M13 Core Sequence Research Articles

Hierarchical genetic variation of Botryosphaeriaceae species associated with decline and dieback of grapevine in south-eastern Australia

Background and Aims Species of Botryosphaeriaceae have been reported in association with various grapevine diseases, such as trunk diseases and fruit rots worldwide. Their distribution, however, has not been thoroughly studied at the genetic level. The aim of this study was to evaluate genetic variation within four species of Botryosphaeriaceae isolated from south-eastern Australian vineyards at several geographical scales. Methods and Results Fungal isolates were taken using a hierarchical methodology. A range of polymerase chain reaction (PCR) primers, including a selected set of six inter-simple sequence repeat primers, the intron splice junction R1 primer and the M13 core sequence microsatellite primer, were selected after preliminary screening. A total of 171 isolates of the Botryosphaeriaceae was assessed, comprising 127 Diplodia seriata, 23 Neofusicoccum parvum, 18 Botryosphaeria dothidea and 3 Lasiodiplodia theobromae isolates from three closely located regions. From these, 280 highly reproducible polymorphic bands were detected across all isolates following PCR. Intra- and inter-specific differences were visualised as a dendrogram and a principle coordinates plot. Cluster analysis with the integrated locus matrix separated the isolates into four distinguishable groups according to species (cophenetic correlation = 0.97). Conclusions Our study indicates that genetic differentiation by region was evident within D. seriata, and there was variation in genotype between vineyards in several instances. Isolates of N. parvum showed greater homogeneity between vineyards and regions, while B. dothidea showed homogeneity between regions from which they were isolated. Significance of the Study The degree of variation in populations of Botryosphaeriaceae in vineyards indicated in this study can be helpful in the future for controlling the spread of the pathogen in Australian vineyards and can be used to guide sampling in future surveys and population studies of the genus. The unique banding patterns of each species detected in this study can be utilised in the future for designing species-specific primers, which would enable in-field real-time detection of Botryosphaeriaceae without destructive sampling.

DNA polymorphism analysis of Xanthomonas campestris pv. campestris using single-strand conformation polymorphism (SSCP) and random amplified polymorphic DNA (RAPD)

The applicability of the random amplified polymorphic DNA (RAPD) and single-strand conformation polymorphism (SSCP) techniques using M13 and 16S rRNA primers, respectively, for genotyping of the phytopathogenic bacterium Xanthomonas campestris pv. campestris was studied. RAPD provided a simple, rapid, and reliable method to identify genetic variation, and SSCP was used to screen for sequence polymorphisms. A 730-bp region amplified using 16S rRNA primers was subjected to restriction digestion followed by SSCP analysis (restriction fragment length polymorphism (RFLP)-SSCP) to identify polymorphisms, and the results were compared with the RAPD profile obtained using the phage M13 core sequence as a single primer. SSCP is a useful tool for the detection of mutations but large amplicon size can hinder secondary structure formation. Therefore, RAPD using an M13 primer is more efficient for phylogeny detection. The use of a novel polymerase chain reaction (PCR)-based DNA fingerprinting method using both RAPD and SSCP to detect DNA sequence diversity is reported. This approach consists of PCR amplification of the 16S ribosomal DNA with universal primers and analysis of the PCR product by SSCP. This is the first report wherein plant pathogens are subjected to RFLP-SSCP combined with M13 primer-based analysis. This report provides a rapid and reliable method for phylogenetic analysis. Keywords: Xanthomonas campestris pv. campestris, polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP), microbial phylogenetics, randomly amplified polymorphic DNA (RAPD). Afr. J. Biotechnol. Vol. 12 No. 50

Genotypic Characterization of Cutaneous Leishmaniasis at al Baha and Al Qasim Provinces (Saudi Arabia)

