Identification of Blastocystis subtypes in clinical stool samples from Sao Paulo City, Brazil

SUMMARY Blastocystis sp. is a protozoan commonly found in human and animal stool samples. Several pathogenic and zoonotic aspects of this organism are still unknown. The aim of the present study was to investigate Blastocystis subtypes (STs) in samples from patients of the Hospital das Clínicas of the Faculdade de Medicina at the Universidade de São Paulo (HC-FMUSP), Brazil. Blastocystis sp.-positive stool samples diagnosed at the Section of Parasitology of the Central Laboratory (HC-FMUSP) were used for DNA isolation. Polymerase chain reaction (PCR) was performed using specific primers targeting the small-subunit rRNA gene. Direct DNA sequencing of the PCR products was performed and the DNA sequences were then aligned and compared with other sequences obtained from the GenBank database. Phylogenetic analysis was used to identify STs and determine the phylogenetic relationships between the sequences. Four STs were identified: ST1 (22·5%), ST2 (12·5%), ST3 (60%) and ST6 (5%). In conclusion, ST3 was the most prevalent ST among the human isolates followed by ST1. The present study is one of the few providing STs data from the human population in South America. Determining ST prevalence in human samples may contribute to the monitoring of Blastocystis sp. infection transmission in endemic regions.

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Development and Application of a Most-Probable-Number–PCR Assay To Quantify Flagellate Populations in Soil Samples

Applied and Environmental Microbiology ◽

10.1128/aem.67.4.1613-1618.2001 ◽

2001 ◽

Vol 67 (4) ◽

pp. 1613-1618 ◽

Cited By ~ 28

Author(s):

Line Fredslund ◽

Flemming Ekelund ◽

Carsten Suhr Jacobsen ◽

Kaare Johnsen

Keyword(s):

Dna Sequences ◽

Sequence Data ◽

Small Subunit ◽

Most Probable Number ◽

Rrna Gene ◽

Heterotrophic Flagellates ◽

Small Subunit Rrna ◽

Pcr Assay ◽

Probable Number ◽

Soil Protozoa

ABSTRACT This paper reports on the first successful molecular detection and quantification of soil protozoa. Quantification of heterotrophic flagellates and naked amoebae in soil has traditionally relied on dilution culturing techniques, followed by most-probable-number (MPN) calculations. Such methods are biased by differences in the culturability of soil protozoa and are unable to quantify specific taxonomic groups, and the results are highly dependent on the choice of media and the skills of the microscopists. Successful detection of protozoa in soil by DNA techniques requires (i) the development and validation of DNA extraction and quantification protocols and (ii) the collection of sufficient sequence data to find specific protozoan 18S ribosomal DNA sequences. This paper describes the development of an MPN-PCR assay for detection of the common soil flagellate Heteromita globosa, using primers targeting a 700-bp sequence of the small-subunit rRNA gene. The method was tested by use of gnotobiotic laboratory microcosms with sterile tar-contaminated soil inoculated with the bacterium Pseudomonas putida OUS82 UCB55 as prey. There was satisfactory overall agreement between H. globosa population estimates obtained by the PCR assay and a conventional MPN assay in the three soils tested.

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Seasonal Shedding of Multiple Cryptosporidium Genotypes in California Ground Squirrels (Spermophilus beecheyi)

Applied and Environmental Microbiology ◽

10.1128/aem.70.11.6748-6752.2004 ◽

2004 ◽

Vol 70 (11) ◽

pp. 6748-6752 ◽

Cited By ~ 27

Author(s):

Edward R. Atwill ◽

Ralph Phillips ◽

Maria Das GraÃ¯Â¿Â½as C. Pereira ◽

Xunde Li ◽

Brenda McCowan

Keyword(s):

