scholarly journals Species Detection within the Echinococcus granulosus sensu lato Complex by Novel Probe-Based Real-Time PCRs

Pathogens ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 791
Author(s):  
Pavlo Maksimov ◽  
Hannes Bergmann ◽  
Marion Wassermann ◽  
Thomas Romig ◽  
Bruno Gottstein ◽  
...  
Keyword(s):  
Real Time ◽  
Echinococcus Granulosus ◽  
Cost Effective ◽  
Sensu Stricto ◽  
Single Step ◽  
Control Measures ◽  
Chain Reactions ◽  
Disease Epidemiology ◽  
Polymerase Chain Reactions ◽  
Echinococcus Granulosus Sensu Lato

Infections 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) 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, E. equinus, E. ortleppi and the E. canadensis cluster. The technique also allowed identification and differentiation of these species from other Echinococcus or Taenia taxa for samples isolated from cysts or faeces.

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

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.

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

2020 ◽  
Author(s):  
Pavlo Maksimov ◽  
Hannes Bergmann ◽  
Marion Wassermann ◽  
Thomas Romig ◽  
Bruno Gottstein ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Real Time ◽  
Echinococcus Granulosus ◽  
Cystic Echinococcosis ◽  
Sensu Stricto ◽  
Single Step ◽  
Sybr Green ◽  
Control Measures ◽  
Analytical Sensitivity ◽  
Chain Reactions

Abstract Background: Infections 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.Methods: Mitochondrial genome sequences from the respective members of E. granulosus s.l. complex were used to identify target regions for the detection and genotyping. The selected primer pairs were first tested in a SYBR-green assay. In the next step, primers discriminating between the genotypes (according to post-amplification melting curves) were tested together with the respective probes in a TaqMan® Real-Time Polymerase Chain Reactions (qPCRs). For the analysis of analytical sensitivity and specificity of the real-time PCRs (SYBR-green and TaqMan), a panel of reference DNAs from various Echinococcus spp. was used.Results: We developed quantitative 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. Conclusions: 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.

scholarly journals Identification of Echinococcus granulosus Genotypes G1 and G3 by SNPs Genotyping Assays

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 125
Author(s):  
Piero Bonelli ◽  
Silvia Dei Giudici ◽  
Angela Peruzzu ◽  
Lorena Mura ◽  
Cinzia Santucciu ◽  
...  
Keyword(s):  
Echinococcus Granulosus ◽  
Animal Species ◽  
Cost Effective ◽  
Sensu Stricto ◽  
Sybr Green ◽  
Hydatid Cysts ◽  
Genotype Identification ◽  
Host Selectivity ◽  
Nad5 Gene ◽  
Echinococcus Granulosus Sensu Lato

Echinococcus granulosus sensu lato (s.l.) is the causative agent of cystic echinococcosis in animals and humans. Different E. granulosuss.l. genotypes exhibit great diversity in their life cycle, host selectivity and pathogenicity. For this reason, the study of genetic variation within Echinococcus species is of importance for their epidemiological implication. We employed two SNP genotyping technologies to distinguish G1 and G3 E. granulosus sensu stricto (s.s.). genotypes. The genotypes of DNA samples (n = 28) extracted from hydatid cysts of different animal species were identified by amplification and sequencing of a fragment of the mitochondrial nad5 gene. Two SYBR green and three TaqMan real time PCR assays were developed for targeting of three nad5 informative positions (SNP758, 1123, and 1380) known to be able to discriminate G1 from G3. Genotyping by SYBR Green PCR based on cycle threshold (Ct) with melting temperature (Tm) analysis and performed on SNP1123 and SNP1380 failed to identify one DNA sample. TaqMan assays for SNP758, 1123 and 1380 effectively confirmed genotype identification obtained by Sanger sequencing. Our results demonstrated that the combination of the three Taqman assays developed in this study represents a valuable and cost effective tool alternative to DNA sequencing for E. granulosus s.s. genotyping.

