD 1% (2mL) 100 mM MgCl2 (1 mL) 6 Sodium phosphate pH 7.0 (50 mL) 7 100mM Sodium acetate pH 4.5 (50 mL) 9 100mM Potassium 100 mM MgCl2 phosphate (1 mL) 6 pH 5.5 (50 mL) 10 NADP 1% (1mL) NAD 1% (2mL) PMS 1% (500 µL) MTT 1.25% (1 mL) o-dianisidine 2HCl (16mg) Black K (30 mg) 55 Rev Bras Epidemiol 2005; 8(1): 51-66 Tampão do eletrodo: Tris-citrato pH 8,0 [83,2g de C4H11NO3 (Tris), 33,09g de C6H8O7 . H2O (Ácido cítrico), 1L de H2O]; Tampão do gel: Tampão do eletrodo diluído 1:29; 1 24,2g de C4H11NO3 (Tris), 1L de H2O (pH ajustado com HCl); 2 6,05g de C4H11NO3 (Tris), 1L de H2O (pH ajustado com HCl); 3 12,1g de C4H11NO3 (Tris), 1L de H2O (pH ajustado com HCl); 4 26,8g de C4H6O5 (DL-ácido málico) e 16g de NaOH em 0,1L de H2O (precaução: reação potencialmente explosiva); 5 29,41g de C6H5O7Na3 . 2H2O (DL-ácido isocítrico) em 0,1L de H2O; 6 2,03g de MgCl2 . 6HCl (Cloreto de magnésio) em 0,1L de H2O; 7 Misturar partes iguais de 27,6g de NaH2PO4 . H2O (Fosfato de sódio monobásico) em 1L de H2O e 53,6g de Na2HPO4 . 7H2O (Fosfato de sódio dibásico heptahidratado) em 1L de H2O, então diluir a mistura 1:25 com H2O; 8 Incubar a fatia do gel por 30 minutos a 0oC em 50mL de tampão de 0,1M fosfato de sódio pH 7, então decantar a solução, e imergir o gel em 50mL de solução de iodeto de potássio 1,5% (KI) por 2 minutos. Por conseguinte, enxaguar a fatia do gel com água, e imergir o gel em 50mL de solução de peróxido de hidrogênio (H2O2) 0,03%. Misturar cuidadosamente e remover a solução corante quando zonas brancas surgirem sobre o fundo azul-escuro; 9 13,61g de C2H3O2Na . 3H2O (Acetato de sódio), 1L de H2O; 10 13,61g de KH2PO4 (Fosfato de potássio), 1L de H2O.Electrode buffer: Tris-citrate pH 8.0 [83.2g of C4H11NO3 (Tris), 33.09g of C6H8O7 . H2O (Citric acid), 1L of H2O]; Gel buffer: Electrode buffer diluted 1:29; 1 24.2g of C4H11NO3 (Tris), 1L of H2O (pH adjusted with HCl); 2 6.05g of C4H11NO3 (Tris), 1L of H2O (pH adjusted with HCl); 3 12.1g of C4H11NO3 (Tris), 1L of H2O (pH adjusted with HCl); 4 26.8g of C4H6O5 (DL-malic acid) and 16g of NaOH in 0.1L of H2O (caution: potentially explosive reaction); 5 29.41g of C6H5O7Na3 . 2H2O (DL-isocitric acid) in 0.1L of H2O; 6 2.03g of MgCl2 . 6HCl (Magnesium chloride) in 0.1L of H2O; 7 Mix equal parts of 27.6g of NaH2PO4 . H2O (Sodium phosphate monobasic monohydrate) in 1L of H2O and 53.6g of Na2HPO4 . 7H2O (Sodium phosphate dibasic heptahydrate) in 1L of H2O, then dilute the mixture 1:25 with H2O; 8 Incubate gel slice for 30 minutes at 0oC in 50mL of 0.1M sodium phosphate pH 7.0 buffer, then pour off solution, and immerse it in 50mL of 1.5% potassium iodide solution (KI) for 2 minutes. Then rinse gel slice with water, and immerse it in 50mL of 0.03% hydrogen peroxide (H2O2) solution. Mix gently and remove stain solution when white zones appear on dark-blue background; 9 13.61g of C2H3O2Na . 3H2O (Sodium acetate), 1L of H2O; 10 13.61g of KH2PO4 (Potassium phosphate), 1L of H2O. Mdh-3, Po and Sdh-2; 3 bands: Mdh-2). Among homozygotes, one allele was observed in the Adh, Gdh, Idh-2, Lap, Po, Sdh1 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). Rev Bras Epidemiol 2005; 8(1): 51-66 56 Multilocus enzyme electrophoresis typing of Candida albicans Boriollo, M.F.G. et al. 