scholarly journals Comparison of Different DNA-Based Methods for Molecular Typing of Histoplasma capsulatum

2010 ◽  
Vol 76 (13) ◽  
pp. 4438-4447 ◽  
Author(s):  
Mauro de Medeiros Muniz ◽  
Patrícia Morais e Silva Tavares ◽  
Wieland Meyer ◽  
Joshua Daniel Nosanchuk ◽  
Rosely Maria Zancope-Oliveira
Keyword(s):  
Molecular Typing ◽  
Histoplasma Capsulatum ◽  
Fatty Acid Desaturase ◽  
Rflp Analysis ◽  
Rdna Locus ◽  
Factor H ◽  
Its2 Region ◽  
Limited Information ◽  
Pcr Fingerprinting ◽  
M13 Pcr Fingerprinting

ABSTRACT Histoplasma capsulatum is very prevalent in the environment and is one of the most common causes of mycoses in humans and diverse animals in Brazil. Multiple typing methods have been developed to study H. capsulatum epidemiology; however, there is limited information concerning comparisons of results obtained with different methods using the same set of isolates. To explore the diversity of H. capsulatum in Brazil and to determine correlations between the results of three different molecular typing techniques, we examined 51 environmental, animal, and human isolates by M13 PCR fingerprinting, PCR-restriction fragment length polymorphism (RFLP) analysis of the internal transcribed region 1 (ITS1)-5.8S-ITS2 region of the rDNA locus, and DNA sequencing and phylogenetic analysis of parts of four protein-encoding genes, the Arf (ADP ribosylation factor), H-anti (H antigen precursor), Ole (delta-9 fatty acid desaturase), and Tub1 (alpha-tubulin) genes. Each method identified three major genetic clusters, and there was a high level of concordance between the results of the typing techniques. The M13 PCR fingerprinting and PCR-RFLP analyses produced very similar results and separated the H. capsulatum isolates included in this study into three major groups. An additional approach used was comparison of our Brazilian ITS1-5.8S-ITS2 sequences with the sequences deposited previously in NCBI data banks. Our analyses suggest that H. capsulatum can be divided into different molecular types that are dispersed around the world. Our results indicate that the three methods used in this study are reliable and reproducible and that they have similar sensitivities. However, M13 PCR fingerprinting has some advantages over the other two methods as it is faster, cheaper, and more user friendly, which especially increases its utility for molecular typing of Histoplasma in situations where laboratory facilities are relatively limited.

An Outbreak of ESBL-producing Klebsiella pneumoniae in an Iranian Referral Hospital: Epidemiology and Molecular Typing

2019 ◽  
Vol 19 (1) ◽  
pp. 46-54 ◽  
Author(s):  
Shima Mahmoudi ◽  
Babak Pourakbari ◽  
Aliakbar Rahbarimanesh ◽  
Mohammad Reza Abdosalehi ◽  
Keyghobad Ghadiri ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Nosocomial Infections ◽  
Molecular Typing ◽  
Genetic Relatedness ◽  
Referral Hospital ◽  
Limited Information ◽  
Hospital Epidemiology ◽  
Rapd Pcr ◽  
Published Report ◽  
Random Amplification

Introduction: Klebsiella pneumoniae is a common cause of nosocomial infections; however, there is limited information in Iran regarding nosocomial outbreaks due to extended-spectrum β–lactamase (ESBL) producing K pneumoniae strains, particularly using molecular methods. The present study focused on the molecular mechanism of ESBL resistance and genetic relatedness in K. pneumoniae isolates causing nosocomial infections in an Iranian referral hospital. Material and Methods: This study evaluated the antimicrobial resistance and molecular epidemiology of K. pneumoniae causing nosocomial infections in children between October 2013 and March 2014. The ESBL detection was carried out for all the isolates by the CLSI method and PCR was carried out for the detection of the blaSHV, blaTEM, and blaCTX-M genes among ESBL-producing K. pneumonia. Molecular typing of the K. pneumoniae was performed using random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR). Results: A total of 30 isolates of K. pneumoniae were used for epidemiological analysis. High rates of resistance to cefotaxime (n=29, 97%), cefazolin (n=29, 97%), cefepime (n=25, 83%) and gentamicin (n=23, 77%) were observed. A total of 29 strains (97%) produced ESBLs. The frequency of blaSHV, blaCTX-M and blaTEM genes among these isolates was 83% (n=25), 70% (n=21) and 57% (n=17), respectively. Surprisingly 11 isolated (37%) carried blaSHV, blaCTX-M and blaTEM genes simultaneously. Moreover, the concurrent presence of “blaSHV and blaCTX-M” and “blaSHV and blaTEM” was seen in 8 (27%) and 4 (13%) isolates, respectively. RAPDPCR analyses revealed that K. pneumoniae isolates belonged to 2 RAPD-PCR types among which one cluster counted for 28 isolates. Conclusion: To our knowledge, this is the first published report of a nosocomial outbreak of ESBL-producing K. pneumoniae in children in Iran. Although the epidemiology of nosocomial infections with ESBL-producing organisms has not yet been explored in depth in Iran, our findings suggest that ESBL-producing organisms are already an established public health threat in our country.

