Absceso frío en una mano por un hongo dematiáceo. Primera comunicación en Chile de Pleurostomophora richardsiae / Hand cold abscess by a dematiaceous fungus. First report in Chile of Pleurostomophora richardsiae

Resumen Los hongos dematiáceos son un grupo heterogéneo de microorganismos capaces de sintetizar melanina. Las infecciones de este grupo que producen hifas en tejidos se denominan feohifomicosis y generalmente afectan la piel y tejidos vecinos. Presentamos el caso de un varón de 86 años con un tumor quístico blando progresivo en su mano y muñeca derecha, no asociado a dolor o signos inflamatorios. Se demostró una tenosinovitis de los flexores con pseudocapsula y sinovitis adherida a los tendones. El cultivo demostró un hongo dematiáceo compatible con Pleurostomophora richardsiae que se confirmó por secuenciación de la región ITS. La biopsia mostró una inflamación crónica granulomatosa e hifas. Después del drenaje quirúrgico, el paciente fue dado de alta sin terapia antifúngica, pero falleció por causas no relacionadas, tres meses después. Esta es la primera descripción de P. richardsiae como causa de feohifomicosis en Chile. Esta patología se puede sospechar cuando una lesión quística cutánea crónica involucra extremidades sin signos inflamatorios. Puede afectar a pacientes inmunocompetentes o inmunocomprometidos. El tratamiento contempla la escisión quirúrgica con o sin terapia antifúngica.

Abstract Dematiaceous fungi are a heterogeneous group of microorganisms able to synthesize melanin. Infections by this group that provoke tissular hyphae are called phaeohyphomycosis and usually involve skin and neighbor tissues. We present the case of a 86 years old men with a progressive soft cystic tumor in his right hand and wrist not associated to pain or inflammatory signs. A surgical intervention demonstrated flexor tenosynovitis with serous secretion, pseudocapsule and synovitis. Fungal culture demonstrated a dematiaceous fungi compatible with Pleurostomophora richardsiae that was confirmed by sequencing of the ITS region. Biopsy showed chronic inflammation with granuloma and hyphae. After surgical drainage, the patient was discharged without antifungal therapy but died of unrelated causes three month later. This is the first description of P. richardsiae as a cause of phaeohyphomycosis in Chile, a country with a template climate. Phaeohyphomycosis can be suspected when a chronic skin cystic lesion involves extremities without inflammatory signs, sometimes with an associated fistula. It may affect immunocompetent or immunosuppressed patients. Treatment involves surgical excision with or without antifungal therapy and prognosis is favorable.

Pandan (Pandanus sp), Rotan (Calamus sp), and Rengas (Gluta sp) from Kajuik Lake, Riau Province, Indonesia

ABSTRACT Pandan (Pandanus sp), Rotan (Calamus sp), and Rengas (Gluta sp) are the three most important plants growing at Kajuik Lake, Langgam, Riau Province, Indonesia; however, their species names have not been identified. This study aimed to identify their species names using nuclear internal transcribed spacer (ITS) and psbA-trnH intergenic spacer sequences. The method employed was DNA isolation from fresh leaves, PCR using primer pairs of ITS region for Pandanus sp and psbA-trnH intergenic spacer for Calamus sp and Gluta sp, electrophoresis, sequencing, and data analysis using BLASTn program and MEGA software version 6.0. Pandanus tectorius was the only one accession that was similar to Pandanus sp with the identity was 90%, however the query cover was too small, only 39%. On the contrary, Calamus sp showed the highest genetic similarity to Calamus travancoricus, but in fact, both were differed morphologically. There was no database of psbA-trnH intergenic spacer sequence available for species in Gluta. In conclusion, the species names for those plants still could not be determined. It because they might be the identified plants but their sequences databases were not available in large quantities or they were new species which had never been identified and published in public database.

