Genotypes at the internal transcribed spacers of the nuclear rRNA operon of Pneumocystis jiroveci in nonimmunosuppressed infants without severe pneumonia

The frequency of Pneumocystis jiroveci (human-derived Pneumocystis) in immunocompetent infants developing acute respiratory syndromes has recently been evaluated and has been shown to be close to 25%. Until now, there have been no data on the genomic characteristics of the fungus in these patients, while molecular typing of P. jiroveci organisms was mostly performed with samples from immunosuppressed patients with pneumocystosis (Pneumocystis carinii pneumonia [PCP]). The present report describes the genotypes of P. jiroveci organisms in 26 nonimmunosuppressed infants developing a mild Pneumocystis infection contemporaneously with an episode of bronchioloalveolitis. The typing was based on sequence analysis of internal transcribed spacers (ITSs) 1 and 2 of the rRNA operon, followed by the use of two typing scores. By use of the first score, 11 P. jiroveci ITS types were identified: 10 were previously reported in immunosuppressed patients with PCP, while 1 was newly described. By use of the second score, 13 types were identified, of which 2 were newly described. The most frequent type was identified as type B(1)a(3) (first score), which corresponds to type Eg (second score). Mixed infections were diagnosed in three infants. The occurrence of such diversity of P. jiroveci ITS types, an identical main type, and mixed infections has previously been reported in immunosuppressed patients with PCP. Thus, the P. jiroveci ITS genotypes detected in immunocompetent infants and immunosuppressed patients developing different forms of Pneumocystis infection share characteristics, suggesting that both groups of individuals make up a common human reservoir for the fungus. Finally, the frequency of P. jiroveci in nonimmunosuppressed infants with acute respiratory syndromes and the genotyping results provide evidence that this infant population is an important reservoir for the fungus.     

中国耶氏肺孢子虫基因型的初步分析

为研究我国耶氏肺孢子虫的基因分型,利用巢式PCR方法扩增24例肺孢子虫肺炎(PCP)患者支气管肺泡灌洗液中的耶氏肺孢子虫(Pneumocystis jiroveci)核糖体DNA内转录间隔区(ITS1-5.8S rDNA-ITS2)基因。纯化回收目的片段,将其克隆到PMD18-T载体,并导入JM109细胞系中,每个标本制备3~5个克隆,提取质粒测序。从19例患者的标本中检出ITS1-5.8S rDNA-ITS2基因,其中12份标本的25个克隆的测序结果可用于基因分型。经与Lee等(1998)的分型标准进行比对,本组病例耶氏肺孢子虫的ITS1分为B、E、I、N4型,最常见的为E型;ITS2分为a、b、e、h、i、n6型,最常见的为i型;ITS基因分9型,以Bi多见,有2份标本存在混合感染。     

RFLP analysis of the internal transcribed spacer regions of Sporothrix schenckii.

Restriction fragment length polymorphism (RFLP) analysis was performed on the internal transcribed spacer regions of 204 Sporothrix schenckii isolates and on one strain each of the related fungi, S. schenckii var. luriei, S. curviconia, S. inflata and Ceratocystis stenoceras. S. schenckii isolates, which have been collected from around the world, have already been typed according to their mitochondrial DNA (mtDNA), and are kept in the Department of Dermatology, Kanazawa Medical University, Japan. Approximately 600 bp of the internal transcribed spacer region 1 (ITS1) of their nuclear ribosomal RNA gene (rDNA), 5.8S rDNA and ITS2 was amplified by PCR. From ITS-RFLP analysis of the PCR products, S. schenckii isolates comprised 4 types, rDNA types I - IV. The rDNA type I - III strains corresponded to the Group A strains (mtDNA types 1 - 3, 11, 14 - 19, 22 and 23), while the rDNA type IV strains corresponded to the Group B strains (mtDNA types 4 - 10, 12, 13, 20 and 21), as previously categorized according to their mtDNA-RFLP. The ITS-RFLP patterns of the above 4 related fungi all differed from those of the 4 rDNA types of S. schenckii. Furthermore, only 22 (3.5%) out of a sequence of about 620 bases of the ITS regions of the rDNA differed among representatives of the mtDNA types 1 - 5, 7, 11, 14 - 19, 22 and 23. This difference in the ITS region is smaller than the 10% difference among isolates when estimated by mtDNA-RFLP. From the phylogenetic tree based on the base sequences, rDNA type I - III strains belong to Group I, while rDNA type IV strains belong to Group II which correspond with Groups A and B based on their mtDNA. The Group I strains are predominant in South America and Africa, while Group II are predominant in Australia and Asia. ITS-RFLP analysis is better than mtDNA-RFLP in allowing faster discrimination and identification, and for its ability to divide the 4 types into groups, which is useful in clinical diagnosis and epidemiological investigations of S. schenckii.     

