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

rbepid Revista Brasileira de Epidemiologia Rev. bras. epidemiol. 1415-790X 1980-5497 Associação Brasileira de Saúde Coletiva Rio de Janeiro, RJ, Brazil 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. 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 RosaII; Wagner Luis de Carvalho BernardoI; Denise Madalena Palomari SpolidorioIII; Reginaldo Bruno GonçalvesI; José Francisco HöflingI 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 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)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 populations26,27, and has provided high discriminatory power and reproducibility12,15,26-29. 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 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 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 force44,45. 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 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. albicansCBS-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 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, 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 (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, 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 > 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 ³ dij ³ 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 variation 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 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 > 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 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 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 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). * Correspondência: Av. Limeira 901, CEP13414-90, Piracicaba, SP, Brasil. E-mail: [e-mail] 1. Segal E., Baum GL. Pathogenic yeasts and yeast infections. Boca Raton, Ann Arbor, London, Tokyo; CRC Press Inc.; 1994 Pathogenic yeasts and yeast infections 1994 Segal E. Baum GL 2. Stenderup A. Oral mycology. Acta Odont Scand 1990; 48: 3-10. Oral mycology Acta Odont Scand 1990 3 10 48 Stenderup A 3. Kwon-Chung KJ, Bennett JE. Medical mycology. Philadelphia; Lea and Febiger; 1992. Medical mycology 1992 Kwon-Chung KJ Bennett JE 4. Rippon JW. Medical mycology. Philadelphia; W.B. Saunders; 1988. Medical mycology 1988 Rippon JW 5. Samaranayake LP. Oral candidosis: an old disease in new guises. Dent Update 1990; 17: 36-8. Oral candidosis: an old disease in new guises Dent Update 1990 36 8 17 Samaranayake LP 6. Samaranayake YH, Samaranayake LP, Pow EH, Beena VT, Yeung KW. Antifungal effects of lysozyme and lactoferrin against genetically similar, sequential Candida albicans isolates from a human immunodeficiency virus-infected southern Chinese cohort. J Clin Microbiol 2001; 39: 3296-302. Antifungal effects of lysozyme and lactoferrin against genetically similar, sequential Candida albicans isolates from a human immunodeficiency virus-infected southern Chinese cohort J Clin Microbiol 2001 3296 302 39 Samaranayake YH Samaranayake LP Pow EH Beena VT Yeung KW 7. Greenspan D, Greenspan JS. HIV related oral disease. Lancet 1996; 348: 729-33. HIV related oral disease Lancet 1996 729 33 348 Greenspan D Greenspan JS 8. Cannon RD, Chaffin WL. Oral colonization by Candida albicans. Crit Rev Oral Biol Med 1999; 10: 359-83. Oral colonization by Candida albicans Crit Rev Oral Biol Med 1999 359 83 10 Cannon RD Chaffin WL 9. Arnavielhe S, Blancark A, Mallié M, Quilici M, Bastide JM. Multilocus enzyme electrophoresis analysis of Candida albicans isolates from three intensive care units. An epidemiological study. Mycoses 1997; 40: 159-67. Multilocus enzyme electrophoresis analysis of Candida albicans