scholarly journals Genotyping, serotyping and determination of mating-type of Cryptococcus neoformans clinical isolates from São Paulo State, Brazil

2007 ◽  
Vol 49 (1) ◽  
pp. 41-47 ◽  
Author(s):  
Marcelo Teruyuki Matsumoto ◽  
Ana Marisa Fusco-Almeida ◽  
Lilian Cristiane Baeza ◽  
Márcia de Souza Carvalho Melhem ◽  
Maria José Soares Medes-Giannini
Keyword(s):  
Cryptococcus Neoformans ◽  
Mating Type ◽  
Rflp Analysis ◽  
Sao Paulo ◽  
Clinical Isolates ◽  
São Paulo ◽  
Paulo State ◽  
Pcr Fingerprinting ◽  
Pcr Rflp ◽  
São Paulo State

The basidiomycetous yeast Cryptococcus neoformans is an important fungal pathogen mainly in immunocompromised patients. In this study, 47 clinical isolates of C. neoformans from regions of São Paulo State were studied serologically by using the Crypto Check Iatron RM 304-K kit, their genetic diversity was estimated by PCR-fingerprinting with a microsatellite-specific sequence (GACA)4, RAPD with primer 6 (Amersham Pharmacia Biotech), PCR-restriction fragment length polymorphism (RFLP) analysis of the phospholipase B gene (PLB1) digested with AvaI and mating type analysis by PCR. All 47 strains isolated from HIV positive patients included in this study were serotype A and MATalpha. The majority of the isolates (45/47) were VNI and only two were VNII by PCR-fingerprinting and PCR-RFLP analysis. High degree of homogeneity was observed when (GACA)4 was used, being highly correlated (> 0.9). In contrast, the RAPD analysis was more heterogeneous with higher number of molecular profiles. By PCR-RFLP, no new molecular type was found, enhancing the suggestion that the differences based on conserved gene as PLB1, can be resultant of ongoing divergent evolution within the C. neoformans complex, into the current eight subtypes. Our results furnish new information on the molecular epidemiology of C. neoformans in the southeast region of Brazil.

Molecular Typing of Histoplasma capsulatum Isolated from Infected Bats, Captured in Mexico

2000 ◽  
Vol 30 (3) ◽  
pp. 207-212 ◽  
Author(s):  
Maria Lucia Taylor ◽  
Catalina B. Chávez-Tapia ◽  
Maria Rocio Reyes-Montes
Keyword(s):  
Molecular Typing ◽  
Histoplasma Capsulatum

scholarly journals Cucurbit aphid-borne yellows virus Is Prevalent in Field-Grown Cucurbit Crops of Southeastern Spain

Plant Disease ◽  
2007 ◽  
Vol 91 (3) ◽  
pp. 232-238 ◽  
Author(s):  
M. A. Kassem ◽  
R. N. Sempere ◽  
M. Juárez ◽  
M. A. Aranda ◽  
V. Truniger
Keyword(s):  
Mosaic Virus ◽  
Zucchini Yellow Mosaic Virus ◽  
Rflp Analysis ◽  
Yellow Mosaic Virus ◽  
Multiple Infections ◽  
Watermelon Mosaic Virus ◽  
Limited Information ◽  
Growing Seasons ◽  
Beet Pseudo Yellows Virus ◽  
The Impact

Despite the importance of field-grown cucurbits in Spain, only limited information is available about the impact of disease on their production. During the 2003 and 2004 growing seasons, systematic surveys were carried out in open field melon (Cucumis melo) and squash (Cucurbita pepo) crops of Murcia Province (Spain). The fields were chosen with no previous information regarding their sanitation status, and samples were taken from plants showing viruslike symptoms. Samples were analyzed using molecular hybridization to detect Beet pseudo-yellows virus (BPYV), Cucurbit aphid-borne yellows virus (CABYV), Cucumber mosaic virus (CMV), Cucumber vein yellowing virus (CVYV), Cucurbit yellow stunting disorder virus (CYSDV), Melon necrotic spot virus (MNSV), Papaya ringspot virus (PRSV), Watermelon mosaic virus (WMV), and Zucchini yellow mosaic virus (ZYMV). We collected 924 samples from 48 field plots. Out of these, almost 90% were infected by at least one of the viruses considered, usually CABYV, which was present in 83 and 66% of the melon and squash samples, respectively. In the case of melon, CYSDV, BPYV, and WMV followed CABYV in relative importance, with frequencies of around 20 to 30%, while in squash, CVYV and BPYY showed frequencies between 28 and 21%. The number of multiple infections was very high, 66 and 56% of the infected samples of melon and squash, respectively, being afflicted. CABYV was present in all multiple infections. The high incidence of CABYV in single and multiple infections suggests that this virus may well become an important threat for cucurbit crops in the region. Restriction fragment length polymorphism (RFLP) analysis revealed that CABYV isolates can be grouped into two genetic types, both of which seemed to be present during the 2003 epidemic episode, but only one of the types was found in 2004.