Dna Sequences ◽

Small Subunit ◽

Ground Squirrels ◽

Rrna Gene ◽

Spermophilus Beecheyi ◽

Small Subunit Rrna ◽

Fecal Shedding ◽

Link Type ◽

The Mean ◽

California Ground Squirrels

ABSTRACT Twelve percent of 853 California ground squirrels (Spermophilus beecheyi) from six different geographic locations in Kern County, Calif., were found to be shedding on average 44,482 oocysts g of feces−1. The mean annual environmental loading rate of Cryptosporidium oocysts was 57,882 oocysts squirrel−1 day−1, with seasonal patterns of fecal shedding ranging from <10,000 oocysts squirrel−1 day−1 in fall, winter, and spring to levels of 2 Ã¯Â¿Â½ 105 oocysts squirrel−1 day−1 in summer. Juveniles were about twice as likely as adult squirrels to be infected and shed higher concentrations of oocysts than adults did, with particularly high levels of infection and shedding being found among juvenile male squirrels. Based on DNA sequencing of a portion of the 18S small-subunit rRNA gene, there existed three genotypes of Cryptosporidium species in these populations of squirrels (Sbey03a, Sbey03b, and Sbey03c; accession numbers AY462231 to AY462233 , respectively). These unique DNA sequences were most closely related (96 to 97% homology) to porcine C. parvum (AF115377) and C. wrairi (AF115378). Inoculating BALB/c neonatal mice with up to 10,000 Sbey03b or Sbey03c fresh oocysts from different infected hosts did not produce detectable levels of infection, suggesting that this common genotype shed by California ground squirrels is not infectious for mice and may constitute a new species of Cryptosporidium.

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Comparison of PCR, Isoenzyme Analysis, and Antigen Detection for Diagnosis of Entamoeba histolyticaInfection

Journal of Clinical Microbiology ◽

10.1128/jcm.36.2.449-452.1998 ◽

1998 ◽

Vol 36 (2) ◽

pp. 449-452 ◽

Cited By ~ 143

Author(s):

Rashidul Haque ◽

I. K. M. Ali ◽

S. Akther ◽

William A. Petri

Keyword(s):

Specific Antigen ◽

Small Subunit ◽

Antigen Detection ◽

Rrna Gene ◽

Small Subunit Rrna ◽

Isoenzyme Analysis ◽

Pcr Product ◽

Stool Samples ◽

Stool Specimens ◽

Fresh Stool

The diagnosis of amebiasis by microscopic identification of the parasite in stool is insensitive and unable to distinguish the invasive parasite Entamoeba histolytica from the commensal parasite E. dispar. In this study, we have tested a PCR technique for the detection of E. histolytica and compared it with isoenzyme analysis and the TechLab E. histolytica-specific antigen detection test. The nested-PCR test we used is based on amplification of the small subunit rRNA gene ofE. histolytica and E. dispar followed by restriction digest analysis of the PCR product. Single stool samples were obtained from 98 patients from Dhaka, Bangladesh, with diarrhea: 88 patients diagnosed by microscopy and/or culture with E. histolytica and/or E. dispar infection and 10 patients without infection. Isoenzyme analysis identified 53 of the infections as E. histolytica and 28 as E. dispar. PCR and isoenzyme identification of E. histolytica agreed in 96% (51 of 53) of amebic cultures. PCR forE. histolytica was negative in all 10 samples that were negative for E. histolytica by isoenzyme and antigen detection. PCR and antigen detection had comparable sensitivities when performed directly on fresh stool specimens, identifying 87% (46 of 53) and 85% (45 of 53), respectively, of E. histolyticainfections identified by isoenzyme analysis. The correlation of results by antigen detection and PCR for identification of E. histolytica in stool was 93% (45 of 48 cases). Mixed infections with E. histolytica and E. dispar were detected by PCR in 14% (12 of 88) of cases. In conclusion, all three techniques for specific identification of E. histolytica in fresh stool showed excellent correlation. Only the TechLab E. histolytica antigen detection test was both rapid and technically simple.