isolates from three intensive care units An epidemiological study. Mycoses 1997 159 67 40 Arnavielhe S Blancark A Mallié M Quilici M Bastide JM 10. Barchiesi F et al. Fluconazole susceptibility and strain variation of Candida albicans isolates from HIV-infected patients with oropharyngeal candidosis. J Antimicrob Chemother 1998; 41: 541-8. Fluconazole susceptibility and strain variation of Candida albicans isolates from HIV-infected patients with oropharyngeal candidosis J Antimicrob Chemother 1998 541 8 41 Barchiesi F 11. Boerlin P et al. Typing Candida albicans oral isolates from human immunodeficiency virus-infected patients by multilocus enzyme electrophoresis and DNA fingerprinting. J Clin Microbiol 1996; 34: 1235-48. Typing Candida albicans oral isolates from human immunodeficiency virus-infected patients by multilocus enzyme electrophoresis and DNA fingerprinting J Clin Microbiol 1996 1235 48 34 Boerlin P 12. Caugant DA, Sandven P. Epidemiological analysis of Candida albicans strains by multilocus enzyme electrophoresis. J Clin Microbiol 1993; 31: 215-20. Epidemiological analysis of Candida albicans strains by multilocus enzyme electrophoresis J Clin Microbiol 1993 215 20 31 Caugant DA Sandven P 13. Lehmann PF, Lin D, Lasker BA. Genotypic identification and characterization of species and strains within the genus Candida by using random amplified polymorphic DNA. J Clin Microbiol 1992; 30: 3249-54. Genotypic identification and characterization of species and strains within the genus Candida by using random amplified polymorphic DNA J Clin Microbiol 1992 3249 54 30 Lehmann PF Lin D Lasker BA 14. Mata AL, Rosa RT, Rosa EAR, Gonçalves RB, Höfling JF. Clonal variability among oral Candida albicans assessed by allozyme electrophoresis analysis. Oral Microbiol Immunol 2000; 15: 350-4. Clonal variability among oral Candida albicans assessed by allozyme electrophoresis analysis Oral Microbiol Immunol 2000 350 4 15 Mata AL Rosa RT Rosa EAR Gonçalves RB Höfling JF 15. Pujol C, Joly S, Lockhart SR, Noel S, Tibayrenc M, Soll DR. Parity among the randomly amplified polymorphic DNA method, multilocus enzyme electrophoresis, and Southern blot hybridization with the moderately repetitive DNA probe Ca3 for fingerprinting Candida albicans. J Clin Microbiol 1997; 35: 2348-58. Parity among the randomly amplified polymorphic DNA method, multilocus enzyme electrophoresis, and Southern blot hybridization with the moderately repetitive DNA probe Ca3 for fingerprinting Candida albicans J Clin Microbiol 1997 2348 58 35 Pujol C Joly S Lockhart SR Noel S Tibayrenc M Soll DR 16. Rosa EAR, Rosa RT, Pereira CV, Höfling JF. Inter and Intra-specific genetic variability of oral Candida species. Rev Iberoam Micol 2001; 18: 60-4. Inter and Intra-specific genetic variability of oral Candida species Rev Iberoam Micol 2001 60 4 18 Rosa EAR Rosa RT Pereira CV Höfling JF 17. Gyanchandani A, Khan ZK, Farooqui N, Goswami M, Ranade SA. RAPD analysis of Candida albicans strains recovered from immunocompromised patients (ICP) reveals an apparently non-random infectivity of the strains. Biochem Mol Biol Int 1998; 44: 19-27. RAPD analysis of Candida albicans strains recovered from immunocompromised patients (ICP) reveals an apparently non-random infectivity of the strains Biochem Mol Biol Int 1998 19 27 44 Gyanchandani A Khan ZK Farooqui N Goswami M Ranade SA 18. Pfaller MA, Cabezudo I, Hollis R, Huston B, Wenzel RP. The use of biotyping and DNA fingerprinting in typing Candida albicans from hospitalized patients. Diag Microbiol Infect Dis 1990; 13: 481-9. The use of biotyping and DNA fingerprinting in typing Candida albicans from hospitalized patients Diag Microbiol Infect Dis 1998 481 9 13 Pfaller