Species identification and strain differentiation of dermatophyte fungi by analysis of ribosomal-DNA intergenic spacer regions

Restriction fragment length polymorphisms (RFLPs) identified in the ribosomal-DNA (rDNA) repeat were used for molecular strain differentiation of the dermatophyte fungus Trichophyton rubrum. The polymorphisms were detected by hybridization of EcoRI-digested T. rubrum genomic DNAs with a probe amplified from the small-subunit (18S) rDNA and adjacent internal transcribed spacer (ITS) regions. The rDNA RFLPs mapped to the nontranscribed spacer (NTS) region of the rDNA repeat and appeared similar to those caused by short repetitive sequences in the intergenic spacers of other fungi. Fourteen individual RFLP patterns (DNA types A to N) were recognized among 50 random clinical isolates of T. rubrum. A majority of strains (19 of 50 [38%]) were characterized by one RFLP pattern (DNA type A), and four types (DNA types A to D) accounted for 78% (39 of 50) of all strains. The remaining types (DNA types E to N) were represented by one or two isolates only. A rapid and simple method was also developed for molecular species identification of dermatophyte fungi. The contiguous ITS and 5.8S rDNA regions were amplified from 17 common dermatophyte species by using the universal primers ITS 1 and ITS 4. Digestion of the amplified ITS products with the restriction endonuclease MvaI produced unique and easily identifiable fragment patterns for a majority of species. However, some closely related taxon pairs, such as T. rubrum-T. soudanense and T. quinkeanum-T. schoenlenii could not be distinguished. We conclude that RFLP analysis of the NTS and ITS intergenic regions of the rDNA repeat is a valuable technique both for molecular strain differentiation of T. rubrum and for species identification of common dermatophyte fungi.     

Genotyping of Pneumocystis jiroveci pneumonia in Italian AIDS patients. Clinical outcome is influenced by dihydropteroate synthase and not by internal transcribed spacer genotype

BACKGROUND: Two Pneumocystis jiroveci independent genomic regions, internal transcribed spacer (ITS) 1 and ITS2, and dihydropteroate synthase (DHPS) gene have been used for typing a cohort of HIV-infected Italian patients with P jiroveci pneumonia (PcP). METHODS: Bronchoalveolar lavage samples isolated from 207 HIV-infected adults were ITS and DHPS genotyped by DNA sequencing and by restriction fragment length polymorphism analysis, respectively. Mutant DHPS samples were cloned and ITS typed. Data on severity, treatment, and outcome of PcP were obtained by chart review. RESULTS: High diversity with 46 different ITS genotypes was observed. At the DHPS locus, 9.1% of samples analyzed were found to be mutated. A correlation was observed between DHPS mutants and greater severity of PcP, as defined by higher lactate dehydrogenase (P = 0.015) and need for intubation (P = 0.002), and worse outcomes, as defined by failure of sulfa treatment (P = 0.04), death, and/or relapse of PcP (P = 0.008). There was a significant difference in ITS genotype patterns between DHPS wild-type and mutants (P = 0.028). CONCLUSIONS: The present data suggest the absence of a correlation between P jiroveci ITS types and specific clinical characteristics. DHPS mutations correlate with possible failure of anti-P jiroveci sulfa therapy, and a trend of association is shown between DHPS mutations and some clinical PcP features.     

Phylogenetic study on Shiraia bambusicola by rDNA sequence analyses

In this study, 18S rDNA and ITS-5.8S rDNA regions of four Shiraia bambusicola isolates collected from different species of bamboos were amplified by PCR with universal primer pairs NS1/NS8 and ITS5/ITS4, respectively, and sequenced. Phylogenetic analyses were conducted on three selected datasets of rDNA sequences. Maximum parsimony, distance and maximum likelihood criteria were used to infer trees. Morphological characteristics were also observed. The positioning of Shiraia in the order Pleosporales was well supported by bootstrap, which agreed with the placement by Amano (1980) according to their morphology. We did not find significant inter-hostal differences among these four isolates from different species of bamboos. From the results of analyses and comparison of their rDNA sequences, we conclude that Shiraia should be classified into Pleosporales as Amano (1980) proposed and suggest that it might be positioned in the family Phaeosphaeriaceae.     

