title: A qPCR-based multiplex assay for the detection of Wuchereria bancrofti, Plasmodium falciparum and Plasmodium vivax DNA.
abstract: The purpose of this study was to develop real-time multiplex quantitative PCR (qPCR) assays for the simultaneous detection of Wuchereria bancrofti (Wb), Plasmodium falciparum (Pf) and P. vivax (Pv) in mosquitoes. We optimized the assays with purified DNA samples and then used these assays to test DNA samples isolated from Anopheles punctulatus mosquitoes collected in villages in Papua New Guinea where these infections are co-endemic. Singleplex assays detected Wb, Pf and Pv DNA in 32%, 19% and 15% of the mosquito pools, respectively, either alone or together with other parasites. Multiplex assay results agreed with singleplex results in most cases. Overall parasite DNA rates in mosquitoes, estimated by PoolScreen 2 software, for Wb, Pf and Pv were 4.9%, 2.7% and 2.1%, respectively. Parasite DNA rates were consistently higher in blood-fed mosquitoes than in host-seeking mosquitoes. Our results show that multiplex qPCR can be used to detect and estimate prevalence rates for multiple parasite species in arthropod vectors. We believe that multiplex molecular xenodiagnosis has great potential as a tool for non-invasively assessing the distribution and prevalence of vector-borne pathogens such as W. bancrofti and Plasmodium spp. in human populations and for assessing the impact of interventions aimed at controlling or eliminating these diseases.
authors.affiliation: Infectious Diseases Division, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, USA. rrao@im.wustl.edu
authors.forename: Ramakrishna U
authors.initials: RU
authors.lastname: Rao
authors.forename: Yuefang
authors.initials: Y
authors.lastname: Huang
authors.forename: Moses J
authors.initials: MJ
authors.lastname: Bockarie
authors.forename: Melinda
authors.initials: M
authors.lastname: Susapu
authors.forename: Sandra J
authors.initials: SJ
authors.lastname: Laney
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Weil
mesh_terms: D000818:Animals; D000852:Anopheles / Q000469:parasitology; D018509:DNA, Helminth / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000302:isolation & purification*; D005260:Female; D006801:Humans; D007303:Insect Vectors / Q000469:parasitology; D010219:Papua New Guinea; D010963:Plasmodium falciparum / Q000235:genetics*; D010966:Plasmodium vivax / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014958:Wuchereria bancrofti / Q000235:genetics*; D020525:Xenodiagnosis / Q000379:methods
chemical_list: D018509:DNA, Helminth; D016054:DNA, Protozoan
references.citation: Trends Parasitol. 2003 Jun;19(6):253-9
references.pmid: 12798082
references.citation: Acta Trop. 2005 Mar;93(3):233-7
references.pmid: 15715996
references.citation: Trans R Soc Trop Med Hyg. 1997 Mar-Apr;91(2):156-60
references.pmid: 9196756
references.citation: Am J Trop Med Hyg. 2000 Mar;62(3):363-7
references.pmid: 11037778
references.citation: Parasitol Res. 2008 Jan;102(2):175-81
references.pmid: 18026992
references.citation: Trans R Soc Trop Med Hyg. 1998 Jan-Feb;92(1):21-3
references.pmid: 9692139
references.citation: Trends Parasitol. 2007 Feb;23(2):78-82
references.pmid: 17174604
references.citation: J Infect Dis. 1995 Nov;172(5):1414-7
references.pmid: 7594692
references.citation: Ann Trop Med Parasitol. 2002 Dec;96 Suppl 2:S41-6
references.pmid: 12625916
references.citation: Ann Trop Med Parasitol. 2002 Dec;96 Suppl 2:S143-52
references.pmid: 12625927
references.citation: Am J Trop Med Hyg. 2007 Oct;77(4):593-600
references.pmid: 17978055
references.citation: J Clin Microbiol. 2004 Dec;42(12):5636-43
references.pmid: 15583293
references.citation: J Clin Microbiol. 2006 Nov;44(11):3887-93
references.pmid: 16957038
references.citation: Mol Cell Probes. 2001 Aug;15(4):201-7
references.pmid: 11513554
references.citation: Trop Med Int Health. 2003 Feb;8(2):140-3
references.pmid: 12581439
references.citation: Exp Parasitol. 2002 Feb;100(2):131-4
references.pmid: 12054703
references.citation: Am J Trop Med Hyg. 2006 May;74(5):826-32
references.pmid: 16687688
references.citation: Filaria J. 2003 Jul 7;2(1):11
references.pmid: 12890288
references.citation: Parasitology. 1990 Jun;100 Pt 3:369-75
references.pmid: 2194153
references.citation: J Clin Microbiol. 2004 Mar;42(3):1214-9
references.pmid: 15004078
references.citation: Int J Parasitol. 2004 Jun;34(7):795-802
references.pmid: 15157762