Ribosomal Intergenic Spacer 1 Based Characterization of Button Mushroom (Agaricus bisporus) Strains

Breeding the button mushroom requires genetic information about its strains. This study was undertaken to genetically characterize four domestically bred button mushroom strains (Saea, Saejung, Saedo, Saeyeon cultivars) and to assess the possibility of using the intergenic spacer 1 (IGS1) region of rDNA as a genetically variable region in the genetic characterization. For the experiment, 34 strains of Agaricus bisporus, two strains of A. bitorquis, and one strain of A. silvaticus, from 17 countries were used. Nucleotide sequence analysis of IGS1 rDNA in these 37 Agaricus strains confirmed that genetic variations exist, not only among the four domestic strains, but also between the four domestic strains and foreign strains. Crossing two different haploid strains of A. bisporus seems to generate genetic variation in the IGS1 region in their off-spring haploid strains. Phylogenetic analysis based on the IGS1 sequence revealed all A. bisporus strains could be differentiated from A. silvaticus and A. bitorquis strains. Five genetic groups were resolved among A. bisporus strains. Saejung and Saeyeon cultivars formed a separate genetic group. Our results suggest that IGS1 could be complementarily applied in the polymorphism analysis of button mushroom.

Diversidad genética de aislamientos de Fusarium graminearum sensu lato asociados con la fusariosis de la espiga en trigo de diversas localidades argentinas / Genetic diversity of Fusarium graminearum sensu lato isolates from wheat associated with Fusarium Head Blight in diverse geographic locations of Argentina

La fusariosis de la espiga de trigo es una importante enfermedad para la región pampeana Argentina; Fusarium graminearum es el principal patógeno asociado. Se estudió el polimorfismo del ADN de un conjunto de aislamientos utilizando las técnicas de IGS-RFLP e ISSR. La técnica de IGS-RFLP produjo 41 bandas, 30 de ellas fueron polimórficas. El análisis de los ISSR mostró 87 bandas con 47 bandas polimórficas. La primera de estas metodologías fue más eficiente, ya que detectó mayor promedio polimórfico (59,91%) que la segunda (44,11%). Los valores promedio del contenido de información polimórfica (PIC) fueron 0,211 y 0,129, respectivamente. Se identificaron 20 haplotipos por IGS-RFLP, mientras que el análisis de los ISSR reveló 15 haplotipos. La agrupación de genotipos obtenida en ambos dendrogramas fue diferente. Los grupos genéticos obtenidos por la técnica de IGS-RFLP mostraron una asociación parcial con el origen geográfico. Este es el primer reporte que analiza la variabilidad genética en poblaciones de F. graminearum de trigo empleando marcadores IGS-RFLP e ISSR en Argentina

Fusarium Head Blight is an important wheat disease in the Argentine Pampas region, being Fusarium graminearum the predominant pathogen. DNA polymorphism of the isolates was analyzed by IGS-RFLP and ISSR. IGS-RFLP and ISSR profiling were carried out using six endonucleases and eight primers, respectively. IGS-RFLP yielded 41 bands, 30 of which were polymorphic while ISSR produced 87 bands with 47 polymorphic bands. Both markers showed genetic variability among the analyzed isolates; however, IGS-RFLP was more efficient than ISSR, showing a higher polymorphic average (59.91%) than the latter (44.11%). The averages of polymorphic information content (PIC) were 0.211 and 0.129, respectively. Twenty haplotypes were identified by IGS-RFLP and 15 haplotypes by ISSR. Genotype clustering within dendrograms was different for both types of markers. The genetic groups obtained by IGS-RFLP showed a partial association to geographic origin. This is the first report on genetic variability of F. graminearum isolates from wheat in Argentina using IGS-RFLP and ISSR markers

Rapid identification and sequence typing of genotype VG Ⅱ of Cryptococcus gattii / 中华临床感染病杂志

