scholarly journals Comparison of algorithms for the detection of enteroviruses in stool specimens from children diagnosed with Acute Flaccid Paralysis

2017 ◽  
Author(s):  
J.A. Adeniji ◽  
F. A. Ayeni ◽  
A. Ibrahim ◽  
K.A. Tijani ◽  
T.O.C. Faleye ◽  
...  
Keyword(s):  
Cell Culture ◽  
Rna Extraction ◽  
Paired Samples ◽  
Fecal Suspension ◽  
Culture Independent ◽  
A Cell ◽  
Increased Sensitivity ◽  
Stool Specimens ◽  
Stool Suspension ◽  
Culture Dependent

ABSTRACTWith poliovirus eradication within reach, the WHO has included in its recommendations a cell-culture independent algorithm for enterovirus surveillance. This study was designed to compare both the cell culture dependent and independent algorithms and assess how either might impact our perception of the diversity of enterovirus types present in a sample.Sixteen paired samples (16 isolates from RD cell culture and their corresponding stool suspension. i.e. 32 samples) from AFP cases in Nigeria were analyzed in this study. One of these 16 sample pairs (the control) was previously identified and confirmed as poliovirus 2 (PV-2). All the samples were subjected to RNA extraction, cDNA synthesis, RT-snPCR (the WHO recommended cell-culture independent algorithm) and its modifications for co-infection detection and resolution. Amplicons were sequenced and strains identified using the enterovirus genotyping tool and phylogenetic analysis.The enterovirus diversity was shown to be the same between RD cell culture isolates and fecal suspension for the control and five (7, 10, 11, 12 & 14) of the samples analyzed. It was however, different for the remaining 10 (62.5%) samples analyzed. Fourteen different enterovirus types were identified in this study. To be precise, 9 (CV-B4, E6, E7, E13, E14, E19, E29, EV-B75 and EV-B77) and 5 (CV-A1, CV-A11, CV-A13, EV-C99 and PV2) EV-B and EV-C types, respectively where detected in this study. It is crucial to mention that E19 and EV-B75were only recovered from RD cell culture isolates while E14, EV-B77, CV-A11 and CV-A13 were only recovered from fecal suspension.The results of this study show that both the cell culture dependent and independent protocols recommended by the WHO for enterovirus detection unavoidably bias our perception of the diversity of enterovirus types present in a sample. Hence, rather than jettison one for the other, effort should be directed at harmonizing both for increased sensitivity.

scholarly journals Comparison of Algorithms for the Detection of Enteroviruses in Stool Specimens from Children Diagnosed with Acute Flaccid Paralysis

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
J. A. Adeniji ◽  
F. A. Ayeni ◽  
A. Ibrahim ◽  
K. A. Tijani ◽  
T. O. C. Faleye ◽  
...  
Keyword(s):  
Cell Culture ◽  
Rna Extraction ◽  
Cdna Synthesis ◽  
Detection Algorithms ◽  
Paired Samples ◽  
Fecal Suspension ◽  
Increased Sensitivity ◽  
Stool Specimens ◽  
Stool Suspension ◽  
Culture Dependent

This study was designed to compare both the cell culture dependent and independent enterovirus detection algorithms recommended by the WHO and assess how either might impact our perception of the diversity of enterovirus types present in a sample. Sixteen paired samples (16 isolates from RD cell culture and their corresponding stool suspension, i.e., 32 samples) from AFP cases in Nigeria were analyzed in this study. All the samples were subjected to RNA extraction, cDNA synthesis, the WHO recommended RT-snPCR, and its modification. Amplicons were sequenced and strains identified. Enterovirus diversity was the same between the isolates and fecal suspension for the control and five of the samples. It was, however, different for the remaining 10 (62.5%) samples. Nine (CV-B4, E6, E7, E13, E14, E19, E29, EV-B75, and EV-B77) and five (CV-A1, CV-A11, CV-A13, EV-C99, and PV2) EV-B and EV-C types, respectively, were detected. Particularly, E19 and EV-B75 were only recovered from the isolates while E14, EV-B77, CV-A11, and CV-A13 were only recovered from fecal suspension. Both the cell culture dependent and independent protocols bias our perception of the diversity of enterovirus types present in a sample. Hence, effort should be directed at harmonizing both for increased sensitivity.

