scholarly journals Differential susceptibility of Onchocerca volvulus microfilaria to ivermectin in two areas of contrasting history of mass drug administration in Cameroon: Relevance of microscopy and molecular techniques for the monitoring of skin microfilarial repopulation within six months of direct observed treatment

2020 ◽  
Author(s):  
Raphael Awah Abong ◽  
Glory N. Amambo ◽  
Patrick W. Chounna Ndongmo ◽  
Abdel Jelil Njouendou ◽  
Ritter Manuel ◽  
...  
Keyword(s):  
Real Time ◽  
Drug Administration ◽  
Mass Drug Administration ◽  
Real Time Pcr ◽  
Differential Susceptibility ◽  
Diagnostic Techniques ◽  
Molecular Techniques ◽  
Onchocerca Volvulus ◽  
Density Reduction ◽  
Low Sensitivity

Abstract Background. Ivermectin is an excellent microfilaricide against Onchocerca volvulus. However, in some regions, long term use of ivermectin has resulted in sub-optimal responses to the treatment. More data to properly document the phenomenon in various contexts of ivermectin mass drug administration (IVM-MDA) is needed. Also, there is a need to accurately monitor a possible repopulation of skin by microfilariae following treatment. Skin snip microscopy is known to have a low sensitivity in individuals with light infections, which can be the case following treatment. This study was designed with two complementary objectives: (i) to assess the susceptibility of O. volvulus microfilariae to ivermectin in two areas undergoing IVM-MDA for different lengths of time, and (ii) to document the repopulation of skin by the O. volvulus microfilariae following treatment, using 3 independent diagnostic techniques. Method. Identified microfilaridermic individuals were treated with ivermectin and re-examined after 1, 3, and 6 months using microscopy, actin real-time PCR (actinqPCR) and O-150 LAMP assays. Susceptibility to ivermectin and trends in detecting reappearance of skin microfilariae were determined using three techniques. Microscopy was used as an imperfect gold standard to determine the performance of actin-qPCR and LAMP. Results. In Bafia with over 20 years of IVM-MDA, 11/51 (21.6%) direct observe treated microfilaridemic participants were still positive for skin microfilariae after 1 month. In Melong, with 10 years of IVM-MDA, 2/29 (6.9%) treated participants were still positive. The microfilarial density reduction per skin biopsy within one month following treatment was significantly lower in participants from Bafia. In both study sites, the molecular techniques detected higher proportions of infected individuals than microscopy at all monitoring time points. LAMP demonstrated the highest levels of sensitivity and real-time PCR was found to have the highest specificity. Conclusion. Patterns in skin mirofilariae clearance and repopulation were established. O. volvulus worms from Bafia with higher number of annual MDA displayed a lower clearance and higher repopulation rate after treatment with ivermectin. Molecular assays displayed higher sensitivity in monitoring O. volvulus microfilaridemia within six months following treatment.

scholarly journals Differential susceptibility of Onchocerca volvulus microfilaria to ivermectin in two areas of contrasting history of mass drug administration in Cameroon: Relevance of microscopy and molecular techniques for the monitoring of skin microfilarial repopulation within six months of direct observed treatment

2020 ◽  
Author(s):  
Raphael Awah Abong ◽  
Glory N. Amambo ◽  
Patrick W. Chounna Ndongmo ◽  
Abdel Jelil Njouendou ◽  
Ritter Manuel ◽  
...  
Keyword(s):  
Real Time ◽  
Drug Administration ◽  
Mass Drug Administration ◽  
Real Time Pcr ◽  
Differential Susceptibility ◽  
Diagnostic Techniques ◽  
Molecular Techniques ◽  
Onchocerca Volvulus ◽  
Density Reduction ◽  
Low Sensitivity