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, Mdh1, 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 in- Tabela 2 – Perfis alélicos em 43 ETs enzimáticos de C. albicans isolada de 75 crianças saudáveis provenientes de cinco categorias socioeconômicas. Table 2 - Allelic profiles in 43 enzymatic ETs of C. albicans isolated from 75 healthy children coming from five socioeconomic categories. ET No. of Isolates Adh Cat bb bb aa cc bb aa aa aa bb ab ab cc ab aa bb A socioeconomic class 1 2 ab 6 1 ab 16 1 bb 17 1 bb 18 1 bb 20 1 bb 21 1 bb 22 1 bb 23 1 bb 24 2 bb 26 1 bb 27 1 bb 29 1 bb 30 1 bb 31 1 bb 33 1 bb 41 1 bb ab ab ab ab ab ab ab ab ab ab ab ab ab bb bb bb cc aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa bb ab cc aa aa bb cc cc cc cc cc cc cc cc bb cc cc bb bb bb bb bb bb ab ab ab bb bb bb bb cd bb bb bb ab aa aa ac aa ac ac ac aa aa aa ac bb aa aa aa ac aa aa aa aa aa aa aa aa aa aa aa aa bc aa aa aa aa aa aa aa aa aa aa aa ab aa aa aa aa aa aa aa aa aa bb bb bb bb bb ab ab bb bb bb bc bb cc bc bb bb ab aa aa aa aa aa aa aa aa aa aa aa aa cc aa aa aa aa ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab cc cc cc cc cc cc cc cc cc bb cc ab bb cc cc ab aa aa ab ab aa ab ab aa ab aa aa ab ab aa aa ab aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa - bb bb bb bb bb bb bb bb bb bb bb bb bb bb bb bb cd B socioeconomic class 7 1 ab 9 1 ab 10 4 ab 11 1 ab 14 1 ab 31 1 bb 32 4 bb 33 1 bb 34 1 bb 35 1 bb 37 1 bb bb bb bb bb bb bb bb bb bb bb bb aa aa aa aa aa aa aa aa aa aa aa bb cc cc cc cc cc cc cc cc cc cc bb bb bb bb bb bb bb bb bb bb bb aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa - aa aa aa aa ab aa aa aa aa aa aa bb bb bb bb bb bb bb bb bb bb bb aa aa aa aa ab aa aa aa aa aa aa ab ab ab ab ab ab ab ab ab ab ab cc cc cc cc cc cc - ab aa ab aa aa aa ab aa ab aa aa aa aa aa aa aa aa aa aa aa aa aa bb bb bb bb bb bb bb bb bb bb bb C socioeconomic class 9 1 ab 13 1 ab 15 1 ab 32 5 bb 33 1 bb 36 1 bb 37 1 bb 38 1 bb 39 1 bb 40 1 bb 42 1 bc bb bb bb bb bb bb bb bb bb bb bb aa aa aa aa aa aa aa aa aa aa aa cc cc cc cc cc cc cc cc cc cc cc bb bb bb bb bb bb bb bb bb bb bb aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa - aa aa aa aa aa aa aa aa aa aa aa bb bb bb bb bb bb bb bb bb bb bb aa aa ab aa aa aa aa aa aa aa aa ab ab bb ab ab ab ab ab ab bb ab cc cc cc cc aa ab ab ab aa ab aa ab ab ab aa aa aa aa aa aa aa aa aa aa aa aa bb bb bb bb bb bb bb aa bb bb bb TS G6pdh Gdh Alleles of 15 enzymatic loci* Idh-1 Idh-2 Lap M1p Mdh-1 Mdh-2 Mdh-3 Asd Multilocus enzyme electrophoresis typing of Candida albicans Boriollo, M.F.G. et al. 57 Po Sdh-1 Sdh-2 Rev Bras Epidemiol 2005; 8(1): 51-66 Tabela 2 – Perfis alélicos em 43 ETs enzimáticos de C. albicans isolada de 75 crianças saudáveis provenientes de cinco categorias socioeconômicas. Table 2 - Allelic profiles in 43 enzymatic ETs of C. albicans isolated from 75 healthy children coming from five socioeconomic categories. ET No. of Isolates Adh Cat bb bb aa cc bb aa aa aa bb ab ab cc ab aa bb D socioeconomic class 3 1 ab 4 1 ab 19 1 bb 23 4 bb 24 3 bb 28 2 bb ab ab ab ab ab ab aa aa aa aa aa aa cc cc bb cc cc cc ab ab bb bb bb bb ac ac ac aa aa ac aa aa aa aa aa aa aa aa aa aa aa aa aa ab bb bb bb bb aa aa aa aa aa aa ab ab ab ab ab ab cc cc cc cc cc cc aa aa ab