Echinococcus granulosus sensu stricto, Echinococcus ortleppi; and E. intermedius (G7) are present in Bolivia

Parasitology ◽  
2020 ◽  
Vol 147 (9) ◽  
pp. 949-956 ◽  
Author(s):  
V. Ali ◽  
E. Martinez ◽  
P. Duran ◽  
M. A. Seláez ◽  
M. Barragan ◽  
...  
Keyword(s):  
Detailed Analysis ◽  
Echinococcus Granulosus ◽  
Cystic Echinococcosis ◽  
Sensu Stricto ◽  
Control Measures ◽  
Santa Cruz ◽  
La Paz ◽  
Echinococcus Granulosus Sensu Stricto ◽  
Faecal Samples ◽  
Echinococcus Granulosus Sensu Lato

AbstractCystic echinococcosis (CE) is a zoonotic disease caused by a complex of species known as Echinococcus granulosus sensu lato. CE is endemic in Argentina, Chile, Peru, Uruguay and the South part of Brazil. In contrast, little is known regarding the presence of CE in Bolivia. In this study, 35 cysts isolated from livestock (mostly from the Department of La Paz) and 3 from humans (La Paz, Oruro and Potosi) were genetically characterized analysing the sequence of the cox1 gene (1609 bp). In total, 30 cysts (from La Paz, Cochabamba and Beni) were characterized as E. granulosus sensu stricto (3 fertile and 4 non-fertile cysts from sheep, 8 fertile and 12 non-fertile cysts from cattle and 3 fertile cysts from humans). A detailed analysis of the cox1 haplotypes of E. granulosus s.s. is included. Echinococcus ortleppi (G5) was found in 5 fertile cysts from cattle (from La Paz and Cochabamba). Echinococcus intermedius (G7) was identified in 3 fertile cysts from pigs (from Santa Cruz). Additionally, E. granulosus s.s. was detected in 4 dog faecal samples, while E. ortleppi was present in other two dog faecal samples. The implications of these preliminary results in the future implementation of control measures are discussed.

scholarly journals Human Cystic Echinococcosis in Lebanon: A Retrospective Study and Molecular Epidemiology

2021 ◽  
Author(s):  
Gaelle Joanny ◽  
Maria Grazia Cappai ◽  
Francesca Nonnis ◽  
Claudia Tamponi ◽  
Giorgia Dessì ◽  
...  
Keyword(s):  
Echinococcus Granulosus ◽  
Cystic Echinococcosis ◽  
Epidemiological Data ◽  
Sensu Stricto ◽  
Control Measures ◽  
Mediterranean Countries ◽  
High Gene ◽  
Human Infections ◽  
And Control ◽  
Human Cystic Echinococcosis

Abstract Purpose Human cystic echinococcosis (CE) is a zoonotic parasitic disease that constitutes a public health challenge and a socio-economic burden in endemic areas worldwide. No specific surveillance system of CE infections in humans exists in Lebanon. The incidence and trends over time have not been documented. The current study aimed to assess the demographic and epidemiologic features of human CE surgical cases over a 14-year period in the five main regions of Lebanon. Methods From 2005 to 2018, a total of 894 surgically confirmed cases of hydatidosis were recorded from five anatomy and pathology laboratories. Results The mean annual surgical incidence was 1.23/100,000 inhabitants. Over the span of these years, the incidence increased from 0.53 to 1.94 cases/100,000 inhabitants in 2005 and 2018, respectively. CE is present in Lebanon with an uneven distribution from one region to the other with higher prevalence in Bekaa (29.0%), a rural area where sheep raising is widespread. Human CE cases were more common in females (60.1%) than in males (39.9%) and a high burden of infection was reported for the age group of 30–39 years. Besides, 66.7% of the cases expressed only liver complications whereas, 20.5% showed predilection towards lungs. The 7.8% of cases presented cysts in other organs, and 1.3% showed multiple localizations. Additionally, predominant involvement of Echinococcus granulosus sensu stricto was recorded in human infections. Comparison of Echinococcus granulosus s.s. populations from different Mediterranean countries also revealed high gene flow among this region and sharing of alleles. Conclusion The current study is a step forward to fill the gap of knowledge for the hydatidosis in Lebanon where the lack of epidemiological data and control measures have resulted in higher incidence of human CE. Graphic Abstract

scholarly journals Echinococcus granulosus sensu stricto: silencing of thioredoxin peroxidase impairs the differentiation of protoscoleces into metacestodes