Objective To establish a PCR method for rapid identification and sequence typing of VG Ⅱ allele of the intergenic spacer region (IGS) of Cryptococcus gattii.Methods Since IGS1 was of high sequence variation,multiple alignments were conducted by ClustalX 2 in IGS1 of Cryptococcus gattii and Cryptococcus neoformans,and then primer sets specific to genotype VG Ⅱ was designed for PCR analysis.The specificity of the primer pair was detected by amplification of the other genotypes in Cryptococcus gattii,Cryptococcus neoformans,and other pathogenic yeasts.The amplified fragments from VG Ⅱ genotype were sequenced and subtyped.Results Using the PCR analysis developed in this study,all VG Ⅱ genotype strains tested were amplified,whereas no amplification was obtained from other genotypes or yeast species involved herein.Three polymorphic nucleotide sites at 72,79 and 104 bp in the fragment amplified could be used to distinguish sub-genotypes within VG Ⅱ genotype.Conclusions The PCR analysis developed in this study can be used for rapid identification of genotype VG Ⅱ of Cryptococcus gattii.The sequence typing based on the amplified fragment from IGS1 may be performed for screening the highly virulent sub-genotype VGⅡ a.

The genomic identification of Colombian Acinetobacter baumannii clinical isolates by RFLP-PCR analysis of the 16S-23S rRNA gene spacer region / Identificación genómica de aislamientos colombianos de Acinetobacter baumannii mediante RFLP-PCR de la región intergénica espaciadora de los genes 16S y 23S rRNA

The 16S-23S rRNA gene intergenic spacer (ITS) was analysed by RFLP in this study to identify A. baumannii from 139 isolates from four hospitals (identified as A, B, C and D). One hundred and twenty of these isolates (86.3%) belonged to the A. baumannii species; those identified as being A. baumannii were found to be polyclonal (19 clone groups) when determining the genetic relationships, 16 of them being found in hospital C. Hospitals A, B and D shared two clone groups isolated during different years. This study describes a rapid and easy method for genospecies identification of Acinetobacter baumannii.

Con el objeto de identificar la genomoespecie Acinetobacter baumannii, se estudiaron 189 aislamientos pertenecientes al Complejo Acinetobacter baumannii-Acinetobacter calcoaceticus provenientes de cuatro hospitales colombianos (denominados A,B,C,D) mediante el análisis por RFLP-PCR de la región intergénica espaciador (ITS) de los genes 16S y 23S rRNA. Se encontraron 120 aislamientos (86.3%) pertenecientes a la especie A. baumannii. La estructura de la población fue policlonal, con 19 grupos clonales, 16 de los cuales se hallaron en el hospital C. En los hospitales A,B y D se encontraron 2 grupos clonales aislados durante diferentes años. En este estudio se propone un método rápido y fácil para la identificación de Acinetobacter baumannii.

Phylogenetic relationships within Chamaecrista sect. Xerocalyx (Leguminosae, Caesalpinioideae) inferred from the cpDNA trnE-trnT intergenic spacer and nrDNA ITS sequences

Chamaecrista belongs to subtribe Cassiinae (Caesalpinioideae), and it comprises over 330 species, divided into six sections. The section Xerocalyx has been subjected to a profound taxonomic shuffling over the years. Therefore, we conducted a phylogenetic analysis using a cpDNA trnE-trnT intergenic spacer and nrDNA ITS/5.8S sequences from Cassiinae taxa, in an attempt to elucidate the relationships within this section from Chamaecrista. The tree topology was congruent between the two data sets studied in which the monophyly of the genus Chamaecrista was strongly supported. Our analyses reinforce that new sectional boundaries must be defined in the Chamaecrista genus, especially the inclusion of sections Caliciopsis and Xerocalyx in sect. Chamaecrista, considered here paraphyletic. The section Xerocalyx was strongly supported as monophyletic; however, the current data did not show C. ramosa (microphyllous) and C. desvauxii (macrophyllous) and their respective varieties in distinct clades, suggesting that speciation events are still ongoing in these specimens.