aa ab ab aa aa aa aa aa aa aa ab bb bb bb bb E socioeconomic class 2 1 4 1 5 1 8 1 12 1 23 1 24 1 25 1 28 1 32 1 34 1 43 1 ab ab ab bb bb ab ab ab ab bb bb bb aa aa aa aa aa aa aa aa aa aa aa aa bb cc cc cc cc cc cc cc cc cc cc cc ab ab bb bb bb bb bb bb bb bb bb bb ac ac aa aa aa aa aa aa ac aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa cc bb ab bb bb bb bb bb bb bb bb bb bb aa aa aa aa aa aa aa aa aa aa aa aa ab ab ab ab ab ab ab ab ab ab ab ab cc cc cc cc cc cc cc cc cc ab aa aa aa ab aa ab ab ab ab ab bb aa aa aa aa aa aa aa aa aa aa aa aa bb ab ab ab ab bb bb bb bb bb bb bb TS ab ab ab ab ab bb bb bb bb bb bb cc G6pdh Gdh Alleles of 15 enzymatic loci* Idh-1 Idh-2 Lap M1p Mdh-1 Mdh-2 Mdh-3 Asd Po Sdh-1 Sdh-2 * Heterozigotos estão presentes como ab, ac, bc e cd. (-) alelo nulo. TS corresponde a linhagem-tipo de C. albicansCBS-562. * Heterozygotes are present as ab, ac, bc and cd. (-) null allele. TS corresponds to C. albicansCBS-562 type-strain. Figura 1 – Subtipos de C. albicans (ETs) coexistentes na maioria das populações de crianças saudáveis. Figure 1 – C. albicans subtypes (ETs) coexisting in most populations of healthy children. Rev Bras Epidemiol 2005; 8(1): 51-66 58 Multilocus enzyme electrophoresis typing of Candida albicans Boriollo, M.F.G. et al. Tabela 3 - Distribuição de 43 ETs enzimáticos de C. albicans em 75 crianças saudáveis provenientes de cinco categorias socioeconômicas. Table 3 - Distribution of 43 enzymatic ETs of C. albicans in 75 healthy children coming from five socioeconomic categories. ET A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 2 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 ∑ 19 Socioeconomic classes B C D 1 1 4 1 1 1 4 1 1 1 1 ∑ 17 1 1 1 5 1 1 1 1 1 1 1 ∑ 15 1 1 1 4 3 2 ∑ 12 E 1 1 1 1 1 1 1 1 1 1 1 1 ∑ 12 n corresponde ao número de ETs de C. albicans entre n crianças saudáveis provenientes de várias classes socioeconômicas e – corresponde a ausência de ET. n correspond to the number of C. albicans ETs among n healthy children coming from several socioeconomic classes and – corresponds to ET absence. distinguishable C. albicans subtypes (0.012 > dij ≥ 0) among some healthy children coming from the same socioeconomic category. However, variations of highly related or indistinguishable (0.012 > dij ≥ 0), and moderately related or non-related (dij ≥ 0.012) isolate numbers were observed in each population of children (Table 4). Thus, the larger percentile index of polymorphism ( dij ≥ 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 ≥ dij ≥ 0, 0.148 ≥ dij > 0, 0.123 ≥ dij > 0, 0.127 ≥ dij > 0, and 0.039 ≥ dij > 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 patients11,12,14,15,32,38,39,50. Like previous results of MLEE studies11,12,38,39, 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 varia- Multilocus enzyme electrophoresis typing of Candida albicans Boriollo, M.F.G. et al. 59 Rev Bras Epidemiol 2005; 8(1): 51-66 Figure 2 – Diversidade genética dentro e entre populações de C. albicans isoladas da cavidade bucal de crianças saudáveis provenientes de cinco categorias socioeconômicas. Dendrogramas UPGMA (0,92980 ≤ rjk ≤ 0,94560 – muito bom ajuste) gerados a partir das matrizes de distância genética dij (Nei, 1972). Figure 2 – Genetic diversity