scholarly journals Diagnosis of Taenia solium infections based on “mail order” RNA-sequencing of single tapeworm egg isolates from stool samples

2021 ◽  
Vol 15 (12) ◽  
pp. e0009787
Author(s):  
Henrik Sadlowski ◽  
Veronika Schmidt ◽  
Jonathan Hiss ◽  
Johannes A. Kuehn ◽  
Christian G. Schneider ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Taenia Solium ◽  
Rna Extraction ◽  
Mitochondrial Rna ◽  
Mail Order ◽  
Laptop Computer ◽  
Diagnostic Laboratory ◽  
Community Health Programs ◽  
Stool Samples ◽  
Sequencing Facility

Combined community health programs aiming at health education, preventive anti-parasitic chemotherapy, and vaccination of pigs have proven their potential to regionally reduce and even eliminate Taenia solium infections that are associated with a high risk of neurological disease through ingestion of T. solium eggs. Yet it remains challenging to target T. solium endemic regions precisely or to make exact diagnoses in individual patients. One major reason is that the widely available stool microscopy may identify Taenia ssp. eggs in stool samples as such, but fails to distinguish between invasive (T. solium) and less invasive Taenia (T. saginata, T. asiatica, and T. hydatigena) species. The identification of Taenia ssp. eggs in routine stool samples often prompts a time-consuming and frequently unsuccessful epidemiologic workup in remote villages far away from a diagnostic laboratory. Here we present “mail order” single egg RNA-sequencing, a new method allowing the identification of the exact Taenia ssp. based on a few eggs found in routine diagnostic stool samples. We provide first T. solium transcriptome data, which show extremely high mitochondrial DNA (mtDNA) transcript counts that can be used for subspecies classification. “Mail order” RNA-sequencing can be administered by health personnel equipped with basic laboratory tools such as a microscope, a Bunsen burner, and access to an international post office for shipment of samples to a next generation sequencing facility. Our suggested workflow combines traditional stool microscopy, RNA-extraction from single Taenia eggs with mitochondrial RNA-sequencing, followed by bioinformatic processing with a basic laptop computer. The workflow could help to better target preventive healthcare measures and improve diagnostic specificity in individual patients based on incidental findings of Taenia ssp. eggs in diagnostic laboratories with limited resources.

Diagnosis of Taenia solium infections based on “mail order” RNA-sequencing of single tapeworm egg isolates from stool samples v1

2021 ◽  
Author(s):  
Henrik Sadlowski ◽  
Veronika Schmidt ◽  
Jonathan Hiss ◽  
Christian G. Schneider ◽  
Gideon Zulu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Rna Sequencing ◽  
Taenia Solium ◽  
Mail Order ◽  
Next Generation ◽  
Sample Collection ◽  
Additional Information ◽  
Stool Samples ◽  
Sequencing Facility ◽  
Generation Sequencing

Here we present a detailed protocol for the identification of Taenia solium based on the few Taenia spp. eggs found in diagnostic stool samples. Our approach is based on "mail order" RNA sequencing of single eggs and can be performed in laboratories equipped with basic tools such as a microscope, a Bunsen burner, and access to an international post office for shipping samples to a next-generation sequencing facility. This protocol describes sample collection and transport, isolation of individual Taenia spp. eggs, reliable disruption of individual Taenia eggs, and important considerations for shipping samples to a next-generation sequencing facility. We provide images and videos to help prepare the tools needed for the protocol. Additional information on our rationale for designing the critical steps can help implement the protocol in new environments.

Identification of Taenia solium based on single tapeworm eggs in diagnostic stool samples using RNA sequencing  v1

2020 ◽  
Author(s):  
Henrik Sadlowski ◽  
Veronika Schmidt ◽  
Jonathan Hiss ◽  
Christian G. Schneider ◽  
Gideon Zulu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Rna Sequencing ◽  
Taenia Solium ◽  
Next Generation ◽  
Sample Collection ◽  
Additional Information ◽  
Stool Samples ◽  
Sequencing Facility ◽  
Generation Sequencing ◽  
Detailed Protocol

