Optimized strategy for real-time qPCR detection of Onchocerca volvulus DNA in pooled Simulium sp. blackfly vectors.

<h4>Background</h4>Onchocerca volvulus is a filarial parasite that is a major cause of dermatitis and blindness in endemic regions primarily in sub-Saharan Africa. Widespread efforts to control the disease caused by O. volvulus infection (onchocerciasis) began in 1974 and in recent years...

Full description

Bibliographic Details
Main Authors: Mary Doherty, Jessica R Grant, Nils Pilotte, Sasisekhar Bennuru, Kerstin Fischer, Peter U Fischer, Sara Lustigman, Thomas B Nutman, Kenneth Pfarr, Achim Hoerauf, Thomas R Unnasch, Hassan K Hassan, Samuel Wanji, Patrick J Lammie, Eric Ottesen, Charles Mackenzie, Steven A Williams
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2023-12-01
Series:PLoS Neglected Tropical Diseases
Online Access:https://doi.org/10.1371/journal.pntd.0011815
_version_ 1797326124014895104
author Mary Doherty
Jessica R Grant
Nils Pilotte
Sasisekhar Bennuru
Kerstin Fischer
Peter U Fischer
Sara Lustigman
Thomas B Nutman
Kenneth Pfarr
Achim Hoerauf
Thomas R Unnasch
Hassan K Hassan
Samuel Wanji
Patrick J Lammie
Eric Ottesen
Charles Mackenzie
Steven A Williams
author_facet Mary Doherty
Jessica R Grant
Nils Pilotte
Sasisekhar Bennuru
Kerstin Fischer
Peter U Fischer
Sara Lustigman
Thomas B Nutman
Kenneth Pfarr
Achim Hoerauf
Thomas R Unnasch
Hassan K Hassan
Samuel Wanji
Patrick J Lammie
Eric Ottesen
Charles Mackenzie
Steven A Williams
author_sort Mary Doherty
collection DOAJ
description <h4>Background</h4>Onchocerca volvulus is a filarial parasite that is a major cause of dermatitis and blindness in endemic regions primarily in sub-Saharan Africa. Widespread efforts to control the disease caused by O. volvulus infection (onchocerciasis) began in 1974 and in recent years, following successful elimination of transmission in much of the Americas, the focus of efforts in Africa has moved from control to the more challenging goal of elimination of transmission in all endemic countries. Mass drug administration (MDA) with ivermectin has reached more than 150 million people and elimination of transmission has been confirmed in four South American countries, with at least two African countries having now stopped MDA as they approach verification of elimination. It is essential that accurate data for active transmission are used to assist in making the critical decision to stop MDA, since missing low levels of transmission and infection can lead to continued spread or recrudescence of the disease.<h4>Methodology/principal findings</h4>Current World Health Organization guidelines for MDA stopping decisions and post-treatment surveillance include screening pools of the Simulium blackfly vector for the presence of O. volvulus larvae using a PCR-ELISA-based molecular technique. In this study, we address the potential of an updated, practical, standardized molecular diagnostic tool with increased sensitivity and species-specificity by comparing several candidate qPCR assays. When paired with heat-stable reagents, a qPCR assay with a mitochondrial DNA target (OvND5) was found to be more sensitive and species-specific than an O150 qPCR, which targets a non-protein coding repetitive DNA sequence. The OvND5 assay detected 19/20 pools of 100 blackfly heads spiked with a single L3, compared to 16/20 for the O150 qPCR assay.<h4>Conclusions/significance</h4>Given the improved sensitivity, species-specificity and resistance to PCR inhibitors, we identified OvND5 as the optimal target for field sample detection. All reagents for this assay can be shipped at room temperature with no loss of activity. The qPCR protocol we propose is also simpler, faster, and more cost-effective than the current end-point molecular assays.
first_indexed 2024-03-08T06:20:00Z
format Article
id doaj.art-07aba128e6634deeb7f43656fec00b66
institution Directory Open Access Journal
issn 1935-2727
1935-2735
language English
last_indexed 2024-03-08T06:20:00Z
publishDate 2023-12-01
publisher Public Library of Science (PLoS)
record_format Article
series PLoS Neglected Tropical Diseases
spelling doaj.art-07aba128e6634deeb7f43656fec00b662024-02-04T05:32:00ZengPublic Library of Science (PLoS)PLoS Neglected Tropical Diseases1935-27271935-27352023-12-011712e001181510.1371/journal.pntd.0011815Optimized strategy for real-time qPCR detection of Onchocerca volvulus DNA in pooled Simulium sp. blackfly vectors.Mary DohertyJessica R GrantNils PilotteSasisekhar BennuruKerstin FischerPeter U FischerSara LustigmanThomas B NutmanKenneth PfarrAchim HoeraufThomas R UnnaschHassan K HassanSamuel WanjiPatrick J LammieEric OttesenCharles MackenzieSteven A Williams<h4>Background</h4>Onchocerca volvulus is a filarial parasite that is a major cause of dermatitis and blindness in endemic regions primarily in sub-Saharan Africa. Widespread efforts to control the disease caused by O. volvulus infection (onchocerciasis) began in 1974 and in recent years, following successful elimination of transmission in much of the Americas, the focus of efforts in Africa has moved from control to the more challenging goal of elimination of transmission in all endemic countries. Mass drug administration (MDA) with ivermectin has reached more than 150 million people and elimination of transmission has been confirmed in four South American countries, with at least two African countries having now stopped MDA as they approach verification of elimination. It is essential that accurate data for active transmission are used to assist in making the critical decision to stop MDA, since missing low levels of transmission and infection can lead to continued spread or recrudescence of the disease.