MA Cabezudo I Hollis R Huston B Wenzel RP 19. Panaka K. Strain-relatedness among different populations of the pathogenic yeast Candida albicans analyzed by DNA-based methods. Nagoya J Med Sci 1997; 60: 1-14. Strain-relatedness among different populations of the pathogenic yeast Candida albicans analyzed by DNA-based methods Nagoya J Med Sci 1997 1 14 60 Panaka K 20. Kleinegger C, Lockhart SR, Vargas K, Soll DR. Frequency, intensity, species and strains of oral yeast vary as a function of host age. J Clin Microbiol 1996; 34: 2246-54. Frequency, intensity, species and strains of oral yeast vary as a function of host age J Clin Microbiol 1996 2246 54 34 Kleinegger C Lockhart SR Vargas K Soll DR 21. Lockhart SR, Joly S, Vargas K, Swails-Wenger J, Enger L, Soll DR. Natural defenses against Candida colonization break down in the oral cavity of the elderly. J Dent Res 1998; 78: 857-68. Natural defenses against Candida colonization break down in the oral cavity of the elderly J Dent Res 1998 857 68 78 Lockhart SR Joly S Vargas K Swails-Wenger J Enger L Soll DR 22. Pfaller MA et al. Hospital specificity, region specificity, and fluconazole resistance of Candida albicans bloodstream isolates. J Clin Microbiol 1998; 36: 1518-29. Hospital specificity, region specificity, and fluconazole resistance of Candida albicans bloodstream isolates J Clin Microbiol 1998 1518 29 36 Pfaller MA 23. Schmid J, Hunter PR, White GC, Nand AK, Soll RD. Physiological traits associated with success of Candida albicans strains as commensal colonizers and pathogens. J Clin Microbiol 1995; 33: 2920-6. Physiological traits associated with success of Candida albicans strains as commensal colonizers and pathogens J Clin Microbiol 1995 2920 6 33 Schmid J Hunter PR White GC Nand AK Soll RD 24. Mehta SK, Stevens DA, Mishra SK, Feroze F, Pierson DL. Distribuition of Candida albicans genotypes among family members. Diagn Microbiol Infect Dis 1999; 34: 19-25. Distribuition of Candida albicans genotypes among family members Diagn Microbiol Infect Dis 1999 19 25 34 Mehta SK Stevens DA Mishra SK Feroze F Pierson DL 25. Lupetti A, Guzzi G, Paladini A, Swart K, Campa M, Senesi S. Molecular typing of Candida albicans in oral candidiasis: karyotype epidemiology with human immunodeficiency virus-seropositive patients in comparison with that with healthy carriers. J Clin Microbiol 1995; 33: 1238-42. Molecular typing of Candida albicans in oral candidiasis: karyotype epidemiology with human immunodeficiency virus-seropositive patients in comparison with that with healthy carriers J Clin Microbiol 1995 1238 42 33 Lupetti A Guzzi G Paladini A Swart K Campa M Senesi S 26. Murphy RW, Sites JW, Buth DG, Haufler CH. In: Hillis DM, Moritz C. Molecular systematics. Sunderland, Mass.: Sinauer Associates Inc. Publishers; 1990. P. 45-126. Molecular systematics 1990 45 126 Murphy RW Sites JW Buth DG Haufler CH Hillis DM Moritz C 27. Pasteur N, Pasteur G, Bonbomme F, Catalan J, Britton-Davidian J. Manuel technique de génétique par électrophorèse dês proteins. Technique et documentation. Paris; Lavoisier; 1987. Manuel technique de génétique par électrophorèse dês proteins: Technique et documentation 1987 Pasteur N Pasteur G Bonbomme F Catalan J Britton-Davidian J 28. Boerlin P. Applications of multilocus enzyme electrophoresis in medical microbiology. J Microbiol Meth 1997; 28: 221-31. Applications of multilocus enzyme electrophoresis in medical microbiology J Microbiol Meth 1997 221 31 28 Boerlin P. 29. Hunter PR. A critical review of typing methods for Candida albicans and their applications. Crit Rev Microbiol 1991; 17: 417-34. A critical review of typing methods for Candida albicans and their applications Crit Rev Microbiol 