Abstract Background. Ivermectin is an excellent microfilaricide against Onchocerca volvulus. However, in some regions, long term use of ivermectin has resulted in sub-optimal responses to the treatment. More data to properly document the phenomenon in various contexts of ivermectin mass drug administration (IVM-MDA) is needed. Also, there is a need to accurately monitor a possible repopulation of skin by microfilariae following treatment. Skin snip microscopy is known to have a low sensitivity in individuals with light infections, which can be the case following treatment. This study was designed with two complementary objectives: (i) to assess the susceptibility of O. volvulus microfilariae to ivermectin in two areas undergoing IVM-MDA for different lengths of time, and (ii) to document the repopulation of skin by the O. volvulus microfilariae following treatment, using 3 independent diagnostic techniques.Method. Identified microfilaridermic individuals were treated with ivermectin and re-examined after 1, 3, and 6 months using microscopy, real-time PCR and LAMP assays. Susceptibility to ivermectin and trends in detecting reappearance of skin microfilariae were determined using three techniques. Microscopy was used as an imperfect gold standard to determine the performance of real-time PCR and LAMP.Results. In Bafia with over 20 years of IVM-MDA, 11/51 (21.6%) treated microfilaridemic participants where still positive for skin microfilariae after 1 month. In Melong, with 10 years of IVM-MDA, 2/29 (6.9%) treated participants were still positive. The microfilarial density reduction per skin biopsy within one month following treatment was significantly lower in participants from Bafia.In both study sites, the molecular techniques detected higher proportions of infected individuals than microscopy at all monitoring time points. LAMP demonstrated the highest levels of sensitivity and real-time PCR was found to have the highest specificity.Conclusion. Patterns in skin mirofilarial clearance and repopulation were established. O. volvulus worms from Bafia with higher number of annual MDA were less susceptible to ivermectin. Molecular assays displayed higher sensitivity in monitoring O. volvulus microfilaridemia within six months following treatment.

scholarly journals Differential susceptibility of Onchocerca volvulus microfilaria to ivermectin in two areas of contrasting history of mass drug administration in Cameroon: relevance of microscopy and molecular techniques for the monitoring of skin microfilarial repopulation within six months of direct observed treatment

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Raphael Awah Abong ◽  
Glory N. Amambo ◽  
Patrick W. Chounna Ndongmo ◽  
Abdel Jelil Njouendou ◽  
Ritter Manuel ◽  
...  
Keyword(s):  
Real Time ◽  
Drug Administration ◽  
Mass Drug Administration ◽  
Real Time Pcr ◽  
Differential Susceptibility ◽  
Diagnostic Techniques ◽  
Molecular Techniques ◽  
Onchocerca Volvulus ◽  
Density Reduction ◽  
Low Sensitivity

Abstract Background Ivermectin is an excellent microfilaricide against Onchocerca volvulus. However, in some regions, long term use of ivermectin has resulted in sub-optimal responses to the treatment. More data to properly document the phenomenon in various contexts of ivermectin mass drug administration (IVM-MDA) is needed. Also, there is a need to accurately monitor a possible repopulation of skin by microfilariae following treatment. Skin snip microscopy is known to have a low sensitivity in individuals with light infections, which can be the case following treatment. This study was designed with two complementary objectives: (i) to assess the susceptibility of O. volvulus microfilariae to ivermectin in two areas undergoing IVM-MDA for different lengths of time, and (ii) to document the repopulation of skin by the O. volvulus microfilariae following treatment, using 3 independent diagnostic techniques. Method Identified microfilaridermic individuals were treated with ivermectin and re-examined after 1, 3, and 6 months using microscopy, actin real-time PCR (actin-qPCR) and O-150 LAMP assays. Susceptibility to ivermectin and trends in detecting reappearance of skin microfilariae were determined using three techniques. Microscopy was used as an imperfect gold standard to determine the performance of actin-qPCR and LAMP. Results In Bafia with over 20 years of IVM-MDA, 11/51 (21.6%) direct observe treated microfilaridemic participants were still positive for skin microfilariae after 1 month. In Melong, with 10 years of IVM-MDA, 2/29 (6.9%) treated participants were still positive. The microfilarial density reduction per skin biopsy within one month following treatment was significantly lower in participants from Bafia. In both study sites, the molecular techniques detected higher proportions of infected individuals than microscopy at all monitoring time points. LAMP demonstrated the highest levels of sensitivity and real-time PCR was found to have the highest specificity. Conclusion Patterns in skin mirofilariae clearance and repopulation were established. O. volvulus worms from Bafia with higher number of annual MDA displayed a lower clearance and higher repopulation rate after treatment with ivermectin. Molecular assays displayed higher sensitivity in monitoring O. volvulus microfilaridemia within six months following treatment.