<h4>Methodology/principal findings</h4>Current World Health Organization guidelines for MDA stopping decisions and post-treatment surveillance include screening pools of the Simulium blackfly vector for the presence of O. volvulus larvae using a PCR-ELISA-based molecular technique. In this study, we address the potential of an updated, practical, standardized molecular diagnostic tool with increased sensitivity and species-specificity by comparing several candidate qPCR assays. When paired with heat-stable reagents, a qPCR assay with a mitochondrial DNA target (OvND5) was found to be more sensitive and species-specific than an O150 qPCR, which targets a non-protein coding repetitive DNA sequence. The OvND5 assay detected 19/20 pools of 100 blackfly heads spiked with a single L3, compared to 16/20 for the O150 qPCR assay.<h4>Conclusions/significance</h4>Given the improved sensitivity, species-specificity and resistance to PCR inhibitors, we identified OvND5 as the optimal target for field sample detection. All reagents for this assay can be shipped at room temperature with no loss of activity. The qPCR protocol we propose is also simpler, faster, and more cost-effective than the current end-point molecular assays.https://doi.org/10.1371/journal.pntd.0011815
spellingShingle Mary Doherty
Jessica R Grant
Nils Pilotte
Sasisekhar Bennuru
Kerstin Fischer
Peter U Fischer
Sara Lustigman
Thomas B Nutman
Kenneth Pfarr
Achim Hoerauf
Thomas R Unnasch
Hassan K Hassan
Samuel Wanji
Patrick J Lammie
Eric Ottesen
Charles Mackenzie
Steven A Williams
Optimized strategy for real-time qPCR detection of Onchocerca volvulus DNA in pooled Simulium sp. blackfly vectors.
PLoS Neglected Tropical Diseases
title Optimized strategy for real-time qPCR detection of Onchocerca volvulus DNA in pooled Simulium sp. blackfly vectors.
title_full Optimized strategy for real-time qPCR detection of Onchocerca volvulus DNA in pooled Simulium sp. blackfly vectors.
title_fullStr Optimized strategy for real-time qPCR detection of Onchocerca volvulus DNA in pooled Simulium sp. blackfly vectors.
title_full_unstemmed Optimized strategy for real-time qPCR detection of Onchocerca volvulus DNA in pooled Simulium sp. blackfly vectors.
title_short Optimized strategy for real-time qPCR detection of Onchocerca volvulus DNA in pooled Simulium sp. blackfly vectors.
title_sort optimized strategy for real time qpcr detection of onchocerca volvulus dna in pooled simulium sp blackfly vectors
url https://doi.org/10.1371/journal.pntd.0011815
work_keys_str_mv AT marydoherty optimizedstrategyforrealtimeqpcrdetectionofonchocercavolvulusdnainpooledsimuliumspblackflyvectors
AT jessicargrant optimizedstrategyforrealtimeqpcrdetectionofonchocercavolvulusdnainpooledsimuliumspblackflyvectors
AT nilspilotte optimizedstrategyforrealtimeqpcrdetectionofonchocercavolvulusdnainpooledsimuliumspblackflyvectors
AT sasisekharbennuru optimizedstrategyforrealtimeqpcrdetectionofonchocercavolvulusdnainpooledsimuliumspblackflyvectors
AT kerstinfischer optimizedstrategyforrealtimeqpcrdetectionofonchocercavolvulusdnainpooledsimuliumspblackflyvectors
AT peterufischer optimizedstrategyforrealtimeqpcrdetectionofonchocercavolvulusdnainpooledsimuliumspblackflyvectors
AT saralustigman optimizedstrategyforrealtimeqpcrdetectionofonchocercavolvulusdnainpooledsimuliumspblackflyvectors
AT thomasbnutman optimizedstrategyforrealtimeqpcrdetectionofonchocercavolvulusdnainpooledsimuliumspblackflyvectors
AT kennethpfarr optimizedstrategyforrealtimeqpcrdetectionofonchocercavolvulusdnainpooledsimuliumspblackflyvectors
AT achimhoerauf optimizedstrategyforrealtimeqpcrdetectionofonchocercavolvulusdnainpooledsimuliumspblackflyvectors
AT thomasrunnasch optimizedstrategyforrealtimeqpcrdetectionofonchocercavolvulusdnainpooledsimuliumspblackflyvectors
AT hassankhassan optimizedstrategyforrealtimeqpcrdetectionofonchocercavolvulusdnainpooledsimuliumspblackflyvectors
AT samuelwanji optimizedstrategyforrealtimeqpcrdetectionofonchocercavolvulusdnainpooledsimuliumspblackflyvectors
AT patrickjlammie optimizedstrategyforrealtimeqpcrdetectionofonchocercavolvulusdnainpooledsimuliumspblackflyvectors
AT ericottesen optimizedstrategyforrealtimeqpcrdetectionofonchocercavolvulusdnainpooledsimuliumspblackflyvectors
AT charlesmackenzie optimizedstrategyforrealtimeqpcrdetectionofonchocercavolvulusdnainpooledsimuliumspblackflyvectors
AT stevenawilliams optimizedstrategyforrealtimeqpcrdetectionofonchocercavolvulusdnainpooledsimuliumspblackflyvectors