1991 417 34 17 Hunter PR 30. Arnavielhe S et al. Suivi mycologique d´infections à Candida albicans dans divers services hospitaliers. Path Biol 1996; 44: 447-51. Suivi mycologique d´infections à Candida albicans dans divers services hospitaliers Path Biol 1996 447 51 44 Arnavielhe S 31. Bertout S, Renaud F, Swinne D, Mallie M, Bastide J-M. Genetic multilocus studies of different strains of Cryptococcus neoformans: taxonomy and genetic structure. J Clin Microbiol 1999; 37: 715-20. Genetic multilocus studies of different strains of Cryptococcus neoformans: taxonomy and genetic structure J Clin Microbiol 1999 715 20 37 Bertout S Renaud F Swinne D Mallie M Bastide J-M 32. Boerlin P et al. Cluster of oral atypical Candida albicans isolates in a group of human immunodeficiency virus-positive drug users. J Clin Microbiol 1995; 33: 1129-35. Cluster of oral atypical Candida albicans isolates in a group of human immunodeficiency virus-positive drug users J Clin Microbiol 1995 1129 35 33 Boerlin P 33. Doebeling BN et al. Comparison of pulsed-field gel electrophoresis with isoenzyme profiles as a typing system for Candida tropicalis. Clin Infect Dis 1993; 16: 377-83. Comparison of pulsed-field gel electrophoresis with isoenzyme profiles as a typing system for Candida tropicalis Clin Infect Dis 1993 377 83 16 Doebeling BN 34. Lehmann PF, Kemker BJ, Hsiao C-B, Dev S. Isoenzyme biotypes of Candida species. J Clin Microbiol 1989; 27: 2514-21. Isoenzyme biotypes of Candida species J Clin Microbiol 1995 2514 21 27 Lehmann PF Kemker BJ Hsiao C-B Dev S 35. Lehmann PF, Wu L-C, Mackenzie DWR. Isozyme changes in Candida albicans domestication. J Clin Microbiol 1991; 29: 2623-5. Isozyme changes in Candida albicans domestication J Clin Microbiol 1991 2623 5 29 Lehmann PF Wu L-C Mackenzie DWR 36. Lehmann PF, Wu L-C, Pruitt WR, Meyer SA, Ahearn DG. Unrelatedness of groups of yeasts within the Candida haemulonii complex. J Clin Microbiol 1993; 31: 1683-7. Unrelatedness of groups of yeasts within the Candida haemulonii complex J Clin Microbiol 1993 1683 7 31 Lehmann PF Wu L-C Pruitt WR Meyer SA Ahearn DG 37. Lin D, Wu L-C, Rinaldi MG, Lehmann PF. Three distinct genotypes within Candida parapsilosis from clinical sources. J Clin Microbiol 1995; 33: 1815-21. Three distinct genotypes within Candida parapsilosis from clinical sources J Clin Microbiol 1995 1815 21 33 Lin D Wu L-C Rinaldi MG Lehmann PF 38. Pujol C, Reynes J, Renaud F, Mallie M, Bastide J-M. Genetic analysis of Candida albicans strains studies by isoenzyme electrophoresis. J Mycol Med 1993; 3: 14-9. Genetic analysis of Candida albicans strains studies by isoenzyme electrophoresis J Mycol Med 1993 14 9 3 Pujol C Reynes J Renaud F Mallie M Bastide J-M 39. Pujol C et al. The yeast Candida albicans has a clonal mode of reproduction in a population of infected human immunodeficiency viry-positive patients. Proc Natl Acad Sci USA 1993; 90: 9456-9. The yeast Candida albicans has a clonal mode of reproduction in a population of infected human immunodeficiency viry-positive patients Proc Natl Acad Sci USA 1993 9456 9 90 Pujol C 40. San Millan RM, Wu LC, Salkin IF, Lehmann PF. Clinical isolates of Candida guilliermondii include Candida fermentati. Int J Syst Bacteriol 1997; 47: 385-93. Clinical isolates of Candida guilliermondii include Candida fermentati Int J Syst Bacteriol 1997 385 93 47 San Millan RM Wu LC Salkin IF Lehmann PF 41. Moreira D et al. Candida spp. Biotypes in the oral cavity of school children from different socioeconomic categories in Piracicaba SP, Brazil. Pesqui Odontol Bras 2001; 15: 187-95. Candida spp. Biotypes in the oral cavity of school children from different socioeconomic categories in Piracicaba - SP, Brazil