Parasite ◽  
2018 ◽  
Vol 25 ◽  
pp. 57 ◽  
Author(s):  
Hui Wang ◽  
Jun Li ◽  
Chuanshan Zhang ◽  
Baoping Guo ◽  
Qin Wei ◽  
...  
Keyword(s):  
Echinococcus Granulosus ◽  
Sensu Stricto ◽  
Antioxidant Defence ◽  
Culture Studies ◽  
Antioxidant Defence System ◽  
Thioredoxin Peroxidase ◽  
Survival And Growth ◽  
Echinococcus Granulosus Sensu Lato

Cystic echinococcosis (CE) is a cosmopolitan parasitic disease caused by infection with the larval stage of Echinococcus granulosus sensu lato. Thioredoxin peroxidase (TPx) may play an essential role in the antioxidant defence system of E. granulosus s.l. as neither catalase nor glutathione peroxidase activities have been detected in the parasite. However, it is not known whether TPx affects the survival and growth of E. granulosus s.l. during development. In this study, three fragments of siRNA specific for EgTPx (siRNA-1/2/3) were designed and transfected into protoscoleces of E. granulosus sensu stricto by electroporation. Quantitative real-time PCR and Western blotting analysis showed that siRNA-3 significantly reduced the expression of EgTPx. Coincidentally, knockdown of EgTPx expression in protoscoleces with siRNA-3 significantly reduced the viability of the parasite under oxidative stress induced by 0.6 mM H2O2. In vitro culture studies showed that protoscoleces treated with siRNA-3 reduced pre-microcyst formation. In vivo experiments showed that injecting mice intraperitoneally with protoscoleces treated with siRNA-3 resulted in a significant reduction in the number, size and weight of CE cysts compared with those of control animals. Silencing of EgTPx led to the impairment of growth of E. granulosus s.s. both in vitro and in vivo, indicating that EgTPx is an important factor for protoscoleces survival and plays an important role in the antioxidant defence against the host during development.

scholarly journals Duplex Methylation-Specific Semi-Quantitative Real-Time PCR for Cost-Effective & Time-Efficient Diagnostic Screening of Chromosome 15 and 14 Imprinted Regions

2015 ◽  
Vol 15 (3) ◽  
pp. 5-12
Author(s):  
E Ruszova ◽  
M Chmelarova ◽  
M Senkerikova ◽  
S Stefackova
Keyword(s):  
Motor Development ◽  
Cpg Islands ◽  
Cost Effective ◽  
Copy Number Variations ◽  
Chromosome 15 ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Effective Time ◽  
Development Delay ◽  
Polymerase Chain

AbstractPurpose: Our goal was to develop two-tier strategy based onin house-designed methylation specific-duplex polymerase chain reactions (MS-PCRs) that could serve as a relatively simple, cost effective, time efficient approach for molecular screening of imprinted regions on chromosomes 15 and 14.Patients and methods: Patients were referred to examination during infancy due to hypotonia and motor development delay. Duplex MS-PCRs were performed that enabled detection of methylated/unmethylated DNA inNDNandMEG3CpG islands via plurality of detection channels on PCR instrument Rotor Gene 6000.Results and discussion: Both, copy number variations as well as methylation changes, were revealed by ourin house-designed methodology by focusingNDNgene. No imprinting aberrations were yet discovered inMEG3gene. Clinical features of the patients were compared. In agree with literature no typical facial features were observed in PWS patient with imprinting defect and AS UPD patient seems to have a relatively better development and language ability in comparison to deletional form of the disease.Conclusion: In conclusion we were able to establish new, throughput and robust diagnostic approach to PWS/AS.

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

Pathogens ◽  
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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