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Effect of Primers Hybridizing to Different Evolutionarily Conserved Regions of the Small-Subunit rRNA Gene in PCR-Based Microbial Community Analyses and Genetic Profiling

Applied and Environmental Microbiology ◽

10.1128/aem.67.8.3557-3563.2001 ◽

2001 ◽

Vol 67 (8) ◽

pp. 3557-3563 ◽

Cited By ~ 193

Author(s):

Achim Schmalenberger ◽

Frank Schwieger ◽

Christoph C. Tebbe

Keyword(s):

Microbial Community ◽

Small Subunit ◽

Sscp Analysis ◽

Rrna Genes ◽

Rrna Gene ◽

Small Subunit Rrna ◽

Genetic Profiling ◽

Evolutionarily Conserved ◽

Pcr Products ◽

Pcr Targeting

ABSTRACT Genetic profiling techniques of microbial communities based on PCR-amplified signature genes, such as denaturing gradient gel electrophoresis or single-strand-conformation polymorphism (SSCP) analysis, are normally done with PCR products of less than 500-bp. The most common target for diversity analysis, the small-subunit rRNA genes, however, are larger, and thus, only partial sequences can be analyzed. Here, we compared the results obtained by PCR targeting different variable (V) regions (V2 and V3, V4 and V5, and V6 to V8) of the bacterial 16S rRNA gene with primers hybridizing to evolutionarily conserved flanking regions. SSCP analysis of single-stranded PCR products generated from 13 different bacterial species showed fewer bands with products containing V4-V5 (average, 1.7 bands per organism) than with V2-V3 (2.2 bands) and V6-V8 (2.3 bands). We found that the additional bands (>1 per organism) were caused by intraspecies operon heterogeneities or by more than one conformation of the same sequence. Community profiles, generated by PCR-SSCP from bacterial-cell consortia extracted from rhizospheres of field-grown maize (Zea mays), were analyzed by cloning and sequencing of the dominant bands. A total of 48 sequences could be attributed to 34 different strains from 10 taxonomical groups. Independent of the primer pairs, we found proteobacteria (α, β, and γ subgroups) and members of the genus Paenibacillus (low G+C gram-positive) to be the dominant organisms. Other groups, however, were only detected with single primer pairs. This study gives an example of how much the selection of different variable regions combined with different specificities of the flanking “universal” primers can affect a PCR-based microbial community analysis.

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Targeted Next-Generation Sequencing and Informatics as an Effective Tool to Establish the Composition of Bovine Piroplasm Populations in Endemic Regions

Microorganisms ◽

10.3390/microorganisms9010021 ◽

2020 ◽

Vol 9 (1) ◽

pp. 21

Author(s):

Abdul Ghafar ◽

Anson V. Koehler ◽

Ross S. Hall ◽

Charles G. Gauci ◽

Robin B. Gasser ◽

...

Keyword(s):

Next Generation Sequencing ◽

Water Buffalo ◽

Hypervariable Region ◽

Small Subunit ◽

Rrna Gene ◽

Next Generation ◽

Small Subunit Rrna ◽

Tropical Theileriosis ◽

Ecological Zones ◽

Generation Sequencing

Protists of the genera Babesia and Theileria (piroplasms) cause some of the most prevalent and debilitating diseases for bovines worldwide. In this study, we established and used a next-generation sequencing-informatic approach to explore the composition of Babesia and Theileria populations in cattle and water buffalo in a country (Pakistan) endemic for these pathogens. We collected individual blood samples from cattle (n = 212) and water buffalo (n = 154), extracted genomic DNAs, PCR-amplified the V4 hypervariable region of 18S small subunit rRNA gene from piroplasms, sequenced amplicons using Illumina technology, and then analysed data using bioinformatic platforms. The results revealed piroplasms in 68.9% (252/366) samples, with overall occurrence being markedly higher in cattle (85.8%) than in water buffaloes (45.5%). Babesia (B.) occultans and Theileria (T.) lestoquardi-like species were recorded for the first time in Pakistan, and, overall, T. annulata was most commonly detected (65.8%) followed by B. bovis (7.1%), B. bigemina (4.4%), and T. orientalis (0.5%), with the genetic variability within B. bovis being pronounced. The occurrence and composition of piroplasm species varied markedly across different agro-ecological zones. The high detection of T. annulata in asymptomatic animals suggested a relatively high level of endemic stability of tropical theileriosis in the bovine population.