scholarly journals Culture-Independent Detection of Poliovirus in Stool Samples by Direct RNA Extraction

2021 ◽  
Author(s):  
Chelsea Harrington ◽  
Hong Sun ◽  
Stacey Jeffries-Miles ◽  
Nancy Gerloff ◽  
Mark Mandelbaum ◽  
...  
Keyword(s):  
Cell Culture ◽  
World Health Organization ◽  
Surveillance System ◽  
Virus Isolation ◽  
Rna Extraction ◽  
World Health ◽  
Culture Independent ◽  
Laboratory Surveillance ◽  
Stool Samples ◽  
Health Organization

The GPLN is a global surveillance system composed of 146 laboratories in 92 countries, in each of the six World Health Organization regions. Laboratory surveillance for PV relies on virus isolation by cell culture to identify PV in stool samples from AFP cases.

scholarly journals Direct Detection and Identification of Enteroviruses from Faeces of Healthy Nigerian Children Using a Cell-Culture Independent RT-Seminested PCR Assay

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Temitope Oluwasegun Cephas Faleye ◽  
Moses Olubusuyi Adewumi ◽  
Bamidele Atinuke Coker ◽  
Felix Yasha Nudamajo ◽  
Johnson Adekunle Adeniji
Keyword(s):  
Cell Culture ◽  
Sequence Data ◽  
Direct Detection ◽  
Clinical Specimen ◽  
Enterovirus 71 ◽  
Healthy Children ◽  
Molecular Sequence Data ◽  
Band Size ◽  
Culture Independent ◽  
A Cell

Recently, a cell-culture independent protocol for detection of enteroviruses from clinical specimen was recommended by the WHO for surveillance alongside the previously established protocols. Here, we investigated whether this new protocol will show the same enterovirus diversity landscape as the established cell-culture dependent protocols. Faecal samples were collected from sixty apparently healthy children in Ibadan, Nigeria. Samples were resuspended in phosphate buffered saline, RNA was extracted, and the VP1 gene was amplified using WHO recommended RT-snPCR protocol. Amplicons were sequenced and sequences subjected to phylogenetic analysis. Fifteen (25%) of the 60 samples yielded the expected band size. Of the 15 amplicons sequenced, 12 were exploitable. The remaining 3 had electropherograms with multiple peaks and were unexploitable. Eleven of the 12 exploitable sequences were identified as Coxsackievirus A1 (CVA1), CVA3, CVA4, CVA8, CVA20, echovirus 32 (E32), enterovirus 71 (EV71), EVB80, and EVC99. Subsequently, the last exploitable sequence was identified as enterobacteriophage baseplate gene by nucleotide BLAST. The results of this study document the first description of molecular sequence data on CVA1, CVA8, and E32 strains present in Nigeria. The result further showed that species A enteroviruses were more commonly detected in the region when cell-culture bias is bypassed.

scholarly journals Nonpolio enteroviruses can also be recovered from non-reproducible cytopathology in L20B cell line

2017 ◽  
Author(s):  
J. A. Adeniji ◽  
U.I. Ibok ◽  
O.T. Ayinde ◽  
A.O. Oragwa ◽  
U.E. George ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Line ◽  
Rna Extraction ◽  
Cdna Synthesis ◽  
Pcr Assay ◽  
Fecal Suspension ◽  
Coxsackievirus A20 ◽  
Culture Supernatants ◽  
Seminested Pcr ◽  
University Of Ibadan