Fifty samples of skin ulcers were collected from the western region of Saudi Arabia Kingdom (Al Baha and Al Qasim) to study genotypic characterization of Cutaneous Leishmaniasis in this area. Thirty-six samples were recorded as Leishmania isolates. The same isolates were subsequently tested with fingerprinting with single arbitrary primers. The primers used derived from the core sequence of the phage M13, and the repeat sequences (GTG)5 and (GACA)4. The 36 isolates were all identified as Leishmania major (n = 25 isolates) or Leishmania tropica (n = 11 isolates). All produced polymorphic patterns, which were grouped depending on the species they belonged to, next to the relevant well-characterized strains of the same species. Within the L. major and L. tropica group the subgroupings formed were mainly related to the geographical origin of the strains. A nested polymerase chain reaction-based schizodeme method for identifying Leishmania kinetoplast minicircle classes was used as a diagnostic tool for L. major and L. tropica.

Genetic variation of Heterobasidion abietinum in Southern Europe and the Mediterranean Basin

Summary Heterobasidion abietinum causes root and butt rot on Abies species occurring in central Europe and the Mediterranean Basin in a distributionextending from Spain in the west to Turkey and Caucasia in the east. To investigate the genetic diversity within H. abietinum, 95 isolates fromdifferent regions were analysedwith 61 polymorphic molecular markers. The markers showed genetic differentiation amongst the H. abietinumpopulations. The most diversified population is that on A. pinsapo in southern Spain, which is isolated from gene flow with other populations.Lower but still significant differences were found amongst other European populations. Isolates from Turkey showed little diversificationcompared with the south European isolates from east of the Alps. The genetic structure of the H. abietinum populations in relation topostglacial recolonization of Abies is discussed. 1 Introduction Heterobasidion abietinum Niemela¨ & Korhonen, a member of the H. annosum complex, is a pathogen or saprotroph on Abiesspecies occurring in central Europe and in the Mediterranean Basin (Niemela¨ and Korhonen 1998; Woodward et al. 1998;Asiegbu et al. 2005). Its main host species is A. alba Mill. H. abietinum has economic and environmental impacts especially inItaly on A. alba and in Greece on A. cephalonica Loud. and A. borisii-regis Mattf. (Tsopelas and Korhonen 1996; Capretti 1998;Korhonen and Dai 2005). In these countries the damage caused by the fungus is related to stress caused by dry summers(Puddu et al. 2003). In Central Europe and the Pyrenees, H. abietinum is mainly a saprotroph, colonizing mostly stumps andlogs of A. alba and has a lower economic impact than in Italy and Greece (Munda 1994; Łakomy 1996; Korhonen et al. 1998;Puddu et al. 2003; Oliva and Colinas 2010).Other host species of H. abietinum are present in western and eastern regions of the Mediterranean Basin. The westernmosthost is A. pinsapo, a species occurring in a small area of Andalucia, southern Spain (Sa`nchez et al. 2005, 2007). In the east, H.abietinum was recorded on A. cilicica Carr. and A. nordmanniana (Steven) Spach in Turkeyand Russian Caucasia (Korhonen andDai 2005; Dog˘mus¸-Lehtija¨rvi et al. 2006).Recent work investigating genetic variability in H. abietinum populations in the Balkan Peninsula, the Pyrenees and theItalian Peninsula indicated that geographical provenances formed separate clusters (Zamponi et al. 2007). In addition, a highlevel of differentiation was found between H. abietinum isolates collected from A. pinsapo and those from A. alba in Italy andFrance (Luchi et al. 2008).These previous population studies were performed using the DNA fingerprinting DAMD-PCR (Direct AmplificationMinisatellite DNA) that includes direct amplification of minisatellite-region DNA by the polymerase chain reaction (PCR) usingthe M13 core sequence. This approach had already been applied to studies of genetic variations in H. annosum (Fr.) Bref. sensustricto and H. abietinum (Petta et al. 2001; Zamponi et al. 2007). The utility of RAMS-PCR in studying genetic variability withinthe H. annosum complex (Vainio and Hantula 1999; Dai et al. 2003; DAmico et al. 2007) and other tree pathogens (Vainio et al.1998; Kasanen et al. 2004) was demonstrated previously. The use of these repetitive DNA elements has been applied to studieson population genetics, ecology and taxonomy of many fungi (Wo¨stemeyer and Kreibich 2002).The aim of this study was to investigate the population structure of H. abietinum collected from different hosts in severalregions in southern Europe and around the Mediterranean Basin. The work was based on molecular analysis using micro- andminisatellites. In addition, the number of mating factors was identified from a sample of isolates collected in each locality.