Pesqui Odontol Bras 2001 187 95 15 Moreira D 42. Asakura K, Iwaguchi S, Homma M, Sukai T, Higashide K, Tanaka K. Electrophoretic karyotypes of clinically isolated yeasts of Candida albicans and C. glabrata. J Gen Microbiol 1991; 137: 2531-8. Electrophoretic karyotypes of clinically isolated yeasts of Candida albicans and C. glabrata J Gen Microbiol 1991 2531 8 137 Asakura K Iwaguchi S Homma M Sukai T Higashide K Tanaka K 43. Casanova M, Chaffin DWL. Cell wall glycoproteins of Candida albicans as released by different methods. J Gen Microbiol 1991; 137: 1045-51. Cell wall glycoproteins of Candida albicans as released by different methods J Gen Microbiol 1991 1045 51 137 Casanova M Chaffin DWL 44. Waters MG, Blobel G. Secretory protein translocation in a yeast cell-free system can occur posttranslationally and requires ATP hydrolysis. J Cell Biol 1986; 102: 1543-50. Secretory protein translocation in a yeast cell-free system can occur posttranslationally and requires ATP hydrolysis J Cell Biol 1986 1543 50 102 Waters MG Blobel G 45. Woontner M, Jaehning JA. Accurate initiation by RNA polimerase II in a whole cell extract from Saccharomyces cerevisiae. J Biol Chem 1990; 265: 8979-82. Accurate initiation by RNA polimerase II in a whole cell extract from Saccharomyces cerevisiae J Biol Chem 1986 8979 82 265 Woontner M Jaehning JA 46. Antonsson B, Montessuit S, Friedli L, Payton MA, Paravicini G. Protein kinase C in yeast. Characteristics of the Saccharomyces cerevisiae PKC1 gene product. J Biol Chem 1994; 269: 16821-8. Protein kinase C in yeast: Characteristics of the Saccharomyces cerevisiae PKC1 gene product J Biol Chem 1994 16821 8 269 Antonsson B Montessuit S Friedli L Payton MA Paravicini G 47. Alfenas AC. Eletroforese de isoenzimas e proteínas afins; fundamentos e aplicações em plantas e microrganismos. Viçosa; Editora UFV; 1998. Eletroforese de isoenzimas e proteínas afins; fundamentos e aplicações em plantas e microrganismos 1994 Alfenas AC 48. Selander RK, Caugant DA, Ochman H, Musser JM, Gilmour MN, Whittam TS. Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl Environ Microbiol 1986; 51: 873-84. Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics Appl Environ Microbiol 1986 873 84 51 Selander RK Caugant DA Ochman H Musser JM Gilmour MN Whittam TS 49. Hartl DL, Clark AG. Principles of population genetics. Inc. Publishers Suderland, Mass.; Sinauer Associates; 1997. Principles of population genetics 1997 Hartl DL Clark AG 50. Le Guennec R, Reynes J, Mallie M, Pujol C, Janbon F, Bastide J-M. Fluconazole- and itraconazole-resistant Candida albicans strain from AIDS patients: multilocus enzyme electrophoresis analysis and antifungal susceptibilities. J Clin Microbiol 1995; 33: 2732-7. Fluconazole- and itraconazole-resistant Candida albicans strain from AIDS patients: multilocus enzyme electrophoresis analysis and antifungal susceptibilities J Clin Microbiol 1995 2732 7 33 Le Guennec R Reynes J Mallie M Pujol C Janbon F Bastide J-M 51. Nébavi F et al. Oropharyngeal candidiasisj in AIDS patients from Abidjan (Ivory Coast): antifungal susceptibilities and multilocus enzyme electrophoresis analysis of Candida albicans isolates. Path Biol 1998; 46: 307-14. Oropharyngeal candidiasisj in AIDS patients from Abidjan (Ivory Coast): antifungal susceptibilities and multilocus enzyme electrophoresis analysis of Candida albicans isolates Path Biol 1998 307 14 46 Nébavi F 52. Nei M. Genetic distances between populations. Am Naturalist 1972; 106, 283-92. Genetic distances between populations Am Naturalist 1972 283 92 106 Nei M. 53. Sneath PHA, Sokal RR. Numerical taxonomy. San Francisco; W.H. Freeman and Company; 1973. Numerical taxonomy 1973 Sneath PHA Sokal RR 