ABSTRACTSamples showing cytopathology (CPE) on initial inoculation into L20B cell line but with no observed or reproducible CPE on passage in L20B or RD are considered negative for both poliovirus and nonpolio enteroviruses (NPEVs). The phenomenon is termed ‘non-reproducible CPE’. Its occurrence is usually ascribed to the likely presence of reoviruses, adenoviruses and other non-enteroviruses. This study aimed to investigate the likelihood that NPEVs are also present in cases with non-reproducible CPE.Twenty-six (26) cell culture suspensions were analyzed in this study. The suspensions were collected from the WHO National Polio Laboratory, Department of Virology, College of Medicine, University of Ibadan. The suspensions emanated from 13 L20B cell culture tubes that showed cytopathology within 5 days of inoculation with fecal suspension from AFP cases. However, on passage into one each of RD and L20B cell lines, the CPE was not reproducible. All samples were subjected to RNA extraction, cDNA synthesis, the WHO recommended VP1 RT-seminestedPCR assay, species resolution PCR assay, sequencing and phylogenetic analysis.Six (6) samples were positive for the VP1 RT-seminested PCR assay. Only four of which were positive by the species resolution PCR assay. The four amplicons were sequenced, however, only three (3) were successfully identified as Coxsackievirus A20 (2 isolates) and Echovirus 29 (1 isolate).The results of this study unambiguously showed the presence of NPEVs (particularly CVA20 and E29) in cell culture supernatants of samples with CPE on initial inoculation into L20B cell line but with no observed or reproducible CPE on passage in RD cell line. Therefore, like reoviruses, adenoviruses and other non-enteroviruses, NPEVs can also be recovered in cases with non-reproducible CPE.

Automated cell counting of astrocytes on patterned substrates containing aliphatic and charged properties

1995 ◽  
Vol 53 ◽  
pp. 974-975
Author(s):  
W. Shain ◽  
H. Ancin ◽  
H.C. Craighead ◽  
M. Isaacson ◽  
L. Kam ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Attachment ◽  
Culture Method ◽  
Cell Counting ◽  
Data Sets ◽  
Nuclear Staining ◽  
Double Positive ◽  
A Cell ◽  
Wafer Test ◽  
Cell Densities

Neural protheses have potential to restore nervous system functions lost by trauma or disease. Nanofabrication extends this approach to implants for stimulating and recording from single or small groups of neurons in the spinal cord and brain; however, tissue compatibility is a major limitation to their practical application. We are using a cell culture method for quantitatively measuring cell attachment to surfaces designed for nanofabricated neural prostheses.Silicon wafer test surfaces composed of 50-μm bars separated by aliphatic regions were fabricated using methods similar to a procedure described by Kleinfeld et al. Test surfaces contained either a single or double positive charge/residue. Cyanine dyes (diIC18(3)) stained the background and cell membranes (Fig 1); however, identification of individual cells at higher densities was difficult (Fig 2). Nuclear staining with acriflavine allowed discrimination of individual cells and permitted automated counting of nuclei using 3-D data sets from the confocal microscope (Fig 3). For cell attachment assays, LRM5 5 astroglial cells and astrocytes in primary cell culture were plated at increasing cell densities on test substrates, incubated for 24 hr, fixed, stained, mounted on coverslips, and imaged with a 10x objective.

Juglone Exerts Cytotoxic, Anti-proliferative and Anti-invasive Effects on Glioblastoma Multiforme in a Cell Culture Model

2016 ◽  
Vol 16 (9) ◽  
pp. 1190-1197 ◽  
Author(s):  
Dziugas Meskelevicius ◽  
Kastytis Sidlauskas ◽  
Ruta Bagdonaviciute ◽  
Julius Liobikas ◽  
Daiva Majiene
Keyword(s):  
Cell Culture ◽  
Glioblastoma Multiforme ◽  
Cell Culture Model ◽  
Culture Model ◽  
A Cell

scholarly journals Microbiota identified from preserved Anopheles

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Bianca E Silva ◽  
Zvifadzo Matsena Zingoni ◽  
Lizette L. Koekemoer ◽  
Yael L. Dahan-Moss
Keyword(s):  
Microbial Diversity ◽  
Vector Competence ◽  
Mosquito Species ◽  
Anopheles Funestus ◽  
Cost Effective ◽  
Diversity Indices ◽  
Malaria Vectors ◽  
Microbial Composition ◽  
Culture Independent ◽  
Culture Dependent