Genospecies Classification of Acinetobacter calcoaceticus-Acinetobacter baumannii Complex by Restriction Fragment Length Polymorphism

Acinetobacter calcoaceticus-Acinetobacter baumannii (A. calcoaceticus-A. baumannii) complex, which includes A. calcoaceticus (genospecies 1), A. baumannii (genospecies 2), Acinetobacter genospecies 3 and 13, has been identified as A. baumannii by automated bacteria identification system. The purpose of this study is to develop rapid genospecies classification of A. calcoaceticus-A. baumannii complex by molecular techniques. Random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) were determined for 4 reference strains and 80 isolates of A. calcoaceticus-A. baumannii complex from clinical sources. Four and eleven RAPD patterns were observed among the reference strains and the isolates, respectively. RAPD might be useful for genomic typing but not for genospecies classification of Acinetobacter spp. RFLP of 16S-23S rRNA intergenic spacer gene with three selected restriction enzymes (ApaLI, SwaI, and SalI) showed only four RFLP patterns in the reference and the isolates. Of 80 isolates, 10 of A. calcoaceticus (12.5%), 50 of A. baumannii (62.5%), 11 of A. genospecies 3 (13.75%), and 9 of A. genospecies 13 (11.25%) were classified by RFLP. This result suggests that RFLP of 16S-23S rRNA intergenic spacer gene of A. calcoaceticus-A. baumannii complex might be useful for genospecies classification.

PCR Amplification of 16S-23S rDNA Intergenic Spacer Regions of Bacteria in Trauma Infection by Universal Primers / 中华医院感染学杂志

OBJECTIVE To examine the feasibility of PCR amplification of 16S-23S rDNA intergenic spacer regions of bacteria in trauma infection by a pair of universal primers for gene diagnosis.METHODS The universal primers were designed at conserved regions of the 3' end of 16S rDNA and the 5' end of 23S rDNA.Bacterial genomic DNA from selected five commom bacteria in trauma infection were amplified by PCR.PCR products were examined using electrophoresis in agarose gel,and futher analyzed by sequencing.RESULTS The PCR products were similar to that we expected on the gel,which were confirmed by the results of sequencing and alignment.CONCLUSIONS Using the universal primers,16S-23S rDNA intergenic spacer regions of bacteria in trauma infection could be amplified by PCR,which lays a solid foundation for gene diagnosis in farther studies.

Sequence analysis of the rDNA intergenic spacer of Metarhizium strains isolated in Brazil

To assess the extent of genetic variability of rDNA intergenic spacer (IGS) in Metarhizium sp., 34 strains (27 isolated in Brazil) were sequenced and analyzed together with an additional 20 Metarhizium anisopliae var. anisopliae sequences retrieved from GenBank. Overall, the global nucleotide diversity for the region under study was of 0.090, while for the Brazilian isolates it was only 0.016. Phylogenetic analyses showed four well-supported groups (A, B, C, and D), one of which (D) has not been previously identified. All but one of the Brazilian strains cluster in this novel D phylogroup, suggesting that the genetic variation found in Brazil is a subset of the worldwide M. anisopiliae var. anisopliae variation.

RNA polymerase I promoter and splice acceptor site recognition affect gene expression in non-pathogenic Leishmania species

Leishmania (Sauroleishmania) tarentolae has biotechnological potential for use as live vaccine against visceral leishmaniasis and as a system for the over expression of eukaryotic proteins that possess accurate post-translational modifications. For both purposes, new systems for protein expression in this non-pathogenic protozoan are necessary. The ribosomal RNA promoter proved to be a stronger transcription driver since its use yielded increased levels of recombinant protein in organisms of both genera Trypanosoma or Leishmania. We have evaluated heterologous expression systems using vectors with two different polypyrimidine tracts in the splice acceptor site by measuring a reporter gene transcribed from L. tarentolae RNA polymerase I promoter. Our data indicate that the efficiency of chloramphenicol acetyl transferase expression changed drastically with homologous or heterologous sequences, depending on the polypyrimidine tract used in the construct and differences in size and/or distance from the AG dinucleotide. In relation to the promoter sequence the reporter expression was higher in heterologous lizard-infecting species than in the homologous L. tarentolae or in the mammalian-infecting L. (Leishmania) amazonensis.