The Genetic Structure of Cypress Canker Fungus in Italy Using RAPD and Minisatellite Markers

Over the past half century a destructive blight of Cupressus spp., caused by Seiridium cardinale, has spread worldwide from North America, devastating forests, plantations, and ornamental cypresses. The epidemic has been particularly severe in the Mediterranean region, on C. sempervirens. Seventy-seven isolates of Seiridium cardinale have been collected for the genetic characterization of the North-Italian populations of the fungus. Five Seiridium spp. isolates from different countries and different hosts growing in different parts of the world were used for comparison. The structure of the population has been analysed by means of Random Amplified Polymorphic DNAs (RAPDs) and Direct Amplification of Minisatellite-Region DNA (DAMD) PCR marker techniques by using the M13 core sequence. The results indicated a very high level of homogeneity in the North-Italian population of the fungus, whereas a certain variability was recognized in isolates from other hosts and other species. The isolates belonged to the North-Italian population appear to be very similar from the molecular comparison with both type of markers. The isolate from Greece was included in the same group of the Italian isolates. Only the S. cardinale from Chile was clustered at significant distance from the other S. cardinale isolates from Italy and Greece. The genetic homogeneity of the fungus in Italy suggests that this population has gone through a recent genetic bottleneck, perhaps from the introduction in Europe of few genotypes of the fungus. This supports the hypothesis that the pathogen was introduced to Europe during World War II on infected wood material from the United States. The results are discussed in relation to the introduction and spread of the fungus in Europe.

RAPD with microsatellite as a tool for differentiation of Candida genus yeasts isolated in brewing

Fifteen wild yeast strains were isolated in two factories of a lager brewing company in Poland. Their identification with API 32C system showed mainly the presence of Candida sake species (7/15). To differentiate the isolates, randomly amplified polymorphic DNA (RAPD) with (GTG) 5, (GAC) 5, (GACA) 4 microsatellite primers and M13 core sequence (5′-GAG GGT GGC GGT TCT-3′) were chosen. The results of patterns similarity are presented as dendrograms for each RAPD analysis and for overall patterns. On the overall patterns, all isolates identified as C. sake, except Strain No. 1, were regrouped in one cluster. Collection strain C. sake CBS 617 was similar in 46% to the cluster with six isolates (Strain Nos. 3, 6, 8, 11, 13, 14). The second reference strain C. sake CBS 159 and the Strain No. 1 were regrouped with other Candida species (collection strains) showing, respectively, only 20% and 42% of similarity to other C. sake strains. The similarity based on the overall dendrogram between isolate Nos. 3, 6, 8, 11, 13, 14 and C. sake CBS 617 was 49%. Between those strains and other Candida, the similarity was only 37%.

PCR-Based Differentiation and Homology of Brewing and Type Strains of the Genus Saccharomyces

Restriction fragment length polymorphism (RFLP) patterns of PCR-amplified ribosomal RNA gene fragments (rDNA) and randomly amplified polymorphic DNA (RAPD) were applied for the analysis of 15 brewing and 6 related yeast strains of the genus Saccharomyces. One five-base (ScrFI) and two four-base cutting (HaeIII, MspI) restriction enzymes were used. The primers 21 and M13 core sequence were selected for RAPD analysis. PCR-RFLP rDNA analysis with HaeIII, ScrFI and MspI differentiated the strains tested into four, five and four types of patterns, respectively and the analyses of the profiles showed 100% homology, between the yeast strains. One strain was an exception. Homological groups were observed for strains used in breweries globally, from a local production strain and from the isolates identified as S. cerevisiae. Using RAPD analysis, and according to discrete differences in the profiles, it was possible to divide twenty one strains into 15 and 20 groups with primer 21 and M13 respectively. RFLP-PCR rDNA analysis was used to show similarities in closely related brewing strains, while RAPD analysis was used for differentiation of strains.