scholarly journals Differential susceptibility of Onchocerca volvulus microfilaria to ivermectin in two areas of contrasting history of mass drug administration in Cameroon: Relevance of microscopy and molecular techniques for the monitoring of skin microfilarial repopulation within six months of direct observed treatment

2020 ◽  
Author(s):  
Raphael Awah Abong ◽  
Glory Ngongeh Amambo ◽  
Patrick Chounna Ndongmo ◽  
Abdel Jelil Njouendou ◽  
Ritter Manuel ◽  
...  
Keyword(s):  
Real Time ◽  
Drug Administration ◽  
Mass Drug Administration ◽  
Real Time Pcr ◽  
Differential Susceptibility ◽  
Diagnostic Techniques ◽  
Molecular Techniques ◽  
Onchocerca Volvulus ◽  
Density Reduction ◽  
Low Sensitivity

Abstract Background. Ivermectin is an excellent microfilaricide against Onchocerca volvulus. However, in some regions, long term use of ivermectin has resulted in sub-optimal responses to the treatment. More data to properly document the phenomenon in various contexts of ivermectin mass drug administration (IVM-MDA) is needed. Also, there is a need to accurately monitor a possible repopulation of skin by microfilariae following treatment. Skin snip microscopy is known to have a low sensitivity in individuals with light infections, which can be the case following treatment. This study was designed with two complementary objectives: (i) to assess the susceptibility of O. volvulus microfilariae to ivermectin in two areas undergoing IVM-MDA for different lengths of time, and (ii) to document the repopulation of skin by the O. volvulus microfilariae following treatment, using 3 independent diagnostic techniques.Method. Identified microfilaridermic individuals were treated with ivermectin and re-examined after 1, 3, and 6 months using microscopy, actin real-time PCR (actin-qPCR) and O-150 LAMP assays. Susceptibility to ivermectin and trends in detecting reappearance of skin microfilariae were determined using three techniques. Microscopy was used as an imperfect gold standard to determine the performance of actin-qPCR and LAMP.Results. In Bafia with over 20 years of IVM-MDA, 11/51 (21.6%) direct observe treated microfilaridemic participants were still positive for skin microfilariae after 1 month. In Melong, with 10 years of IVM-MDA, 2/29 (6.9%) treated participants were still positive. The microfilarial density reduction per skin biopsy within one month following treatment was significantly lower in participants from Bafia.In both study sites, the molecular techniques detected higher proportions of infected individuals than microscopy at all monitoring time points. LAMP demonstrated the highest levels of sensitivity and real-time PCR was found to have the highest specificity.Conclusion. Patterns in skin mirofilariae clearance and repopulation were established. O. volvulus worms from Bafia with higher number of annual MDA displayed a lower clearance and higher repopulation rate after treatment with ivermectin. Molecular assays displayed higher sensitivity in monitoring O. volvulus microfilaridemia within six months following treatment.

scholarly journals Differential susceptibility of Onchocerca volvulus microfilaria to ivermectin in two areas of contrasting history of mass drug administration in Cameroon: Relevance of microscopy and molecular techniques for the monitoring of skin microfilarial repopulation within six months of direct observed treatment

2020 ◽  
Author(s):  
Raphael Awah Abong ◽  
Glory N. Amambo ◽  
Patrick W. Chounna Ndongmo ◽  
Abdel Jelil Njouendou ◽  
Ritter Manuel ◽  
...  
Keyword(s):  
Real Time ◽  
Drug Administration ◽  
Mass Drug Administration ◽  
Real Time Pcr ◽  
Differential Susceptibility ◽  
Lamp Assay ◽  
Molecular Techniques ◽  
Onchocerca Volvulus ◽  
History Of ◽  
Higher Sensitivity