Molecular phylogeny of the fungi of the Iceman's grass clothing

To investigate the origin of the fungal hyphae that cover the grass clothing (cloak, boots) found near the neolithic mummy known as the Tyrolean Iceman, two radiocarbon-dated samples of grass were submitted to DNA extraction. The DNA was then PCR amplified using, respectively, primers specific for the region containing the internal transcribed spacers and the 5.8s rDNA (ITS), and primers specific for an approximately 600-bp long fragment of the nuclear small-subunit ribosomal DNA (SSU rDNA) repeat units of eukaryotes. The amplification products were cloned and sequenced. Sequence analysis of 20 individual ITS clones and of ten SSU rDNA clones indicated that three types of fungal DNA can be extracted from the grass. Phylogenetic analyses, using 5.8s and SSU rDNA fungal reference sequences from EMBL and GenBank databases, suggest that the DNAs come, respectively, from a psychrophilic basidiomycetous yeast, phylogenetically close to Leucosporidium scottii, and from two ascomycetes, one of which is possibly related to the Eurotiales     

Pneumocystis jirovecii multilocus genotyping profiles in patients from Portugal and Spain.

Pneumonia caused by the opportunistic organism Pneumocystis jirovecii is a clinically important infection affecting AIDS and other immunocompromised patients. The present study aimed to compare and characterise the frequency pattern of DNA sequences from the P. jirovecii mitochondrial large-subunit rRNA (mtLSU rRNA) gene, the dihydropteroate synthase (DHPS) gene and the internal transcribed spacer (ITS) regions of the nuclear rRNA operon in specimens from Lisbon (Portugal) and Seville (Spain). Total DNA was extracted and used for specific molecular sequence analysis of the three loci. In both populations, mtLSU rRNA gene analysis revealed an overall prevalence of genotype 1. In the Portuguese population, genotype 2 was the second most common, followed by genotype 3. Inversely, in the Spanish population, genotype 3 was the second most common, followed by genotype 2. The DHPS wild-type sequence was the genotype observed most frequently in both populations, and the DHPS genotype frequency pattern was identical to distribution patterns revealed in other European studies. ITS types showed a significant diversity in both populations because of the high sequence variability in these genomic regions. The most prevalent ITS type in the Portuguese population was Eg, followed by Cg. In contrast to other European studies, Bi was the most common ITS type in the Spanish samples, followed by Eg. A statistically significant association between mtLSU rRNA genotype 1 and ITS type Eg was revealed.     

Molecular characterization of <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis> isolates from the Philippines

Trichomonas vaginalis is a human urogenital pathogen that causes trichomoniasis, the most common nonviral parasitic sexually transmitted infection in the world. Presently, there are no reports on comparative sequence analysis as well as on the identification of phylogenetic positions of T. vaginalis isolates from the Philippines relative to known trichomonads. In this study, 5.8S rDNA and the flanking internal transcribed spacer (ITS) regions of 57 T. vaginalis isolates were sequenced. The phylogenetic positions of the isolates relative to known trichomonads were determined using the model-based (GTR+Γ+I) neighbor-joining, maximum likelihood, and Bayesian-inference analyses and the nonmodel-based maximum parsimony analysis. Construction of a phylogenetic tree showed the clustering of all the sequences in one branch together with other T. vaginalis strains obtained through basic local alignment search tool search. Sequencing of the 5.8S rDNA gene and the flanking ITS1and ITS2 regions of T. vaginalis isolates from the Philippines demonstrated low genetic polymorphism. However, comparison of the ribosomal DNA sequences may have implications on some phenotypic characteristics of T. vaginalis.     

Comparison of the 5.8s rDNA and internal transcribed spacer sequences of isolates of Leptosphaeria maculans from different pathogenicity groups

The regions coding for the 5.8s rRNA and the flanking internal transcribed spacers (ITS1 and ITS2) from nine isolates of the blackleg pathogen Leptosphaeria maculans and one isolate of Sclerotinia sclerotiorum were amplified by the polymerase chain reaction and sequenced. Five of the L. maculans isolates were highly virulent to Brassica plants, two were weakly virulent and two were isolated from the cruciferous weed Thlaspi arvense. The 5.8s DNA sequences of all L. maculans isolates were identical. However, there were major differences in both ITS1 and ITS2 sequences that correlated with the pathogenicity grouping. Phylogenetic analysis of the ITS sequences by both parsimony and maximum-likelihood methods indicated that each pathogenicity group was statistically different from each other with the weaklyvirulent isolates being more closely related to the Thlaspi than to the highly-virulent isolates. The relationships of L. maculans to other fungi, based on a comparison of the 5.8s rDNA sequences, are discussed.     