scholarly journals Variability in molecular typing of Coxsackie A viruses by RFLP analysis and sequencing

2002 ◽  
Vol 16 (1) ◽  
pp. 59-69 ◽  
Author(s):  
Nikolaos Siafakas ◽  
Panayotis Markoulatos ◽  
Glyn Stanway
Keyword(s):  
Molecular Typing ◽  
Rflp Analysis

scholarly journals Malassezia arunalokei sp. nov., a Novel Yeast Species Isolated from Seborrheic Dermatitis Patients and Healthy Individuals from India

2016 ◽  
Vol 54 (7) ◽  
pp. 1826-1834 ◽  
Author(s):  
Prasanna Honnavar ◽  
Gandham S. Prasad ◽  
Anup Ghosh ◽  
Sunil Dogra ◽  
Sanjeev Handa ◽  
...  
Keyword(s):  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Novel Species ◽  
Seborrheic Dermatitis ◽  
Rflp Analysis ◽  
Tween 20 ◽  
Its2 Region ◽  
Polymorphism Analysis ◽  
Content Type

The majority of species within the genusMalasseziaare lipophilic yeasts that colonize the skin of warm-blooded animals. Two species,Malassezia globosaandMalassezia restricta, are implicated in the causation of seborrheic dermatitis/dandruff (SD/D). During our survey of SD/D cases, we isolated several species ofMalasseziaand noticed vast variations within a few lipid-dependent species. Variations observed in the phenotypic characteristics (colony morphology, absence of catalase activity, growth at 37°C, and precipitation surrounding wells containing Tween 20 or Cremophor EL) suggested the possible presence of a novel species. Sequence divergence observed in the internal transcribed spacer (ITS) region, the D1/D2 domain, and the intergenic spacer 1 (IGS1) region of rDNA and theTEF1gene, PCR-restriction fragment length polymorphism (RFLP) analysis of the ITS2 region, and fluorescent amplified fragment length polymorphism analysis support the existence of a novel species. Based on phenotypic and molecular characterization of these strains, we propose a new species, namely,M. arunalokeisp. nov., and we designate NCCPF 127130 (= MTCC 12054 = CBS 13387) as the type strain.

scholarly journals The use of PCR-RFLP to genetically distinguish the morphologically close species: Ceriodaphnia dubia Richard, 1894 and Ceriodaphnia silvestrii Daday, 1902 (Crustacea Cladocera)

2010 ◽  
Vol 70 (1) ◽  
pp. 121-124 ◽  
Author(s):  
MJ. Abreu ◽  
MJ. Santos-Wisniewski ◽  
O. Rocha ◽  
TC. Orlando
Keyword(s):  
Exotic Species ◽  
Native Species ◽  
Rflp Analysis ◽  
Ceriodaphnia Dubia ◽  
Molecular Techniques ◽  
Rrna Genes ◽  
Its2 Region ◽  
State Of Water ◽  
Pcr Rflp ◽  
Rna Genes

The cladocerans are important components of planktonic and benthic freshwater and good indicators of the trophic state of water bodies. The morphological taxonomy of many species of Cladocera is considered complex with minor differences separating some species. Nowadays, molecular techniques provide a powerful tool to identify and classify different taxonomical levels, using mainly ribosomal RNA genes (rRNA) as molecular markers. In the present work we performed PCR-RFLP to separate Ceriodaphnia dubia, an exotic species in Brazil and the native species Ceriodaphnia silvestrii, widely distributed in Brazilian freshwater. The RFLP analysis of the ITS1-5.8S-ITS2 region of rRNA genes showed to be different between C. dubia and C. silvestrii when using enzymes EcoRI, ApaI and SalI. Thus, the ITS1-5.8S-ITS2 region proved to be a useful molecular marker to differentiate the studied Ceriodaphnia species, which makes the task easier of telling apart species that are morphologically very similar. Also, this methodology might be interesting in determining the distribution of the exotic species C. dubia in Brazilian freshwaters, particularly in cases when C. dubia occurs in the absence of C. silvestrii, a particularly difficult task for ecologists who are not taxonomy specialists.

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