High resolution melting technique for molecular epidemiological studies of cystic echinococcosis: differentiating G1, G3, and G6 genotypes of Echinococcus granulosus sensu lato

Abstract Background Cystic echinococcosis (CE) is a zoonosis caused by Echinococcus granulosus (sensu lato). It is a neglected tropical disease with a global distribution, affecting an estimated 2–3 million people globally. Official reporting systems in Spain lack information concerning imported cases and their country of origin. Methods This is a systematic review of the literature that was performed to obtain published cases of immigrant patients diagnosed with CE in Spain. Results From the 21 included articles, a total of 84 cases of CE imported into Spain were documented from 1995 to 2018, with an average age of 33.2 years. The main countries of origin of the patients were Morocco with 30 cases (35.7%), Romania with 12 cases (14.3%) and Peru with 8 cases (9.5%). The most involved organ was the liver (28 cases [33.3%]). We found discrepancies between the published cases of imported CE in Spain and those reported by official authorities. Conclusions This review of the literature shows the lack of information and clarity in the mechanisms of CE notification in Spain. The disparity between these systems and the cases documented in the literature highlights a failure or shortcoming of the current reporting system.

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Cystic Echinococcosis: Clinical, Immunological, and Biomolecular Evaluation of Patients from Sardinia (Italy)

Pathogens ◽

10.3390/pathogens9110907 ◽

2020 ◽

Vol 9 (11) ◽

pp. 907

Author(s):

Cinzia Santucciu ◽

Piero Bonelli ◽

Angela Peruzzu ◽

Alessandro Fancellu ◽

Vincenzo Marras ◽

...

Keyword(s):

Molecular Biology ◽

Echinococcus Granulosus ◽

Cystic Echinococcosis ◽

Public Health Problem ◽

Sensu Stricto ◽

Surgical Removal ◽

Neighbor Joining ◽

Intermediate Hosts ◽

High Prevalence ◽

Echinococcus Granulosus Sensu Lato

Cystic echinococcosis (CE), a zoonotic disease caused by the larval stage of the tapeworm Echinococcus granulosus sensu lato (s.l.), is a worldwide public health problem. Echinococcus granulosus sensu stricto (s.s.), associated with G1 and G3 genotypes, is endemic with high prevalence in the Mediterranean basin. The parasite’s life cycle comprises definitive hosts (canids) and intermediate hosts (ruminants) and can occasionally involve humans. The main aim of this research was to confirm the diagnosis of 13 patients suspected of CE who presented different complications and needed the surgical removal of the cysts. We also wanted to understand and clarify more the diagnosis of echinococcosis in humans. For this purpose, the patients first underwent cyst evaluation by ultrasound (US), immunological analysis, and then total pericystectomy, followed by parasitological, histopathological, and molecular biology examinations of the cysts. US stadiated one CE1, one CE2, eight CE3b, one CE4, and two CE5; immunology evidenced nine positives; histopathology confirmed 11 CE cysts, of which 8 fertile presenting protoscoleces were identified as E. granulosus s.s. by molecular biology, genotyped as three G1 and four G3 by neighbor-joining (NJ) phylogenetic tree. In conclusion, the results showed that 11 patients were affected by E. granulosus s.s. G1 orG3, and 2 cystic neoformations were of non-parasitic origin.

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High-resolution melting analysis for rapid detection of the internationally spreading ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkz395 ◽

2019 ◽

Vol 75 (1) ◽

pp. 106-109 ◽

Cited By ~ 3

Author(s):

Leshan Xiu ◽

Chi Zhang ◽

Yamei Li ◽

Feng Wang ◽

Junping Peng

Keyword(s):

High Resolution ◽

Neisseria Gonorrhoeae ◽

Internal Control ◽

High Resolution Melting ◽

Direct Detection ◽

High Specificity ◽

Dual Therapy ◽

Epidemiological Studies ◽

Cross Reactivity ◽

Clinical Samples

Abstract Objectives Increased awareness of the international spread of the ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone, which threatens recommended dual therapy, is essential. The objective of the present study was to develop and evaluate a rapid, simple and cost-effective method based on high-resolution melting (HRM) analysis for direct detection of the FC428 clone from clinical isolates and specimens. Methods The singleplex HRM assay was designed to identify the FC428 clone by using specific primers, which flank the alteration A311V in the penA-60.001 allele. Analytical performance was initially evaluated by testing 623 isolates and a panel of non-gonococcal strains. To ensure the method can be directly applied in clinical samples, two internal control targets (opa and porA) were also designed and included in the final multiplex HRM assay. Two hundred and eighty-two clinical samples (94 urine and 188 urethral/genital swabs) were then analysed using this multiplex HRM assay. Results The FC428 clone was easily differentiated from the non-mosaic alleles and other mosaic alleles without A311 mutations by comparing the differences in melt curves. Cross-reactivity was not observed for the penA-60.001 allele when testing 15 non-gonococcal Neisseria strains. When applied to the 623 isolates, the HRM assay successfully characterized one isolate as an FC428 clone (MLST1903, NG-MAST3435, NG-STAR233). Our data show that the multiplex HRM assay with high specificity can be directly applied in clinical samples. Conclusions This method can generate results within 90 min at a cost of less than US$0.5 per isolate or sample, making this assay an ideal tool for large epidemiological studies to enhance surveillance of the internationally transmitted ceftriaxone-resistant N. gonorrhoeae FC428 clone.