Detection and genotyping of Giardia intestinalis isolates using intergenic spacers(IGS)-based PCR

Giardia intestinalis infections arise primarily from contaminated food or water. Zoonotic transmission is possible, and at least 7 major assemblages including 2 assemblages recovered from humans have been identified. The determination of the genotype of G. intestinalis is useful not only for assessing the correlation of clinical symptoms and genotypes, but also for finding the infection route and its causative agent in epidemiological studies. In this study, methods to identify the genotypes more specifically than the known 2 genotypes recovered from humans have been developed using the intergenic spacer (IGS) region of rDNA. The IGS region contains varying sequences and is thus suitable for comparing isolates once they are classified as the same strain. Genomic DNA was extracted from cysts isolated from the feces of 5 Chinese, 2 Laotians and 2 Koreans infected with G. intestinalis and the trophozoites of WB, K1, and GS strains cultured in the laboratory, respectively. The rDNA containing the IGS region was amplified by PCR and cloned. The nucleotide sequence of the 3' end of IGS region was determined and examined by multiple alignment and phylogenetic analysis. Based on the nucleotide sequence of the IGS region, 13 G. intestinalis isolates were classified to assemblages A and B, and assemblage A was subdivided into A1 and A2. Then, the primers specific to each assemblage were designed, and PCR was performed using those primers. It detected as little as 10 pg of DNA, and the PCR amplified products with the specific length to each assemblage (A1, 176 bp; A2, 261 bp; B, 319 bp) were found. The PCR specific to 3 assemblages of G. intestinalis did not react with other bacteria or protozoans, and it did not react with G. intestinalis isolates obtained from dogs and rats. It was thus confirmed that by applying this PCR method amplifying the IGS region, the detection of G. intestinalis and its genotyping can be determined simultaneously.

Nucleotide Divergence Analysis of IGS Region in Fusarium oxysporum and its formae speciales Based on the Sequence

The intergenic spacer(IGS) sequence of Fusarium oxysporum have been reported to provide reliable information concerning intraspecific variation and phylogeny of fungal species. The eleven strains of Fusarium oxysporum and its formae speciales belonging to section Elegans were compared with sequencing analysis. The direct sequencing of partial IGS was carried out using PCR with primer NIGS1(5'-CTTCGCCTCGATTTCCCCAA-3')/NIGS2(5'-TCGTCGCCGACAGTTTTCTG-3') and internal primer NIGS3(5'-TCGAGGATCGATTCGAGG-3')/NIGS4(5'-CCTCGAATCGATCCTCGA-3'). A single PCR product was found for each strain. The PCR fragments were sequenced and revealed a few within species polymorphisms at the sequence level. The size of partial IGS sequencing of F. oxysporum was divided into three groups; 526~527 bp including F. o. f. sp. chrysanthemi, cucumerinum, cyclaminis, lycopersici, and fragariae; 514~516 bp including F. o. f. sp. lilii, conglutinans, and raphani; 435 bp for F. o. f. sp. cucumerinum from Korea. Sequence analysis of PCR products showed that transitions were more frequent than transversions as well as the average numbers of substitution per site were range 0.41% to 3.54%.

Strain Differentiation of Trichophyton rubrum Using Polymorphism of rDNA Intergenic Spacer / 대한의진균학회지

BACKGROUND: In Korea, Trichophyton (T.) rubrum is occupied more than 80% of causative fungi of dermatophytosis. Strain differentiation of T. rubrum is essential for epidermiologic study. OBJECTIVE: The aim of this study is to develope the primers for amplification of rDNA intergenic spacer (IGS) to detect polymorphism of T. rubrum. METHODS: The primers were designed from 25S and 18S of rDNA, and were applied to 20 strains of T. rubrum, which included 1 standard strain and 19 clinical isolates. RESULTS: Primers for amplification of polymorphic rDNA IGS were designed from the 3'end of the 25S (primer ANID25-3, 5'-GACAGGTTAGTTTTACCCTACTGA-3') and the 5'end of the 18S (TR18-2R, 5'- ATCTAATAAATACACCCCTTCCGA-3'). PCR condition was adjusted for detecting polymorphism. Best results were obtained at 55degrees C for annealing temperature and 3 minutes for extension time. Eight bands sized as 1.1, 2.4, 2.7, 2.9, 3.2, 3.8, 5 kb were amplified with PCR using the primers. With 4 bands sized 2.7, 2.9, 3.2 and 3.8 kb, 20 strains of T. rubrum could be grouped into 6 subtypes. CONCLUSION: The PCR fingerprinting with the primers for rDNA IGS was able to differentiated strains of T. rubrum, and it can be applied in clinical and epidemiologic studies. The primer could be applied to other fungi with unknown sequences.