Molecular analysis of fungal microbiota in samples from healthy human skin and psoriatic lesions

Psoriasis, a common cutaneous disease of unknown etiology, may be triggered by infections, including those due to fungi. Since the fungal community of human skin is poorly characterized, we aimed to analyze the mycological microbiota in healthy skin and psoriatic lesions. Twenty-five skin samples from five healthy subjects (flexor forearm) and three patients with psoriasis were analyzed using broad-range 18S ribosomal DNA (rDNA) and 5.8S rDNA/internal transcribed spacer 2 (ITS2) Malassezia-specific PCR primers. Broad-range PCR analysis indicated that most organisms resembled Malassezia. Malassezia-specific 5.8S/ITS2 analysis of 1,374 clones identified five species and four unknown phylotypes, potentially representing new species. The species distribution appears largely host specific and conserved in different sites of healthy skin. In three subjects, the Malassezia microbiota composition appeared relatively stable over time. Samples of Malassezia microbiota from healthy skin and psoriatic lesions were similar in one patient but substantially different in two others. These data indicate the predominance of Malassezia organisms in healthy human skin, host-specific variation, stability over time, and as yet, no consistent patterns differentiating psoriatic skin from healthy skin.     

Phylogenetic characterization of Histoplasma capsulatum strains based on ITS region sequences, including two new strains from Thai and Chinese patients in Japan.

Two strains of Histoplasma capsulatum var. capsulatum were isolated in Japan: one from a Thai AIDS patient and the other from a Chinese non-immunocompromised patient. The phylogenetical relationship among the two isolates and reference strains of H. capsulatum from other geographical populations was investigated. Random amplified polymorphic DNA (RAPD) analysis of the two H. capsulatum strains showed that they had RAPD band patterns similar to those of the reference Thai isolates and North American strains, although the patterns differed slightly from those of the reference strains. Phylogeny of thirty geographically diverse H. capsulatum isolates representing the three varieties, var. capsulatum, var. duboisii and var. farciminosum were evaluated using nucleotide sequences of the internal transcribed spacer (ITS) region (ITS1-5.8S rDNA -ITS2). We found that the ITS region contained sufficient information to resolve the phylogenetic relationship among the fungal isolates. An unrooted dendrogram constructed from the ITS sequences showed that thirty strains of H. capsulatum could be classified into eight geographic clades; Asia type (i), South America types A (ii) and B (iii), North American types 1 (iv) and 2 (v), H. duboisii types A (vi) and B (vii), and East Asia type (viii). Based on the ITS region sequence analysis, the two strains isolated from the Thai and Chinese patients in Japan were found to be distinct from Asia type (i) in which eight Thai, one Chinese, one English and one Indonesian isolate were included. Some extent of DNA polymorphism was observed between the North America type 1 isolates and the Thai and Chinese strains isolated in Japan. We believe that the Thai and Chinese isolates were unique and propose a new clade, East Asia type (viii) for the two strains. DNA sequence analysis of the ITS region provided useful information to understand the epidemiology and evolution of H. capsulatum.     

Internal transcribed spacer regions of rRNA genes of Pneumocystis carinii from monkeys

Analysis of sequence variations among isolates of Pneumocystis carinii f. sp. macacae from 14 Indian rhesus monkeys (Macaca mulatta) at the internal transcribed spacer (ITS) regions of the nuclear rRNA gene was undertaken. Like those from P. carinii f. sp. hominis, the ITS sequences from various P. carinii f. sp. macacae isolates were not identical. Two major types of sequences were found. One type of sequence was shared by 13 isolates. These 13 sequences were homologous but not identical. Variations were found at 13 of the 180 positions in the ITS1 region and 28 of the 221 positions in the ITS2 region. These sequence variations were not random but exhibited definite patterns when the sequences were aligned. According to this sequence variation, ITS1 sequences were classified into three types and ITS2 sequences were classified into five types. The remaining specimen had ITS1 and ITS2 sequences substantially different from the others. Although some specimens had the same ITS1 or ITS2 sequence, all 14 samples exhibited a unique whole ITS sequence (ITS1 plus ITS2). The 5.8S rRNA gene sequences were also analyzed, and only two types of sequences that differ by only one base were found. Unlike P. carinii f. sp. hominis infections in humans, none of the monkey lung specimens examined in this study were found to be infected by more than one type of P. carinii f. sp. macacae. These results offer insights into the genetic differences between P. carinii organisms which infect distinct species.     