54. Vancanneyt M, Pot B, Hennebert G, Kersters K. Differentiation of yeast species based on electrophoretic whole-cell protein patterns. Syst Appl Microbiol 1991; 14: 23-32. Differentiation of yeast species based on electrophoretic whole-cell protein patterns Syst Appl Microbiol 1991 23 32 14 Vancanneyt M Pot B Hennebert G Kersters K 55. Scherer S, Magee PT. Genetics of Candida albicans. Microbiol Rev 1990; 54: 226-41. Genetics of Candida albicans Microbiol Rev 1990 226 41 54 Scherer S Magee PT 56. Soll DR, Galask R, Schmid J, Hanna C, Mac K, Morrow B. Genetic dissimilarity of commensal strains of Candida spp. carried in different anatomical localizations of the same healthy women. J Clin Microbiol 1991; 29: 1702-10. Genetic dissimilarity of commensal strains of Candida spp. carried in different anatomical localizations of the same healthy women J Clin Microbiol 1991 1702 10 29 Soll DR Galask R Schmid J Hanna C Mac K Morrow B 57. Jorge AOC, Almeida NQ, Unterkircher CS, Shimizu MT. Influence of the use of orthodontic apparatus in presence of Candida albicans in oral cavity. Rev Assoc Paul Cir Dent 1987; 41: 308-10. Influence of the use of orthodontic apparatus in presence of Candida albicans in oral cavity Rev Assoc Paul Cir Dent 1987 308 10 41 Jorge AOC Almeida NQ Unterkircher CS Shimizu MT 58. Selander RK, Levin BR. Genetic diversity and structure in Escherichia coli populations. Science 1980; 210: 545-7. Genetic diversity and structure in Escherichia coli populations Science 1980 545 7 210 Selander RK Levin BR 59. Selander RK, Musser JM, Caugant DA, Gilmour MN, Whittam TS. Population genetics of pathogenic bacteria. Microb Pathog 1987; 3: 1-7. Population genetics of pathogenic bacteria Microb Pathog 1987 1 7 3 Selander RK Musser JM Caugant DA Gilmour MN Whittam TS 60. Tibayrenc M, Ward P, Moya A, Ayala FJ. Natural populations of Trypanosoma cruzi, the agent of Chagas disease, have a complex multiclonal structure. Proc Natl Acad Sci USA 1986; 83: 115-9. Natural populations of Trypanosoma cruzi, the agent of Chagas disease, have a complex multiclonal structure Proc Natl Acad Sci USA 1986 115 9 83 Tibayrenc M Ward P Moya A Ayala FJ 61. Tibayrenc M, Kjellberg F, Ayala FJ. A clonal theory of parasitic protozoa: the population structures of Entamoeba, Giardia, Leishmania, Naegleria, Plasmodium, Trichomonas, and Trypanosoma and their medical and taxonomical consequences. Proc Natl Acad Sci USA 1990; 87: 2414-8. A clonal theory of parasitic protozoa: the population structures of Entamoeba, Giardia, Leishmania, Naegleria, Plasmodium, Trichomonas, and Trypanosoma and their medical and taxonomical consequences Proc Natl Acad Sci USA 1990 2414 8 87 Tibayrenc M Kjellberg F Ayala FJ 62. Tibayrenc M et al. Are eukaryotic microorganisms clonal or sexual? A population genetics vantage. Proc Natl Acad Sci USA 1991; 88: 5129-33. Are eukaryotic microorganisms clonal or sexual? A population genetics vantage Proc Natl Acad Sci USA 1991 5129 33 88 Tibayrenc M 63. Reynes J et al. Simultaneous carriage of Candida albicans strains from HIV-infected patients with oral candidiasis: multilocus enzyme electrophoresis analysis. FEMS Microbiol Lett 1996; 137: 269-73. Simultaneous carriage of Candida albicans strains from HIV-infected patients with oral candidiasis: multilocus enzyme electrophoresis analysis FEMS Microbiol Lett 1996 269 73 137 Reynes J 64. Schmid J et al. Evidence for a general-purpose genotype in Candida albicans, highly prevalent in multiple geographical regions, patient types and types of infection. Microbiology 1999; 145: 2405-13. Evidence for a general-purpose genotype in Candida albicans, highly prevalent in multiple geographical regions, patient types and types of infection