Abstract Background Ivermectin is an excellent microfilaricide against Onchocerca volvulus. However, in some regions, long term use of ivermectin has resulted in sub-optimal responses to the treatment. More data to properly document the phenomenon in various contexts of ivermectin mass drug administration (IVM-MDA) is needed. Also, there is a need to accurately monitor a possible repopulation of skin by microfilariae following treatment. Microscopy is known to have low sensitivity to detect skin microfilariae, when their density is low following treatment. This study was designed with two complementary objectives: (i) to assess the susceptibility of O. volvulus microfilariae to ivermectin in situations of contrasting history of treatments, and (ii) to document the repopulation of skin by the O. volvulus microfilariae following treatment, comparing the performances of 3 independent diagnostic techniques. Method Identified microfilaridermic individuals were treated with ivermectin and re-examined after 1, 3, and 6 months using microscopy, real-time PCR and LAMP assays. Susceptibility to ivermectin and trends in detecting reappearance of skin microfilariae were reported for the three techniques. Microscopy was used as an imperfect gold standard to determine the performance characteristics of real-time PCR and LAMP assay. Results In Bafia with over 20 years of IVM-MDA, 11/51 (21.6%) treated microfilaridemic participants where still positive for skin microfilariae after 1 month. In Melong, with 10 years of IVM-MDA, 2/29 (6.9%) treated participants were still positive. The microfilarial density reduction per skin biopsy within one month following treatment was significantly lower in participants from Bafia, but the pattern in the two sites was similar. In both study sites, the molecular techniques detected higher proportions of infected individuals than microscopy at all monitoring time points. LAMP assay showed lower specificity compared to real-time PCR and microscopy, but had comparable sensitivity to real-time PCR and significantly higher sensitivity than microscopy. Conclusion Patterns in skin mirofilarial clearance and repopulation were established. O . volvulus worms from Bafia with higher number of annual MDA were less susceptible to ivermectin. Molecular assays displayed higher sensitivity in monitoring O. volvulus microfilaridemia within six months following treatment.

scholarly journals Detection of Peanut Allergen Traces with a Real Time PCR Assay - The Challenge to Protect Food-Allergic Consumers

2017 ◽  
Vol 7 (1) ◽  
pp. 32 ◽  
Author(s):  
Dimitra Houhoula ◽  
Stamatios Koussissis ◽  
Vladimiros Lougovois ◽  
John Tsaknis ◽  
Dimitra Kassavita ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Food Products ◽  
Molecular Techniques ◽  
Food Allergens ◽  
Pcr Assay ◽  
Peanut Allergen ◽  
Pcr Method ◽  
Detection And Quantification

The aim of the present study was the implementation of molecular techniques in the detection and quantification of allergic substances of peanut in various kinds of food products, e.g., breakfast cereals, chocolates and biscuits that are frequently related to allergies. In some cases, the presence of peanuts can be due to contamination during production and are not declared on the label. A total of 152 samples were collected from supermarkets and were analysed by a Real Time PCR method. The results indicated that 125 samples (83,3%) were found positive in peanut traces but the most important finding is that from the 84 samples that had no allergen declaration for peanuts, 48 (57,1%) of them were found positive. In conclusion, Real Time PCR can be a very important tool for the rapid detection and quantification of food allergens.

Detection of Leishmania infantum DNA by real-time PCR in canine oral and conjunctival swabs and comparison with other diagnostic techniques

2012 ◽  
Vol 184 (1) ◽  
pp. 10-17 ◽  
Author(s):  
Gabriella Lombardo ◽  
Maria Grazia Pennisi ◽  
Tiziana Lupo ◽  
Antonella Migliazzo ◽  
Alessandra Caprì ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Leishmania Infantum ◽  
Diagnostic Techniques

scholarly journals Primers designed for the detection of grapevine pathogens spreading with propagating material by quantitative real-time PCR

2015 ◽  
Vol 21 (1-2) ◽  
Author(s):  
N. Czotter ◽  
E. Manduláné Farkas ◽  
R. Lózsa ◽  
I. Ember ◽  
G. Szûcsné Varga ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dna Amplification ◽  
Molecular Techniques ◽  
Latent Infections ◽  
Quantitative Real Time Pcr ◽  
Detection And Identification

Several grapevine pathogens are disseminated by propagating material as systemic, but latent infections. Their detection and identification have a basic importance in the production and handling of propagating stocks. Thus several sensitive and reliable diagnostic protocols mostly based on molecular techniques have been developed. Of these methods quantitative real-time PCR (q-PCR) has recently got an emerging importance. Here we collected primer data for the detection and identification of grapevine pathogens which are important in the production of propagating stocks by q-PCR. Additional novel techniques that use DNA amplification, hybridization and  sequencing are also briefly reviewed.