Analysis of the Italian Dutch Elm Disease Fungal Population

AbstractSixty‐two Ophiostoma ulmi sensu lato strains have been collected from symptomatic trees in seven areas of Central Italy. Isolates were compared with 10 reference strains, belonging to the species O. ulmi and to the two subspecies of O. novo‐ulmi, in order to establish the genetic variability within the Italian population of this fungal pathogen. The structure of the population has been analysed by means of morpho‐physiological features and of the direct amplification of minisatellite‐region DNA polymerase chain reaction (DAMD‐PCR) by using the M13 core sequence. The DNA profiles have been compared with taxonomic parameters (growth rate, culture aspect and fertility barriers). Taxa could thus be well separated. None of the isolates collected was recognized as O. ulmi. Isolates assigned to the two subspecies of O. novo‐ulmi (novo ulmi and americana) by means of the fertility test, showed short genetic distances with the respective reference strains and they constituted subgroups according to their geographical origin. The high level of variation detected indicates a postepidemic situation in Italy. Some inconsistency was found within the subspecies clusters. Several isolates, assigned to subspecies americana using fertility test, were in the novo‐ulmi cluster and vice versa. A possible explanation is that these isolates are americana–novo‐ulmi hybrids.

Genetic characterization of the nonconventional yeast Hansenula anomala

We describe genetic, molecular and taxonomic characteristics of the yeast Hansenula anomala. Pulsed-field gel electrophoresis of chromosomal DNAs from 19 H. anomala strains and related species indicated that H. anomala had a clearly different karyotype. Chromosome length polymorphism of the H. anomala strains was independent of their geographic origin and source of isolation. The strains were classified into four groups of similar karyotypes and one strain showed a unique profile. The sizes of chromosomes ranged from 850 to 3500 kb in different strains. The haploid chromosome number of H. anomala is at least nine. We have found RAPD primers discriminating at both the species and strain levels. All the primers tested except the M13 core sequence generated unique patterns with most strains. The results indicate the usefulness of PCR analysis with primer M13 for identification of the H. anomala species. Screening of the CBS (Utrecht) collection strains of H. anomala showed that they are rather difficult objects for genetic hybridization analysis. The strains have low fertility, viz. very poor sporulation, low mating type activities and, as a rule, nonviable ascospores. The majority of the hybrids obtained are polyploid, probably tetraploid, as judged by the segregation of control auxotrophic markers. Nevertheless, some monosporic cultures of the strains studied, including the biocontrol yeast J121, formed diploid hybrids with regular meiotic segregation of control auxotrophic markers. As a rule, H. anomala isolates are homothallic, showing delayed self-diploidization. Rare stable heterothallic strains of H. anomala also occur.

Species and genotypic diversities and similarities of pathogenic yeasts colonizing women.

We examined the patterns of strain relatedness among pathogenic yeasts from within and among groups of women to determine whether there were significant associations between genotype and host condition or body site. A total of 80 yeast strains were isolated, identified, and genotyped from 49 female volunteers, who were placed in three groups: (i) 19 women with AIDS, (ii) 11 pregnant women without human immunodeficiency virus (HIV) infection, and (iii) 19 women who were neither pregnant nor infected with HIV. Seven yeast species were recovered, including 59 isolates of Candida albicans, 9 isolates of Candida parapsilosis, 5 isolates of Candida krusei, 3 isolates of Candida glabrata, 2 isolates of Saccharomyces cerevisiae, and 1 isolate each of Candida tropicalis and Candida lusitaniae. Seventy unique genotypes were identified by PCR fingerprinting with the M13 core sequence and by random amplified polymorphic DNA analysis. Of the nine shared genotypes, isolates from three different hosts were of one genotype and pairs of strains from different body sites of the same host shared each of the other eight genotypes. Genetic similarities among groups of strains were calculated and compared. We found no significant difference in the patterns of relatedness of strains from the three body sites (oral cavity, vagina, and rectum), regardless of host conditions. The yeast microflora of all three host groups had similar species and genotypic diversities. Furthermore, a single host can be colonized with multiple species or multiple genotypes of the same species at the same or different body sites, indicating dynamic processes of yeast colonization on women.