Partial nucleotide sequence of a single ribosomal RNA coding region and secondary structure of the large subunit 25 s rRNA of Candida albicans

A rDNA cistron of Candida albicans strain WO-1 was cloned and the ITS1, ITS2, 5.8 s rDNA and 25 s rDNA coding regions sequenced in their entirety. These sequences were compared to those of three related yeast species (Saccharomyces cerevisiae, Saccharomyces carlsbergensis, and Thermomyces lanuginosus), and the 5.8 s rDNA was compared to seven additional 5.8 s rDNAs from organisms ranging in complexity from D. discoideum to H. sapiens. The C. albicans ITS regions are shorter than those of most other eukaryotes. The 25 s and 5.8 s rDNA sequences were folded into a secondary structure model based on comparative methods. In a comparison of regional similarities between the large subunit rDNAs of C. albicans, the three related yeasts and other eukaryotes, it is demonstrated that the additional sequences not present in the E. coli 23 s rDNA are more variable than the regions present in both prokaryotes and eukaryotes.     

Sequence variation of the rDNA ITS regions within and between anastomosis groups in Rhizoctonia solani

Sequence analysis of the rDNA region containing the internal transcribed spacer (ITS) regions and the 5.8s rDNA coding sequence was used to evaluate the genetic diversity of 45 isolates within and between anastomosis groups (AGs) in Rhizoctonia solani. The 5.8s rDNA sequence was completely conserved across all the AGs examined, whereas the ITS rDNA sequence was found to be highly variable among isolates. The sequence homology in the ITS regions was above 96% for isolates of the same subgroup, 66 – 100% for isolates of different subgroups within an AG, and 55 – 96% for isolates of different AGs. In neighbor-joining trees based on distances derived from ITS-5.8s rDNA sequences, subgroups IA, IB and IC within AG-1 and subgroups HG-I and HG-II within AG-4 were placed on statistically significant branches as assessed by bootstrap analysis. These results suggest that sequence analysis of ITS rDNA regions of R. solani may be a valuable tool for identifying AG subgroups of biological significance. Received: 11 February /16 May 1997     

Identification of tissue-embedded ascarid larvae by ribosomal DNA sequencing

Polymerase chain reaction (PCR) was applied to identify tissue-embedded ascarid nematode larvae. Two sequences of the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA), ITS1 and ITS2, of the ascarid parasites were amplified and compared with those of ascarid-nematodes registered in a DNA database (GenBank). The ITS sequences of the PCR products obtained from the ascarid parasite specimen in our laboratory were compatible with those of registered adult Ascaris and Toxocara parasites. PCR amplification of the ITS regions was sensitive enough to detect a single larva of Ascaris suum mixed with porcine liver tissue. Using this method, ascarid larvae embedded in the liver of a naturally infected turkey were identified as Toxocara canis. These results suggest that even a single larva embedded in tissues from patients with larva migrans could be identified by sequencing the ITS regions.     

Detection and identification of fungal pathogens by PCR and by ITS2 and 5.8S ribosomal DNA typing in ocular infections

The goal of this study was to determine whether sequence analysis of internal transcribed spacer/5.8S ribosomal DNA (rDNA) can be used to detect fungal pathogens in patients with ocular infections (endophthalmitis and keratitis). Internal transcribed spacer 1 (ITS1) and ITS2 and 5.8S rDNA were amplified by PCR and seminested PCR to detect fungal DNA. Fifty strains of 12 fungal species (yeasts and molds) were used to test the selected primers and conditions of the PCR. PCR and seminested PCR of this region were carried out to evaluate the sensitivity and specificity of the method. It proved possible to amplify the ITS2/5.8S region of all the fungal strains by this PCR method. All negative controls (human and bacterial DNA) were PCR negative. The sensitivity of the seminested PCR amplification reaction by DNA dilutions was 1 organism per PCR, and the sensitivity by cell dilutions was fewer than 10 organisms per PCR. Intraocular sampling or corneal scraping was undertaken for all patients with suspected infectious endophthalmitis or keratitis (nonherpetic), respectively, between November 1999 and February 2001. PCRs were subsequently performed with 11 ocular samples. The amplified DNA was sequenced, and aligned against sequences in GenBank at the National Institutes of Health. The results were PCR positive for fungal primers for three corneal scrapings, one aqueous sample, and one vitreous sample; one of them was negative by culture. Molecular fungal identification was successful in all cases. Bacterial detection by PCR was positive for three aqueous samples and one vitreous sample; one of these was negative by culture. Amplification of ITS2/5.8S rDNA and molecular typing shows potential as a rapid technique for identifying fungi in ocular samples.     