Analyzing Gene Expression through Real Time PCR while Neo-tissue Regeneration using Developed Tissue Constructs

2020 ◽  
pp. 15-34
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Tissue Regeneration ◽  
Real Time Pcr ◽  
Molecular Level ◽  
Northern Blotting ◽  
Tissue Constructs ◽  
Low Sensitivity

Real-time PCR offers a wide area of application to analyze the role of gene activity in various biological aspects at the molecular level with higher specificity, sensitivity and the potential to troubleshoot with post-PCR processing and difficulties. With the recent advancement in the development of functional tissue graft for the regeneration of damaged/diseased tissue, it is effective to analyze the cell behaviour and differentiation over tissue construct toward specific lineage through analyzing the expression of an array of specific genes. With the ability to collect data in the exponential phase, the application of Real-Time PCR has been expanded into various fields such as tissue engineering ranging from absolute quantification of gene expression to determine neo-tissue regeneration and its maturation. In addition to its usage as a research tool, numerous advancements in molecular diagnostics have been achieved, including microbial quantification, determination of gene dose and cancer research. Also, in order to consistently quantify mRNA levels, Northern blotting and in situ hybridization (ISH) methods are less preferred due to low sensitivity, poor precision in detecting gene expression at a low level. An amplification step is thus frequently required to quantify mRNA amounts from engineered tissues of limited size. When analyzing tissue-engineered constructs or studying biomaterials–cells interactions, it is pertinent to quantify the performance of such constructs in terms of extracellular matrix formation while in vitro and in vivo examination, provide clues regarding the performance of various tissue constructs at the molecular level. In this chapter, our focus is on Basics of qPCR, an overview of technical aspects of Real-time PCR; recent Protocol used in the lab, primer designing, detection methods and troubleshooting of the experimental problems.

scholarly journals Evaluation of Two DNA Extraction Methods for Detection of Strongyloides stercoralis Infection

2018 ◽  
Vol 56 (4) ◽  
Author(s):  
Beatrice Barda ◽  
Rahel Wampfler ◽  
Somphou Sayasone ◽  
Khampheng Phongluxa ◽  
Syda Xayavong ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Strongyloides Stercoralis ◽  
Extraction Methods ◽  
Molecular Techniques ◽  
Diagnostic Methods ◽  
Content Type ◽  
Stool Samples ◽  
Dna Extraction Methods ◽  
Baermann Method

ABSTRACT Strongyloides stercoralis is present worldwide, but its prevalence is still uncertain, mainly due to the lack of sensitivity of diagnostic methods. Molecular techniques are under development, but a standardized protocol is still unavailable. We compared the sensitivity of real-time PCR, using two extraction protocols, with that of the Baermann technique. Samples were collected in the framework of the baseline screening of a randomized clinical trial evaluating moxidectin against S. stercoralis in Lao People's Democratic Republic. Two stool samples from each participant were processed by the Baermann method, and one subsample was processed by PCR. DNA was extracted using the QIAamp DNA stool minikit based on the standard protocol for the QIAamp DNA minikit (QIA) and using a modification of the QIA procedure (POL). Subsequently, all extracted samples were analyzed by real-time PCR. Overall, 95 samples were analyzed by the three diagnostic methods. Sixty-nine (72.6%) samples were positive according to the Baermann method, 25 (26.3%) by the QIA method, and 62 (65.3%) by the POL method. The sensitivities were 86% (95% confidence interval [CI], 76.7 to 92.9), 31.0% (95% CI, 21.3 to 42.6), and 78.0% (95% CI, 66.8 to 86.1) for the Baermann, QIA, and POL methods, respectively. The sensitivities calculated for each day of the Baermann method separately were 60% (48.4 to 70.8%) and 64% (52.2 to 74.2%) for days 1 and 2, respectively. In conclusion, the POL method revealed a good performance and was comparable to the Baermann test performed on two stool samples and superior to the Baermann method performed on one stool sample. Additional studies are needed to standardize a PCR protocol for S. stercoralis diagnosis.

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