Genotypic relatedness of yeast isolates from women infected with human immunodeficiency virus and their children.

The objective of this study was to compare polymerase chain reaction (PCR) fingerprinting with other molecular typing methods as an epidemiologic tool to investigate the transmission of Candida strains between HIV-positive mothers and their children. Forty-nine yeast strains (including Candida albicans, Candida glabrata, Rhodotorula rubra, Candida tropicalis, Candida famata, Candida dubliniensis, Saccharomyces cerevisiae) from 30 individuals (15 children and 15 HIV-infected mothers or accompanying person) were isolated. Colonization/infection with yeast was observed in 80% of all individuals in the oral cavity, and in 33% from hand cultures, respectively. Thirteen out of 15 children (86%) and 12 out of 15 adults (80%) were colonized/infected with yeasts. Candida dubliniensis strains were found in four HIV-infected women but not in children. The results with an arbitrarily primed (AP)-PCR mediated genotyping assay using phage M13 core sequence were compared with the hybridization patterns using the species-specific DNA probe CARE-2 for the C. albicans isolates. Typing of non-C. albicans strains was done using AP-PCR in comparison with pulsed-field gel electrophoresis (PFGE). Twenty-six C. albicans strains gave two different genotypes by AP-PCR but 16 genotypes by CARE-2 hybridization. The CARE-2 probe appeared to have a higher discriminatory power compared with the primer 'M13'-mediated AP-PCR in typing C. albicans isolates.

Population structure and genetic variation in Cylindrobasidium evolvens

A total of 34 isolates of Cylindrobasidium evolvens were obtained from wounded Picea abies in five sample plots in Sweden and Lithuania. Population structure and genetic variation within the species were assessed by somatic incompatibility and arbitrary primed DNA (AP-DNA) analysis, using the M13 core sequence as a primer. All somatic incompatibility pairings between different C. evolvens isolates resulted in weak mycelial antagonism. The AP-DNA profiles indicate genetic variation was high in the species with no differentiation among isolates being found either on a large (more than 700 km) ( R 2 = 0·108) or a fine scale (200 m) ( R 2 = 0·024). In a plot, genetic similarity was significantly higher ( P

Genetic diversity within and among vegetative compatibility groups of Stereum sanguinolentum determined by arbitrary primed PCR

Genetic variation within and among vegetative compatibility groups (VCGs) of Stereum sanguinolentum isolates was investigated with various geographical distances. DNA fingerprints were made using the M13 core sequence as a primer. A total of 113 isolates from 12 plots in Sweden, Finland and Lithuania were studied. Each VCG produced a discrete group of banding patterns. Among 20 isolates from the largest VCG, G1, the incidence of identical banding patterns was 24% within a sample plot, 8% among sample plots within a country, and 0% among countries. In the other 15 VCGs that comprised two and more isolates, corresponding percentages were 42%, 30% and 0%. Average band‐sharing indices (ABSIs) within VCGs decreased with increasing geographical distance: for isolates from VCG G1, ABSI was 91.3% within sample plots, 85.4% among sample plots within a country, and 79.2% among countries. Corresponding figures for the other 15 VCGs were 97.0%, 92.7% and 80.4%, respectively. Among VCGs similarities were significantly lower (P < 0.001) and the trend of decreasing similarity with larger geographical distances was less pronounced: ABSI within sample plots was 73.7%, among sample plots within countries 73.8%, and among countries 71.1%. However, the similarity of populations as compared within and between countries differed significantly (P < 0.001), suggesting geographical differentiation between S. sanguinolentum populations separated by 400 km and the Baltic sea. Calculations of GST clearly indicated geographical population subdivision within the large VCG but not among the total sample. In summary, the results show population structure of inbred lines within VCGs containing closely related strains in local populations and more distant relationships among populations.