Here we present a detailed protocol for the identification of Taenia solium based on the few Taenia spp. eggs found in diagnostic stool samples. Our approach is based on "mail order" RNA sequencing of single eggs and can be performed in laboratories equipped with basic tools such as a microscope, a Bunsen burner, and access to an international post office for shipping samples to a next-generation sequencing facility. This protocol describes sample collection and transport, isolation of individual Taenia spp. eggs, reliable disruption of individual Taenia eggs, and important considerations for shipping samples to a next-generation sequencing facility. We provide images and videos to help prepare the tools needed for the protocol. Additional information on our rationale for designing the critical steps can help implement the protocol in new environments.

scholarly journals Prevalence and Genetic Characterisation of Human Sapovirus from Children with Diarrhoea in the Rural Areas of Vhembe District, South Africa, 2017–2020

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 393
Author(s):  
Mpho Magwalivha ◽  
Jean-Pierre Kabue Ngandu ◽  
Afsatou Ndama Traore ◽  
Natasha Potgieter
Keyword(s):  
South Africa ◽  
Rural Communities ◽  
Rural Areas ◽  
Rna Extraction ◽  
Diarrhoeal Disease ◽  
Positive Sample ◽  
Prevalent Strain ◽  
Stool Samples ◽  
Vhembe District ◽  
Pcr Targeting

Diarrhoeal disease is considered an important cause of morbidity and mortality in developing areas, and a large contributor to the burden of disease in children younger than five years of age. This study investigated the prevalence and genogroups of human sapovirus (SV) in children ≤5 years of age in rural communities of Vhembe district, South Africa. Between 2017 and 2020, a total of 284 stool samples were collected from children suffering with diarrhoea (n = 228) and from children without diarrhoea (n = 56). RNA extraction using Boom extraction method, and screening for SV using real-time PCR were done in the lab. Positive samples were subjected to conventional RT-PCR targeting the capsid fragment. Positive sample isolates were genotyped using Sanger sequencing. Overall SV were detected in 14.1% (40/284) of the stool samples (16.7% (38/228) of diarrhoeal and 3.6% (2/56) of non-diarrhoeal samples). Significant correlation between SV positive cases and water sources was noted. Genogroup-I was identified as the most prevalent strain comprising 81.3% (13/16), followed by SV-GII 12.5% (2/16) and SV-GIV 6.2% (1/16). This study provides valuable data on prevalence of SV amongst outpatients in rural and underdeveloped communities, and highlights the necessity for further monitoring of SV circulating strains as potential emerging strains.

scholarly journals The impact of RNA extraction method on accurate RNA sequencing from formalin-fixed paraffin-embedded tissues

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Michal Marczyk ◽  
Chunxiao Fu ◽  
Rosanna Lau ◽  
Lili Du ◽  
Alexander J. Trevarton ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Rna Sequencing ◽  
Rna Extraction ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
The Impact ◽  
Formalin Fixed

Abstract Background Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. Methods Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. Results Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63–0.66) and between technical replicates (median expression difference 0.13–0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31–0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91–0.96). Conclusions The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

scholarly journals Combination of Culture, Antigen and Toxin Detection, and Cytotoxin Neutralization Assay for OptimalClostridium difficileDiagnostic Testing

2013 ◽  
Vol 24 (2) ◽  
pp. 89-92 ◽  
Author(s):  
Michelle J Alfa ◽  
Shadi Sepehri
Keyword(s):  
Diagnostic Algorithm ◽  
Laboratory Diagnosis ◽  
Neutralization Assay ◽  
Confirmatory Test ◽  
Toxin A ◽  
Diagnostic Laboratory ◽  
Toxigenic Culture ◽  
Stool Samples ◽  
Step Algorithm ◽  
One Year