Pneumocystis jiroveci internal transcribed spacer types in patients colonized by the fungus and in patients with pneumocystosis from the same French geographic region

Pneumocystis jiroveci (human-derived Pneumocystis) infections can display a broad spectrum of clinical presentations, of which pulmonary colonization with the fungus may represent an important part, occurring frequently in patients with various underlying diseases and presenting alternative diagnoses of acute pneumocystosis (Pneumocystis carinii pneumonia [PCP]). There are few data concerning the P. jiroveci genotypes involved in pulmonary colonization, whereas several genotypes responsible for PCP in immunocompromised patients have been described. In this study, P. jiroveci genotypes have retrospectively been investigated and compared in 6 colonized patients and in 11 patients with PCP who were in the same hospital. Seventeen archival bronchoalveolar lavage samples were genotyped at internal-transcribed spacer 1 (ITS1) and ITS2 of the nuclear rRNA operon. Fourteen different genotypes were identified, of which 1 was found only in colonized patients, 10 were found only in patients with PCP, and 3 were found in both patient populations. Mixed infections were diagnosed in 2 of the 6 colonized patients and in 6 of the 11 patients with PCP. The results show that similar genotypes can be responsible for PCP as well as pulmonary colonization. There is a high diversity of genotypes in colonized patients and in patients with PCP. Mixed infections may occur in these two patient populations. These shared features of P. jiroveci ITS genotypes in colonized patients and patients with PCP suggest that human populations infected by P. jiroveci, whatever the clinical manifestation, may play a role as a common reservoir for the fungus.     

Genetic diversity of Pneumocystis carinii f. sp. hominis based on variations in nucleotide sequences of internal transcribed spacers of rRNA genes

A variety of genes have been used to type Pneumocystis carinii. In the present study, nucleotide sequence variations in the ITS1 and ITS2 internal transcribed spacer (ITS) regions of the rRNA genes were used to type Pneumocystis carinii f. sp. hominis DNA obtained from the lungs of 60 human immunodeficiency virus-infected individuals. These regions were amplified by PCR, cloned, and sequenced. Multibase polymorphisms were identified among samples. Several new genotypes are reported on the basis of the nucleotide sequence variations at previously unreported positions of both the ITS1 and the ITS2 regions. Twelve new ITS1 sequences were observed, in addition to the nine sequence types reported previously. The most common was type E, which was observed in 60.5% of the samples. The sequence variations in the ITS1 region were mainly located at positions 5, 12, 23, 24, 45, 53, and 54. Sixteen new ITS2 types were also identified, in addition to the 13 types reported previously. The most common was type g (26.6%). The sequences of the ITS2 regions in most specimens were different from the previously published sequence at bases 120 and 166 through 183. The most common variations observed were deletions at positions 177 through 183. The presence of more than one sequence type in some patients (60%) suggested the occurrence of coinfection with multiple P. carinii strains. The genetic polymorphism observed demonstrates the degree of diversity of Pneumocystis strains that infect humans. Furthermore, the high degree of polymorphism suggests that these genes are evolving faster than other genes. Consequently, the sequence information derived is useful for purposes such as examination of the potential of person-to-person transmission and recurrent infections but perhaps not for other genotyping applications that rely on more stable genetic loci.     

Genetic diversity of the internal transcribed spacers (ITS) and 5.8S rRNA genes among the clinical isolates of Candida parapsilosis in Brazil and Japan.

The internal transcribed spacer (ITS) region including 5.8S rDNA sequences of 58 isolates of Candida parapsilosis in Brazil and Japan was analyzed. Although most of the C. parapsilosis strains tested were confirmed to belong to three already reported genetically distinct groups (I, II and III) based on their ITS region sequences, 5 strains of the Brazilian isolates showed different sequences from those heretofore reported and suggested a presence of new genotype. For these strains of C. parapsilosis, we proposed a new genetic group (IV). The sequence similarities of this new group of IV to I, II and III were 87.4%, 94.7% and 87.3% in the ITS1 region, respectively. Genetic diversity in ITS regions of the remaining C. parapsilosis strains in Brazil and Japan was also discussed.