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A powerful qPCR-high resolution melting assay with taqman probe in plasmodium species differentiation

Malaria Journal ◽

10.1186/s12936-021-03662-w ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Aline Lamien-Meda ◽

Hans-Peter Fuehrer ◽

David Leitsch ◽

Harald Noedl

Keyword(s):

High Resolution ◽

High Resolution Melting ◽

Limit Of Detection ◽

Plasmodium Species ◽

Malaria Diagnosis ◽

Epidemiological Studies ◽

Taqman Probe ◽

Species Differentiation ◽

Resource Limited ◽

Highly Sensitive

Abstract Background The use of highly sensitive molecular tools in malaria diagnosis is currently largely restricted to research and epidemiological settings, but will ultimately be essential during elimination and potentially eradication. Accurate diagnosis and differentiation down to species levels, including the two Plasmodium ovale species and zoonotic variants of the disease, will be important for the understanding of changing epidemiological patterns of the disease. Methods A qPCR-high resolution melting (HRM) method was to detect and differentiate all human Plasmodium species with one forward and one reverse primer set. The HRM detection method was further refined using a hydrolysis probe to specifically discriminate Plasmodium falciparum. Results Out of the 113 samples tested with the developed HRM-qPCR- P. falciparum probe assay, 96 (85.0 %) single infections, 12 (10.6 %) mixed infections, and 5 (4.4 %) were Plasmodium negative. The results were concordant with those of the nested PCR at 98.2 %. The assay limit of detection was varied from 21.47 to 46.43 copies /µl, equivalent to 1–2.11 parasites/µl. All P. falciparum infections were confirmed with the associated Taqman probe. Conclusions Although the dependence on qPCR currently limits its deployment in resource-limited environments, this assay is highly sensitive and specific, easy to perform and convenient for Plasmodium mono-infection and may provide a novel tool for rapid and accurate malaria diagnosis also in epidemiological studies.

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Species detection within the Echinococcus granulosus sensu lato complex by novel probe-based Real-Time PCRs

10.1101/2020.07.24.220756 ◽

2020 ◽

Author(s):

Pavlo Maksimov ◽

Hannes Bergmann ◽

Marion Wassermann ◽

Thomas Romig ◽

Bruno Gottstein ◽

...

Keyword(s):

Real Time ◽

Echinococcus Granulosus ◽

Cystic Echinococcosis ◽

Cost Effective ◽

Sensu Stricto ◽

Single Step ◽

Control Measures ◽

Chain Reactions ◽

Disease Epidemiology ◽

Echinococcus Granulosus Sensu Lato

AbstractInfections with eggs of Echinococcus granulosus sensu lato (s.l.) can cause cystic echinococcosis in intermediate host animals and humans. Upon ingestion of viable eggs, oncospheres hatch from the eggs and subsequently develop into fluid-filled larval cysts, most frequently in the liver or the lungs. The slowly growing cysts progressively interfere with organ function. The risk of infection is determined by the host range of the parasite, its pathogenicity and other epidemiologically relevant parameters, which differ significantly among the five species within the E. granulosus s.l. complex. It is therefore essential to diagnose the correct species within E. granulosus s.l. to help understand specific disease epidemiology and to facilitate effective implementation of control measures. For this purpose, simple, fast and cost-effective typing techniques are needed.We developed quantitative Real-Time Polymerase Chain Reactions (qPCRs) and corresponding sequence-specific hydrolysis DNA probes to target polymorphic regions in the mitochondrial genome of E. granulosus s.l.. In a single-step typing approach, we distinguished E. granulosus s.l. members in four epidemiologically relevant subgroups. These were E. granulosus sensu stricto (G1, G3), E. equinus (G4), E. ortleppi (G5) and the E. canadensis cluster (G6 to G8 and G10). The technique also allowed identification and differentiation of these species from other Echinococcus or Taenia taxa for samples isolated from cysts or faeces.Single-step genotyping techniques for the molecular diagnosis of Echinococcus spp. by qPCRs may not only improve diagnostic performance, but also our knowledge on the epidemiology of the parasites and help controlling the various agents of cystic echinococcosis.

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MeltingPlot: a user-friendly online tool for epidemiological investigation using High Resolution Melting data

10.1101/2020.06.16.20132142 ◽

2020 ◽

Cited By ~ 2

Author(s):

Matteo Perini ◽

Gherard Batisti Biffignandi ◽

Domenico Di Carlo ◽

Ajay Ratan Pasala ◽

Aurora Piazza ◽

...