Clonality and genetic variation in Amylostereum areolatum and A. chailletii from northern Europe

Genetic variation within and between vegetative compatibility groups (VCGs) of Amylostereum areolatum (Fr.) Boid. and Amylostereum chailletii (Pers.: Fr.) Boid. isolates was investigated. DNA fingerprints were made using the M13 core sequence as a primer. A total of 53 isolates of A. areolatum and 57 isolates of A. chailletii from Lithuania, Sweden, Denmark and Great Britain were studied. In all cases isolates belonging to the same VCG showed identical DNA banding patterns, suggesting that VCGs correspond to clones. In A. areolatum the vast majority of the isolates (spread by Sirex juvencus L.) were assigned to dispersive clones, that have wide geographical distribution (i.e. the same genotypes were detected in Lithuania, Sweden and Denmark), with low genetic variation between the different clones. By contrast, A. chailletii population structure was consistent with the spread of airborne basidiospores produced by outcrossing. Only a small fraction of A. chailletii isolates studied, could be assigned to dispersal clones with a local distribution, spread by Urocerus gigas L. Overall, M13 fingerprinting detected low genetic differentiation in both species in the samples we studied.

Identification of common dermatophytes (Trichophyton, Microsporum, Epidermophyton) using polymerase chain reactions.

Polymerase chain reaction (PCR) fingerprinting detected DNA polymorphisms among frequently isolated species and strains of the genera Trichophyton, Microsporum and Epidermophyton. The patterns generated by this DNA-based method permitted species and strains to be identified. The conventional methods to identify dermatophytes rely on the expression of characteristic morphological features, as well as several physiological properties. Identification is often delayed or problematic because isolates may be slow to form conidia or produce atypical microscopic structures or colony appearances. Using non-specific primers such as (AC)10, (GTG)5, M13 core sequence and AP3, characteristic PCR profiles were generated for 17 species. Intraspecies variables were also observed for four of six varieties of T. mentagrophytes, whereas no detectable DNA variability was found within the three varieties of T. tonsurans. Comparing species-specific PCR fingerprints of clinical isolates with those of type strains, species could be identified by their PCR fingerprints, even if they could not be identified by the accepted phenotypic characteristics.

Molecular subtyping of clinical and environmental strains of Cryptococcus neoformans variety neoformans serotype A isolated from southern Italy.

Analysis of ribosomal DNA (rDNA) restriction fragment-length polymorphism (RFLP) and random amplification of polymorphic DNA (RAPD) was used to investigate the genetic variability and biogeographic distribution of clinical and environmental strains of Cryptococcus neoformans isolated from a limited area of southern Italy, where the selection of a predominant cryptococcal genotype could be expected. All isolates belonged to the species Cr. neoformans variety neoformans serotype A. RFLP analysis of a specific rDNA fragment allowed the distinction of strains of Cr. neoformans from closely related fungal reference species, but neither intraspecies nor intravarieties polymorphism was detected. On the contrary, RAPD fingerprints produced by priming with four different primers [(GTG)5, (GACA)4, M13 core sequence and the 8-mer oligonucleotide (GCGGACGG)] were able to characterize the isolates up to the individual level, indicating the presence of marked heterogeneity among Cr. neoformans serotype A strains in southern Italy.