Abstract Background Mosquito species from the Anopheles gambiae complex and the Anopheles funestus group are dominant African malaria vectors. Mosquito microbiota play vital roles in physiology and vector competence. Recent research has focused on investigating the mosquito microbiota, especially in wild populations. Wild mosquitoes are preserved and transported to a laboratory for analyses. Thus far, microbial characterization post-preservation has been investigated in only Aedes vexans and Culex pipiens. Investigating the efficacy of cost-effective preservatives has also been limited to AllProtect reagent, ethanol and nucleic acid preservation buffer. This study characterized the microbiota of African Anopheles vectors: Anopheles arabiensis (member of the An. gambiae complex) and An. funestus (member of the An. funestus group), preserved on silica desiccant and RNAlater® solution. Methods Microbial composition and diversity were characterized using culture-dependent (midgut dissections, culturomics, MALDI-TOF MS) and culture-independent techniques (abdominal dissections, DNA extraction, next-generation sequencing) from laboratory (colonized) and field-collected mosquitoes. Colonized mosquitoes were either fresh (non-preserved) or preserved for 4 and 12 weeks on silica or in RNAlater®. Microbiota were also characterized from field-collected An. arabiensis preserved on silica for 8, 12 and 16 weeks. Results Elizabethkingia anophelis and Serratia oryzae were common between both vector species, while Enterobacter cloacae and Staphylococcus epidermidis were specific to females and males, respectively. Microbial diversity was not influenced by sex, condition (fresh or preserved), preservative, or preservation time-period; however, the type of bacterial identification technique affected all microbial diversity indices. Conclusions This study broadly characterized the microbiota of An. arabiensis and An. funestus. Silica- and RNAlater®-preservation were appropriate when paired with culture-dependent and culture-independent techniques, respectively. These results broaden the selection of cost-effective methods available for handling vector samples for downstream microbial analyses.

A New In Vitro Model for Predicting the Toxicity of Cosmetic Products

1992 ◽  
Vol 20 (1) ◽  
pp. 138-143
Author(s):  
Maria Carrara ◽  
Lorenzo Cima ◽  
Roberto Cerini ◽  
Maurizio Dalle Carbonare
Keyword(s):  
Cell Culture ◽  
Cultured Cells ◽  
Neutral Red ◽  
Total Protein Content ◽  
Cosmetic Products ◽  
Cell Culture Insert ◽  
A Cell ◽  
Vitro Model ◽  
Cosmetic Emulsions

A method has been developed whereby cosmetic products which are not soluble in water or in alcohol can be brought into contact with cell cultures by being placed in a cell culture insert, which is then placed in the cell culture well. Preliminary experiments were carried out with L929 cells, and cytotoxicity was evaluated by measuring neutral red uptake and the total protein content of treated cultured cells. Encouraging results were obtained in comparisons of three cosmetic emulsions and of one emulsion containing a range of concentrations of two preservatives, Kathon CG and Bronopol.

scholarly journals Self-Assembling Polypeptide Hydrogels as a Platform to Recapitulate the Tumor Microenvironment

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3286
Author(s):  
Dariusz Lachowski ◽  
Carlos Matellan ◽  
Ernesto Cortes ◽  
Alberto Saiani ◽  
Aline F. Miller ◽  
...  
Keyword(s):  
Cell Culture ◽  
Tumor Microenvironment ◽  
Cancer Cell ◽  
Critical Role ◽  
Hypoxia Inducible Factor ◽  
Pancreatic Cancer Cell Line ◽  
Cancer Cell Line ◽  
Culture Systems ◽  
A Cell

The tumor microenvironment plays a critical role in modulating cancer cell migration, metabolism, and malignancy, thus, highlighting the need to develop in vitro culture systems that can recapitulate its abnormal properties. While a variety of stiffness-tunable biomaterials, reviewed here, have been developed to mimic the rigidity of the tumor extracellular matrix, culture systems that can recapitulate the broader extracellular context of the tumor microenvironment (including pH and temperature) remain comparably unexplored, partially due to the difficulty in independently tuning these parameters. Here, we investigate a self-assembled polypeptide network hydrogel as a cell culture platform and demonstrate that the culture parameters, including the substrate stiffness, extracellular pH and temperature, can be independently controlled. We then use this biomaterial as a cell culture substrate to assess the effect of stiffness, pH and temperature on Suit2 cells, a pancreatic cancer cell line, and demonstrate that these microenvironmental factors can regulate two critical transcription factors in cancer: yes-associated protein 1 (YAP) and hypoxia inducible factor (HIF-1A).

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