BACKGROUND: There has been a growing interest in developing an appropriate laboratory diagnostic algorithm forClostridium difficile, mainly as a result of increases in both the number and severity of cases ofC difficileinfection in the past decade. AC difficilediagnostic algorithm is necessary because diagnostic kits, mostly for the detection of toxins A and B or glutamate dehydrogenase (GDH) antigen, are not sufficient as stand-alone assays for optimal diagnosis ofC difficileinfection. In addition, conventional reference methods forC difficiledetection (eg, toxigenic culture and cytotoxin neutralization [CTN] assays) are not routinely practiced in diagnostic laboratory settings.OBJECTIVE: To review the four-step algorithm used at Diagnostic Services of Manitoba sites for the laboratory diagnosis of toxigenicC difficile.RESULT: One year of retrospectiveC difficiledata using the proposed algorithm was reported. Of 5695 stool samples tested, 9.1% (n=517) had toxigenicC difficile. Sixty per cent (310 of 517) of toxigenicC difficilestools were detected following the first two steps of the algorithm. CTN confirmation of GDH-positive, toxin A- and B-negative assays resulted in detection of an additional 37.7% (198 of 517) of toxigenicC difficile. Culture of the third specimen, from patients who had two previous negative specimens, detected an additional 2.32% (12 of 517) of toxigenicC difficilesamples.DISCUSSION: Using GDH antigen as the screening and toxin A and B as confirmatory test forC difficile, 85% of specimens were reported negative or positive within 4 h. Without CTN confirmation for GDH antigen and toxin A and B discordant results, 37% (195 of 517) of toxigenicC difficilestools would have been missed. Following the algorithm, culture was needed for only 2.72% of all specimens submitted forC difficiletesting.CONCLUSION: The overview of the data illustrated the significance of each stage of this four-stepC difficilealgorithm and emphasized the value of using CTN assay and culture as parts of an algorithm that ensures accurate diagnosis of toxigenicC difficile.

scholarly journals Parasite prevalence in a village in Burkina Faso: the contribution of new techniques

2014 ◽  
Vol 8 (05) ◽  
pp. 670-675 ◽  
Author(s):  
Simona Fortunato ◽  
Barbara Castagna ◽  
Maria Rita Monteleone ◽  
Raffaela Pierro ◽  
Giuseppe Cringoli ◽  
...  
Keyword(s):  
Burkina Faso ◽  
Pediatric Population ◽  
Adult Population ◽  
Taenia Solium ◽  
Public Health Problem ◽  
Parasite Prevalence ◽  
Diagnostic Methods ◽  
Major Public Health Problem ◽  
Number Of Patients ◽  
Stool Samples

Introduction: Parasites are a major public health problem in developing countries. A coproparasitological and immunoparasitological study was conducted in Burkina Faso, in the rural village of Touguri, in November and December 2011. The coproparasitologic analysis was conducted in the pediatric population and seroprevalence surveys were conducted in the adult population to research intestinal, blood, and helminth parasites. Methodology: The coproparasitologic study was performed on stool samples using two diagnostic methods – standard microscopy and the FLOTAC technique. The total of 49 stool samples analyzed were obtained from children between two months and eleven years of age. The serology study was carried out to evaluate the prevalence of P. falciparum, Echinococcus spp., Tenia solium, and A. lumbricoides using different immunological techniques such as ELISA and Western Blot techniques. The study population included 85 adult patients between 15 and 70 years of age. Results: Results of coproparasitological analyses showed Hymenolepis nana as the only helminth found, in 28.6% of the total number of patients. Results of serological evaluation revealed a practically null prevalence of Echinococcus, Taenia solium, and Ascaris lumbricoides, and a 77.64% prevalence of Plasmodium falciparum. Conclusions: Despite the small number (especially in terms of coprological samples) of individuals examined, this study showed that the parasite prevalence in a rural area of Burkina Faso has a significant impact in the general population, particularly in children. Another finding was that FLOTAC had a higher sensitivity than the widely used ethyl ether-based concentration technique for coprological sample analysis.

scholarly journals A simple RNA preparation method for SARS-CoV-2 detection by RT-qPCR

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Aniela Wozniak ◽  
Ariel Cerda ◽  
Catalina Ibarra-Henríquez ◽  
Valentina Sebastian ◽  
Grace Armijo ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Molecular Diagnostic ◽  
Nasopharyngeal Swab ◽  
Diagnostic Laboratory ◽  
Public Health Decision ◽  
Health Decision Making ◽  
Health Decision ◽  
Analytical Step ◽  
Commercial Kits ◽  
Novel Coronavirus