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Species-specific duplex PCR for the detection of Pratylenchus penetrans

Nematology ◽

10.1163/156854109x428016 ◽

2009 ◽

Vol 11 (6) ◽

pp. 847-857 ◽

Cited By ~ 21

Author(s):

Lieven Waeyenberge ◽

Nicole Viaene ◽

Maurice Moens

Keyword(s):

Internal Control ◽

Dna Sequences ◽

Rrna Gene ◽

Duplex Pcr ◽

Specific Primers ◽

5.8S Rrna Gene ◽

5.8S Rrna ◽

Wide Range ◽

Pcr Products ◽

Species Specific

Abstract ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20 different Pratylenchus species. Additionally, the same region was sequenced from seven populations of P. penetrans. After purifying, cloning and sequencing the PCR products, all sequences were aligned in order to find unique sites suitable for the design of species-specific primers for P. penetrans. Since ITS regions showed variability between and even within populations of P. penetrans, only three small DNA sequences were suitable for the construction of three potentially useful species-specific primers. New species-specific primers were paired with existing universal ITS primers and tested in all possible primer combinations. The best performing primer set, supplemented with a universal 28S rDNA primer set that served as an internal control, was tested in duplex PCR. The ideal annealing temperature, Mg2+ concentration and primer ratios were then determined for the most promising primer set. The optimised duplex PCR was subsequently tested on a wide range of different Pratylenchus spp. and 25 P. penetrans populations originating from all over the world. To test the sensitivity, the duplex PCR was conducted on DNA extracted from a single P. penetrans nematode mixed with varying amounts of nematodes belonging to another Pratylenchus species. Results showed that a reliable and sensitive P. penetrans species-specific duplex PCR was constructed.

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Differences in Microbial Activity and Microbial Populations of Peat Associated with Suppression of Damping-Off Disease Caused by Pythium sylvaticum

Applied and Environmental Microbiology ◽

10.1128/aem.00313-06 ◽

2006 ◽

Vol 72 (10) ◽

pp. 6452-6460 ◽

Cited By ~ 43

Author(s):

Paul J. Hunter ◽

Geoff M. Petch ◽

Leo A. Calvo-Bado ◽

Tim R. Pettitt ◽

Nick R. Parsons ◽

...

Keyword(s):

Microbial Activity ◽

Denaturing Gradient Gel Electrophoresis ◽

Small Subunit ◽

Disease Suppression ◽

Rrna Gene ◽

Damping Off ◽

Small Subunit Rrna ◽

Pythium Sylvaticum ◽

Gradient Gel Electrophoresis ◽

Microbial Groups

ABSTRACT The microbiological characteristics associated with disease-suppressive peats are unclear. We used a bioassay for Pythium sylvaticum-induced damping-off of cress seedlings to identify conducive and suppressive peats. Microbial activity in unconditioned peats was negatively correlated with the counts of P. sylvaticum at the end of the bioassay. Denaturing gradient gel electrophoresis (DGGE) profiling and clone library analyses of small-subunit rRNA gene sequences from two suppressive and two conducive peats differed in the bacterial profiles generated and the diversity of sequence populations. There were also significant differences between bacterial sequence populations from suppressive and conducive peats. The frequencies of a number of microbial groups, including the Rhizobium-Agrobacterium group (specifically sequences similar to those for the genera Ochrobactrum and Zoogloea) and the Acidobacteria, increased specifically in the suppressive peats, although no single bacterial group was associated with disease suppression. Fungal DGGE profiles varied little over the course of the bioassay; however, two bands associated specifically with suppressive samples were detected. Sequences from these bands corresponded to Basidiomycete yeast genera. Although the DGGE profiles were similar, fungal sequence diversity also increased during the bioassay. Sequences highly similar to those of Cryptococcus increased in relative abundance during the bioassay, particularly in the suppressive samples. This study highlights the importance of using complementary approaches to molecular profiling of complex populations and provides the first report that basidiomycetous yeasts may be associated with the suppression of Pythium-induced diseases in peats.