Intraspecific Variations of Epimedium koreanum by Randomly and Specifically Amplified Polymorphic DNA Markers

Randomly and specifically amplified polymorphic DNA banding patterns based on polymerase chain reaction (PCR) analysis were used to assess the intraspecific genetic variations and relationships within Epimedium koreanum populations. A collection of 21 individuals were classified as different accessions by morphological characters such as leaflet number, shape of leaf base, cauline length, plant height, and leaf area. PCR amplification using 12 primers out of 62 [60 random (10-mer) primers, one 15-mer primer (M13 core sequence), and (GGAT)4] resulted in 89 amplified DNA fragments with polymorphisms (80.9%) in all of the tested plants. Similarity indices between accessions were computed from PCR data, and genetic relationships among intraspecific variations were closely related at the levels ranging from 0.66 to 0.93. These DNA data were not matched well with those of morphological characters because they were divided into two major groups at the similarity coefficient value of 0.74. Primers (VII, VIII) gave rise to monomorphic bands in all of examined plants, but specific primers (M13 core and (GGAT)4 sequences) were found to be very valuable molecular markers to evaluate the interspecific variations in Epimedium koreanum.

Comparison of four molecular typing methods for evaluating genetic diversity among Candida albicans isolates from human immunodeficiency virus-positive patients with oral candidiasis

Candida albicans strain delineation by karyotyping. NotI restriction pattern analysis, hybridization with specific probe 27A, and PCR fingerprinting with the phage M13 core sequence were performed with 30 isolates from the oral cavities of 30 human immunodeficiency virus (HIV)-infected patients and 8 reference strains. Within the panel of clinical isolates, 20 were geographically related, although 10 isolates were susceptible to fluconazole and 10 isolates were resistant to fluconazole. The remaining isolates used in this study were fluconazole resistant and geographically unrelated. A composite DNA type was defined for each of the strains as the combination of types obtained by the four molecular methods. By this procedure, a great diversity of DNA types was found among isolates from the oropharynges of HIV-infected individuals with oral candidiasis. This diversity was not reduced when isolates were evaluated on the basis of whether they came from the same geographical locale and whether they were fluconazole resistant. These data refute the idea of a clonal origin for fluconazole-resistant strains among HIV-positive patients. Karyotyping was the least discriminatory method, yielding 19 DNA types among the 38 strains analyzed. Conversely, hybridization with the 27A probe showed a unique DNA pattern for each of the strains examined in this study. Our results demonstrate that at least two different molecular methods are needed for Candida albicans typing and that there is a great deal of strain variation within the species, irrespective of place of origin or antifungal resistance patterns.

Genetic dissimilarity of two fluconazole-resistant Candida albicans strains causing meningitis and oral candidiasis in the same AIDS patient

We describe a patient with AIDS who simultaneously developed Candida meningitis with three positive cerebrospinal fluid cultures and oral candidiasis. This patient also had a history or recurrent episodes of oral candidiasis treated with fluconazole. The patient did not respond to this therapy but was cured with amphotericin B and flucytosine. In vitro susceptibility tests revealed that each infection was caused by fluconazole-resistant Candida albicans isolates. Strain delineation by karyotyping, NotI restriction pattern analysis, hybridization with the specific probe 27A, and PCR fingerprinting with the phage M13 core sequence clearly demonstrated that meningitis and oral thrush were caused by two genetically different isolates.

Identification and determination of the relationships of species and strains within the genus Leishmania using single primers in the polymerase chain reaction

DNA polymorphisms were assessed in different species and strains within the genus Leishmania by amplifying genomic DNA with single non-specific primers. This polymerase chain reaction (PCR) method employed non-random primers which anneal to mini- and microsatellite DNA sequences like the M13 core sequence and the simple repeat sequences (GTG) 5 and (GACA) 4, and the T3B primer derived from an intergenic spacer for tRNA genes. Distinctive and reproducible sets of amplified DNA fragments were obtained for all Leishmania isolates tested. The number and size of amplification products were found to be characteristic for a given taxon. Highly similar PCR profiles were observed when genomic DNA of representatives of the L. donovani, L. mexicana or L. braziliensis complexes was amplified. By comparing PCR patterns of unidentified Leishmania isolates with those obtained from reference strains it was possible to identify these isolates at the species level. The information of the amplification patterns was used for the construction of phylogenetic trees to measure the genetic relatedness within the genus Leishmania.