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      Oligonucleotide Based Magnetic Bead Capture of Onchocerca volvulus DNA for PCR Pool Screening of Vector Black Flies


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          Entomological surveys of Simulium vectors are an important component in the criteria used to determine if Onchocerca volvulus transmission has been interrupted and if focal elimination of the parasite has been achieved. However, because infection in the vector population is quite rare in areas where control has succeeded, large numbers of flies need to be examined to certify transmission interruption. Currently, this is accomplished through PCR pool screening of large numbers of flies. The efficiency of this process is limited by the size of the pools that may be screened, which is in turn determined by the constraints imposed by the biochemistry of the assay. The current method of DNA purification from pools of vector black flies relies upon silica adsorption. This method can be applied to screen pools containing a maximum of 50 individuals (from the Latin American vectors) or 100 individuals (from the African vectors).

          Methodology/Principal Findings

          We have evaluated an alternative method of DNA purification for pool screening of black flies which relies upon oligonucleotide capture of Onchocerca volvulus genomic DNA from homogenates prepared from pools of Latin American and African vectors. The oligonucleotide capture assay was shown to reliably detect one O. volvulus infective larva in pools containing 200 African or Latin American flies, representing a two-four fold improvement over the conventional assay. The capture assay requires an equivalent amount of technical time to conduct as the conventional assay, resulting in a two-four fold reduction in labor costs per insect assayed and reduces reagent costs to $3.81 per pool of 200 flies, or less than $0.02 per insect assayed.


          The oligonucleotide capture assay represents a substantial improvement in the procedure used to detect parasite prevalence in the vector population, a major metric employed in the process of certifying the elimination of onchocerciasis.

          Author Summary

          The absence of infective larvae of Onchocerca volvulus in the black fly vector of this parasite is a major criterion used to certify that transmission has been eliminated in a focus. This process requires screening large numbers of flies. Currently, this is accomplished by screening pools of flies using a PCR-based assay. The number of flies that may be included in each pool is currently limited by the DNA purification process to 50 flies for Latin American vectors and 100 flies for African vectors. Here, we describe a new method for DNA purification that relies upon a specific oligonucleotide to capture and immobilize the parasite DNA on a magnetic bead. This method permits the reliable detection of a single infective larva of O. volvulus in pools containing up to 200 individual flies. The method described here will dramatically improve the efficiency of pool screening of vector black flies, making the process of elimination certification easier and less expensive to implement.

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          Most cited references25

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          Elimination of human onchocerciasis: history of progress and current feasibility using ivermectin (Mectizan(®)) monotherapy.

          We review and analyze approaches over a 65 year period that have proven successful for onchocerciasis control in several different epidemiological settings. These include vector control with the goal of transmission interruption versus the use of mass drug administration using ivermectin (Mectizan(®)) monotherapy. Ivermectin has proven exceedingly effective because it is highly efficacious against Onchocerca volvulus microfilariae, the etiological agent of onchocercal skin and ocular disease and the infective stage for the vector. For these reasons, the drug was donated by the Merck Company for regional control programs in Africa and the Americas. Recurrent treatment with ivermectin at semi-annual intervals also impacts adult worms and result in loss of fecundity and increased mortality. Using a strategy of 6-monthly treatments with high coverage rates, the Onchocerciasis Elimination Program for the Americas has interrupted transmission in seven of the thirteen foci in the Americas and is on track to eliminate onchocerciasis in the region by 2015. Treatments given annually or semi-annually for 15-17 years in three hyperendemic onchocerciasis foci in Mali and Senegal also have resulted in a few infections in the human population with transmission levels below thresholds postulated for elimination. Follow-up evaluations did not detect any recrudescence of infection or transmission, suggesting that onchocerciasis elimination could be feasible with Mectizan(®) treatment in some endemic foci in Africa. Copyright © 2010 Elsevier B.V. All rights reserved.
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            Determining the prevalence of Onchocerca volvulus infection in vector populations by polymerase chain reaction screening of pools of black flies.

            An important variable in the epidemiology of arthropodborne diseases is the intensity of transmission, which is a function of host-vector contact and the prevalence of infection in the vector population. This latter value is often difficult to estimate. It is possible to envision the application of polymerase chain reaction (PCR) assays to this problem. To accomplish this, the assay must detect a single infected vector in a pool containing a large number of uninfected individuals. It must also be possible to calculate the prevalence of infection from the number of positive pools. A PCR assay for detecting Onchocerca volvulus in pools of vector black flies is described, and an algorithm is presented to calculate the prevalence of infection in the vector population, based upon the proportion of PCR-positive pools. This algorithm should be applicable to any disease for which a PCR assay is available.
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              Elimination of onchocerciasis from Africa: possible?

              Human onchocerciasis, a parasitic disease found in 28 African countries, six Latin American countries and Yemen, causes blindness and severe dermatological problems. In 1987, efforts to control this infection shifted from vector approaches to include the mass distribution of ivermectin - a drug donated by Merck & Co. for disease control in Africa and for disease elimination in the Americas. Currently, almost 25 years later, with the Americas being highly successful and now approaching elimination, new evidence points towards the possibility of successful elimination in Africa. We suggest several major changes in the programmatic approach that through focused goal-directed effort could achieve global elimination of onchocerciasis by 2025. Copyright © 2011 Elsevier Ltd. All rights reserved.

                Author and article information

                Role: Editor
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, USA )
                June 2012
                19 June 2012
                : 6
                : 6
                : e1712
                [1 ]Centro de Biotecnología Genómica, Instituto Politécnico Nacional, Reynosa, Tamaulipas, México
                [2 ]Department of Global Health, Global Health Infectious Disease Research Program, University of South Florida, Tampa, Florida, United States of America
                [3 ]Multi-Disease Surveillance Centre, Ouagadougou, Burkina Faso
                [4 ]Laboratory of Molecular Parasitology, Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York, United States of America
                University of Pittsburgh, United States of America
                Author notes

                Conceived and designed the experiments: MAR TRU. Performed the experiments: HG HKH. Analyzed the data: HG HKH MAR TRU. Contributed reagents/materials/analysis tools: LDT SL. Wrote the paper: HG MAR LDT SL TRU.

                Gopal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                : 9 April 2012
                : 14 May 2012
                Page count
                Pages: 5
                Research Article
                Infectious Diseases
                Neglected Tropical Diseases

                Infectious disease & Microbiology
                Infectious disease & Microbiology


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