Abstract The technique RT-qPCR for viral RNA detection is the current worldwide strategy used for early detection of the novel coronavirus SARS-CoV-2. RNA extraction is a key pre-analytical step in RT-qPCR, often achieved using commercial kits. However, the magnitude of the COVID-19 pandemic is causing disruptions to the global supply chains used by many diagnostic laboratories to procure the commercial kits required for RNA extraction. Shortage in these essential reagents is even more acute in developing countries with no means to produce kits locally. We sought to find an alternative procedure to replace commercial kits using common reagents found in molecular biology laboratories. Here we report a method for RNA extraction that takes about 40 min to complete ten samples, and is not more laborious than current commercial RNA extraction kits. We demonstrate that this method can be used to process nasopharyngeal swab samples and yields RT-qPCR results comparable to those obtained with commercial kits. Most importantly, this procedure can be easily implemented in any molecular diagnostic laboratory. Frequent testing is crucial for individual patient management as well as for public health decision making in this pandemic. Implementation of this method could maintain crucial testing going despite commercial kit shortages.

scholarly journals Nested PCR for the detection of Taenia solium DNA in stool samples

2020 ◽  
Author(s):  
C Franco-Muñoz ◽  
A Arévalo ◽  
S Duque-Beltran
Keyword(s):  
Detection Limit ◽  
Nested Pcr ◽  
Intestinal Parasites ◽  
Taenia Solium ◽  
Cross Reaction ◽  
Public Health Importance ◽  
Alkaline Lysis ◽  
Pcr Products ◽  
Stool Samples ◽  
Dna Pcr

ABSTRACTThe traditional parasitological method to diagnose taeniasis is the microscopic observation of eggs in stool samples. However, this method does not allow differentiation between Taenia saginata and Taenia solium. This aim of this study was to achieve the detection of T. solium DNA by polymerase chain reaction (PCR) and to evaluate the cross-reaction with other species of the genus Taenia and other intestinal parasites. DNA was extracted from adult T. solium cestodes by cryolysis in liquid nitrogen and with the DNA stool extraction kit from Qiagen. The detection limit of the test was evaluated by DNA dilutions in water and in stool samples. DNA was extracted from proglottids of T. saginata and T. crassiceps and from stool samples containing other intestinal parasites using ethanol treatment, alkaline lysis, and the DNA stool extraction kit. Nested PCR was used to amplify a previously described fragment of the Tso31 gene, and the PCR products were analyzed by electrophoresis in 2% agarose gels followed by staining with GelRed. The nested PCR of the Tso31 gene allowed the detection of T. solium DNA in stool samples with a detection limit of 20 pg of parasite DNA. PCR showed no cross-reaction with T. saginata, T. crassiceps, or other intestinal parasites of public health importance in Colombia.

scholarly journals Human Taeniasis and Cysticercosis and Related Factors in Phu Tho Province, Northern Vietnam

2021 ◽  
Vol 59 (4) ◽  
pp. 369-376
Author(s):  
Vu Thi Lam Binh ◽  
Do Trung Dung ◽  
Hoang Quang Vinh ◽  
Van Hul Anke ◽  
Praet Nicolas ◽  
...  
Keyword(s):  
Public Health ◽  
Taenia Solium ◽  
Cross Sectional Study ◽  
Sectional Study ◽  
Related Factors ◽  
Cross Sectional ◽  
Raw Meat ◽  
Northern Vietnam ◽  
Circulating Antigens ◽  
Stool Samples

Several factors presumed to facilitate the transmission of Taenia spp. were reported in Vietnam. We conducted a cross-sectional study taking questionnaires from 1,185 participants, and collecting 1,151 sera and 1,036 stool samples in northern Vietnam. Sera were examined for circulating antigens of Taenia solium cysticerci using ELISA, stools for Taenia eggs by Kato-Katz smear, and copro-antigens by ELISA. Ag-ELISA revealed 4.6% antigen positivity, indicating infection with viable cysticerci. Taenia eggs were detected in 1.5% of participants. Copro-antigens were found in 2.8% of participants. Eating raw meat and/or vegetables was significantly associated with the presence of copro-antigen (OR=8.6, 95% CI: 1.16-63.9, P=0.01). Considering the high taeniasis prevalence and the associated threat, public health attention should be given to treat the tapeworm carriers in the projected areas.

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