Keyword(s):

High Resolution ◽

High Resolution Melting ◽

Epidemiological Studies ◽

Supplementary Information ◽

Web Interface ◽

Web Tool ◽

Online Tool ◽

Link Type ◽

Surveillance Programs ◽

User Friendly

AbstractSummaryMeltingPlot is an open source web tool for pathogen typing and epidemiological investigations using High Resolution Melting (HRM) data. The tool implements a graph-based algorithm designed to discriminate pathogen clones on the basis of HRM data, producing portable typing results. MeltingPlot also merges typing information with isolates and patients metadata to create graphical and tabular outputs useful in epidemiological studies. HRM technique allows pathogen typing in less than 5 hours with ~5 euros per sample. MeltingPlot is the first tool specifically designed for HRM-based epidemiological studies and it can analyse hundreds of isolates in a few seconds. Thus, the use of MeltingPlot makes HRM-based typing suitable for large surveillance programs as well as for rapid outbreak reconstructions.Availability and implementationMeltingPlot is implemented in R.The web interface is available at https://skynet.unimi.it/index.php/tools/meltingplot.The source code is also available at https://github.com/MatteoPS/[email protected] informationSupplementary data are available at Bioinformatics online.

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The ectodomains of the lymphocyte scavenger receptors CD5 and CD6 interact with tegumental antigens from Echinococcus granulosus sensu lato and protect mice against secondary cystic echinococcosis

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0006891 ◽

2018 ◽

Vol 12 (11) ◽

pp. e0006891 ◽

Cited By ~ 4

Author(s):

Gustavo Mourglia-Ettlin ◽

Sebastián Miles ◽

María Velasco-De-Andrés ◽

Noelia Armiger-Borràs ◽

Marcela Cucher ◽

...

Keyword(s):

Echinococcus Granulosus ◽

Cystic Echinococcosis ◽

Scavenger Receptors ◽

Echinococcus Granulosus Sensu Lato

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CRISPR 2 PCR and high resolution melting profiling for identification and characterization of clinically-relevant Salmonella enterica subsp. enterica

PeerJ ◽

10.7717/peerj.9113 ◽

2020 ◽

Vol 8 ◽

pp. e9113 ◽

Cited By ~ 1

Author(s):

Nuttachat Wisittipanit ◽

Chaiwat Pulsrikarn ◽

Sudarat Srisong ◽

Rungthiwa Srimora ◽

Nattinee Kittiwan ◽

...

Keyword(s):

High Resolution ◽

High Resolution Melting ◽

Learning Algorithm ◽

Food Poisoning ◽

Hospital Setting ◽

Epidemiological Studies ◽

Detection And Identification ◽

Appropriate Treatment ◽

Signature Sequences ◽

Evolutionary Trajectories

Background Nontyphoidal Salmonella spp. constitute a major bacterial cause of food poisoning. Each Salmonella serotype causes distinct virulence to humans. Method A small cohort study was conducted to characterize several aspects of Salmonella isolates obtained from stool of diarrheal patients (n = 26) admitted to Phayao Ram Hospital, Phayao province, Thailand. A simple CRISPR 2 molecular analysis was developed to rapidly type Salmonella isolates employing both uniplex and high resolution melting (HRM) curve analysis. Results CRISPR 2 monoplex PCR generated a single Salmonella serotype-specific amplicon, showing S. 4,[5],12:i:- with highest frequency (42%), S. Enteritidis (15%) and S. Stanley (11%); S. Typhimurium was not detected. CRISPR 2 HRM-PCR allowed further classification of S. 4,[5],12:i:- isolates based on their specific CRISPR 2 signature sequences. The highest prevalence of Salmonella infection was during the summer season (April to August). Additional studies were conducted using standard multiplex HRM-PCR typing, which confirmed CRISPR 2 PCR results and, using a machine-learning algorithm, clustered the majority of Salmonella serotypes into six clades; repetitive element-based (ERIC) PCR, which clustered the serotypes into three clades only; antibiogram profiling, which revealed the majority resistant to ampicillin (69%); and test for extended spectrum β-lactamase production (two isolates) and PCR-based detection of bla alleles. Conclusion CRISPR 2 PCR provided a simple assay for detection and identification of clinically-relevant Salmonella serotypes. In conjunction with antibiogram profiling and rapid assay for β-lactamase producers, this approach should facilitate detection and appropriate treatment of Salmonellosis in a local hospital setting. In addition, CRISPR 2 HRM-PCR profiling enabled clustering of S. 4,[5],12:i:-isolates according to CRISPR 2 locus signature sequences, indicative of their different evolutionary trajectories, thereby providing a powerful tool for future epidemiological studies of virulent Salmonella serotypes.

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