Microbiology 1999 2405 13 145 Schmid J 65. Rustchenko EP, Howard DH, Sherman F. Chromosomal alterations of Candida albicans are associated with the gain and loss of assimilating functions. J Bacteriol 1994; 176: 3231-41. Chromosomal alterations of Candida albicans are associated with the gain and loss of assimilating functions J Bacteriol 1994 3231 41 176 Rustchenko EP Howard DH Sherman F 66. Rustchenko-Bulgac EP, Sherman F, Hicks JB. Chromosomal rearrangements associated with morphological mutants provide a means for genetic variation of Candida albicans. J Bacteriol 1990; 172: 1276-83. Chromosomal rearrangements associated with morphological mutants provide a means for genetic variation of Candida albicans J Bacteriol 1990 1276 83 172 Rustchenko-Bulgac EP Sherman F Hicks JB 67. Chibana H, Iwaguchi S-I, Homma M, Chindamporn A, Nakagawa Y, Tanaka K. Diversity of tandemly repetitive sequences due to short periodic repetitions in the chromosomes of Candida albicans. J Bacteriol 1994; 176: 3851-8. Diversity of tandemly repetitive sequences due to short periodic repetitions in the chromosomes of Candida albicans J Bacteriol 1994 3851 8 176 Chibana H Iwaguchi S-I Homma M Chindamporn A Nakagawa Y Tanaka K 68. Sadhu C, McEachern MJ, Rustchenko-Bulgac EP, Schmid J, Soll DR, Hicks JB. Telomeric and dispersed repeat sequences in Candida yeasts and their use in strain identification. J Bacteriol 1991; 173: 842-50. Telomeric and dispersed repeat sequences in Candida yeasts and their use in strain identification J Bacteriol 1991 842 50 173 Sadhu C McEachern MJ Rustchenko-Bulgac EP Schmid J Soll DR Hicks JB 69. Powderly WG, Robinson K, Keath EJ. Molecular typing of Candida albicans isolated from oral lesions of HIV-infected individuals. AIDS 1992; 6: 81-4. Molecular typing of Candida albicans isolated from oral lesions of HIV-infected individuals AIDS 1992 81 4 6 Powderly WG Robinson K Keath EJ 70. Whelan WL, Kirsch DR, Know-Chung KJ, Wahl SM, Smith PD. Candida albicans in patients with the aquired immunodeficiency syndrome: absence of a novel or hypervirulent strain. J Infect Dis 1990; 162: 513-8. Candida albicans in patients with the aquired immunodeficiency syndrome: absence of a novel or hypervirulent strain J Infect Dis 1990 513 8 162 Whelan WL Kirsch DR Know-Chung KJ Wahl SM Smith PD 71. Lockhart S et al. Colonizing populations of Candida albicans are clonal in origin but undergo microevolution through C1 fragment reorganization as demonstrated by DNA fingerprinting and C1 sequencing. J Clin Microbiol 1995; 33: 1501-9. Colonizing populations of Candida albicans are clonal in origin but undergo microevolution through C1 fragment reorganization as demonstrated by DNA fingerprinting and C1 sequencing J Clin Microbiol 1995 1501 9 33 Lockhart S 72. Pujol C, Joly S, Notan B, Srikantha T, Soll DR. Microevolutionary changes in Candida albicans identified by the complex Ca3 fingerprinting probe involve insertions and deletions of the full-length repetitive RPS at specific genomic sites. Microbiology 1999; 145: 2635-46. Microevolutionary changes in Candida albicans identified by the complex Ca3 fingerprinting probe involve insertions and deletions of the full-length repetitive RPS at specific genomic sites Microbiology 1999 2635 46 145 Pujol C Joly S Notan B Srikantha T Soll DR 73. Schroeppel K, Rotman M, Galask R, Mac K, Soll DR. The evolution and replacement of Candida albicans strains during recurrent vaginitis demonstrated by DNA fingerprinting. J Clin Microbiol 1994; 32: 2646-54. The evolution and replacement of Candida albicans strains during recurrent vaginitis demonstrated by DNA fingerprinting J Clin Microbiol 1994 2646 54 32 Schroeppel K Rotman M Galask R Mac K Soll DR