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Detection of Kobe-typeBabesia microtiassociated with Japanese human babesiosis in field rodents in central Taiwan and southeastern mainland China

Parasitology ◽

10.1017/s0031182008004356 ◽

2008 ◽

Vol 135 (6) ◽

pp. 691-699 ◽

Cited By ~ 12

Author(s):

A. SAITO-ITO ◽

N. TAKADA ◽

F. ISHIGURO ◽

H. FUJITA ◽

Y. YANO ◽

...

Keyword(s):

Mainland China ◽

Small Subunit ◽

Human Infection ◽

Babesia Microti ◽

Rrna Gene ◽

Microscopical Examination ◽

Small Subunit Rrna ◽

Central Taiwan ◽

Field Rodents ◽

Human Babesiosis

SUMMARYField rodent surveys forBabesiainfection were performed from 2002 to 2005 in the vicinities of human babesiosis occurrences in Taiwan and mainland China.Babesia microtiwas identified by microscopical examination and/or PCR in 1Rattus coxingaand 1Crocidura horsfieldiiin central Taiwan and in 13Niviventer confucianusand 1Apodemus agrariusin Zhejiang and Fujian Provinces of southeastern China. Of 15B. microtisamples detected by PCR, all except 1 were shown to be the Kobe-type, the aetiological small subunit rRNA gene-type of the first Japanese patient; the exception was also a Kobe-related type. The Kobe-type had been found in rodents only in a few places including the human infection occurrence place in Japan. The internal transcribed spacer 1 to 2 sequences of the Taiwanese and Chinese Kobe-types were very similar to each other but considerably different (approx. 94% pairwise identities) from that of the Japanese Kobe-type. A Taiwanese Kobe-type strain was serologically differentiated from the Kobe strain originating from the Japanese first patient. The distribution of the Kobe-type in the vicinities of human babesiosis occurrences in Taiwan and China as well as in Japan is suggestive of involvement of the Kobe-type in Asian human babesiosis.

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Genotypic identification of Cryptosporidium spp. isolated from hiv-infected patients and immunocompetent children of São Paulo, Brazil

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46652008005000003 ◽

2008 ◽

Vol 50 (3) ◽

pp. 139-143 ◽

Cited By ~ 18

Author(s):

Ana Julia Urias dos Santos Araújo ◽

Herminia Yohko Kanamura ◽

Marcos Eduardo de Almeida ◽

Aparecida Helena de Souza Gomes ◽

Thais Helena Lemos Pinto ◽

...

Keyword(s):

Fragment Length Polymorphism ◽

Genetic Characterization ◽

Cryptosporidium Oocyst ◽

Sao Paulo ◽

São Paulo ◽

Oocyst Wall ◽

Cryptosporidium Hominis ◽

Stool Samples ◽

Dna Fragment ◽

Genotypic Identification

Cryptosporidium isolates identified in fourteen stool samples, collected from five HIV-infected patients and nine immunocompetent children, living in the Sate of São Paulo, Brazil, were submitted to a molecular analysis using a nested PCR followed of restriction fragment length polymorphism (RFLP), for genetic characterization. The analysis was based on digestion with RsaI restriction enzyme of a DNA fragment amplified from the Cryptosporidium oocyst wall protein (COWP) gene. Based on this analysis, four samples were identified as Cryptosporidium parvum, eight as Cryptosporidium hominis and two presented a profile that correspondedto Cryptosporidium meleagridis when compared to the standards used in the analysis. The use of molecular methods can be helpful to identify source of infections and risk factors related to Cryptosporidium infection in our communities.

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