within and between C. albicans populations isolated from the oral cavity of healthy children coming from five socioeconomic categories. UPGMA dendrograms (0.92980 ≤ rjk ≤ 0.94560 – very good fit) generated from matrices of genetic distance dij (Nei, 1972). Tabela 4 – Relação do número de isolados altamente relacionados ou indistingüíveis (0.012 > dij ≥ 0) e moderadamente relacionados ou não relacionados (dij ≥ 0.012), obtidos pela análise de agrupamento de populações de C. albicans. Table 4 – List of the number of highly related or indistinguishable (0.012 > dij ≥ 0) and moderately related or non related (dij ≥ 0.012) isolates, obtained by clustering analysis of C. albicans populations. Socioconomic clategories A B C D E Rev Bras Epidemiol 2005; 8(1): 51-66 60 Isolates 0.012 > dij ≥ 0 n % 10 14 13 11 8 52.6 82.4 86.7 91.7 66.7 number of clusters (0.012 > dij ≥ 0) 3 2 4 2 3 Multilocus enzyme electrophoresis typing of Candida albicans Boriollo, M.F.G. et al. Isolates dij ≥ 0.012 n % 9 3 2 1 4 47.4 17.6 13.3 8.3 33.3 Total 19 17 15 12 12 Figure 3 – Convergência ou divergência ancestral (>1 e <2,3 substituição alélica para cada 100 locos) entre isolados de C. albicans provenientes de populações de crianças saudáveis que apresentam uma base socioeconômica. Figure 3 – Ancestral convergence or divergence (>1 and <2.3 allelic substitutions for each 100 loci) among C. albicans isolates coming from healthy children populations with a common socioeconomic background (Fig. 3). tion of subtypes (ETs) in the healthy children populations suggesting the existence of high genetic diversity of C. albicans (A = 17 ETs89,5%, B = 11 ETs64,7%, C = 11 ETs73,3%, D = 6 ETs50%, and E = 12 ETs100%). 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 individuals2,41,57 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 bacteria58,59 and protozoa60,61,62. 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 Multilocus enzyme electrophoresis typing of Candida albicans Boriollo, M.F.G. et al. 61 Rev Bras Epidemiol 2005; 8(1): 51-66 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 HIVseropositive 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 Rev Bras Epidemiol 2005; 8(1): 51-66 62 Multilocus enzyme electrophoresis typing of Candida albicans Boriollo, M.F.G. et al. 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 > dij ≥ 0) was observed in some healthy children of the same socioeconomic category, probably emerging from a common ancestral strain55,63. 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 control50,65,66. Besides, repetitive sequences in tandem and subtelomeric and telomeric sequences can be involved in organization and chromosomal rearrangements67,68. 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 regions11,15,50,51. 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 researchers69,70, 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 param- eters (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 Ca320-23,64,71-73. 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 Multilocus enzyme electrophoresis typing of Candida albicans Boriollo, M.F.G. et al. 63 Rev Bras Epidemiol 2005; 8(1): 51-66 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. 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