Cloning and sequencing of the 16s rRNA intergenic spacer region of proteus mirabilis and acinetobacter iwoffii / 重庆医科大学学报

Objective:To clone and sequence of 16S~23S r RNA intergenic spacer region of Proteus mirabilis and Acinetobacter lowffii for further identification of these two bacteria by means of molecular probe.Methods: The PCR universal clinical common pathogenic bacteria primers for 16S and 23S rRNA's conserved sequences were designed.Then the genomes of these two bacteria were amplified,T Vector clones were constructed with PMD-18 T Vector ad PCR products.The vectors were sequenced and the sequence was submitted to Genbank.Results: T Vector clones were constructed with products amplified by using universal primers;according to Blast analyses,the results of sequencing were determined as ISR sequences of the two kinds of bacteria.And the reports to Genbank were accepted.Conclusion: Genbank accepted the cloning and sequencing results of Proteus mirabilis and Acinetobacter lwoffii as new sequences.And these sequences can be used to identify the two bacteria by universal primers.

Identification of Acinetobacter Genospecies by Analysis of rRNA Spacer Regions / 대한임상병리학회지

BACKGROUND: Recently, the genus Acinetobacter have become increasingly important as nosocomial pathogens. Colonization and infection caused by multiresistant epidemic strains are common manifestations in intensive care units. Up to now 21 genomic species have been described, of which 7 have been named. However, there is no approach about distribution of Acinetobacter species in Korea. The objective of this study was to evaluate the performance of PCR analysis for the accurate identification and distribution of Acinetobacter species. METHODS: tRNA intergenic spacer length polymorphism(tRNA-ILP), 16S-23S rRNA spacer length polymorphism(16S-23S ILP) and restriction analysis of the 16S-23S rRNA intergenic spacer sequences(RA 16S-23S) were performed to identify the 7 type strains and 83 clinical isolates of Acinetobacter. RESULTS: 1. In the 7 Acinetobacter type strains, tRNA-ILP data could be easily analyzed by visual comparision. Eighty one of 83(97.6%) clinical isolates of Acinetobacter were identified as A. baumannii and the others were identified as A. haemolyticus and A. calcoaceticus. 2. The 16S-23S ILP patterns were not distinguished among the species, except A. haemolyticus, A, johnonii and A. lwoffii. The three species could be discriminated by a number of specific DNA fragments. 3. In RA 16S-23S, restriction endonuclease Alu I was able to not only digest the amplification products but also gave characteristic restriction patterns for the 7 type strains. CONCLUSION: The tRNA-ILP analysis and RA 16S-23S can be used as a tool for the rapid identification of Acinetobacter species with a high degree of specificity.

Detection of Bacterial Pathogens by Nanogold-based Gene Chip Combining with One-time PCR with Common Primers / 中华医院感染学杂志

OBJECTIVE To develop a preparation technique of sample of one-time PCR with common primers based on ribotyping which was combined with the detection system of nanogold-based gene chip to detect clinical bacterial pathogens.METHODS According to the highly conserved regions of rDNA,the common primers were designed and used to amplify each target bacterial ISRs by one-time PCR,and the specific oligonucleotide probes for each target ISRs were designed,utilized to establish the new nanogold-based gene chips.After the characteristics of the chip such as sensitivity,specificity and reliability were determined,the chip was used to detect clinical samples.RESULTS The designed common primers could amplify the 12 target bacteria successfully by one-time PCR.All selected probes were of strong specificity and great reliability.The chip had high sensitivity,specificity and reliability,reaching 50 fmol/L of detection sensibility.Clinical detection results showed the chip had a great accuracy.CONCLUSIONS Compared to multi-PCR chip detection,the detection procedure and complexity of the chip are decreased significantly,and have more practical value in clinical pathogens detection.