title: Rapid molecular assays for specific detection and quantitation of Loa loa microfilaremia.
abstract: BACKGROUND
  Accurate diagnosis of Loa loa infection is essential to the success of mass drug administration efforts to eliminate onchocerciasis and lymphatic filariasis, due to the risk of fatal encephalopathic reactions to ivermectin occurring among highly microfilaremic Loa-infected individuals living in areas co-endemic for multiple filarial species.
  
  
  RESULTS
  From a pool of over 1,800 L. loa microfilaria (mf) expressed sequence tags, 18 candidate L. loa mf-specific PCR targets were identified. Real-time PCR (qPCR) assays were developed for two targets (LLMF72 and LLMF269). The qPCR assays were highly specific for L. loa compared with related filariae and also highly sensitive, with detection limits of 0.1 pg genomic DNA, or 1% of DNA extracted from normal blood spiked with a single L. loa microfilaria. Using various DNA extraction methods with dried blood spots obtained from Cameroonian subjects with parasitologically proven loiasis, the LLMF72 qPCR assay successfully estimated mf burden in 65 of 68 samples (50-96,000 mf/mL by microscopy), including all 12 samples subjected to a simple 10-minute boiling extraction. Additionally, the assay detected low-level microfilaremia among 5 of 16 samples from patients thought to be amicrofilaremic by microscopy.
  
  
  CONCLUSIONS
  This novel, rapid, highly sensitive and specific qPCR assay is an important step forward in the laboratory diagnosis of L. loa infection.
authors.affiliation: Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Doran L
authors.initials: DL
authors.lastname: Fink
authors.forename: Joseph
authors.initials: J
authors.lastname: Kamgno
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Nutman
mesh_terms: D000818:Animals; D017931:DNA Primers; D020224:Expressed Sequence Tags; D005260:Female; D006805:Human Experimentation; D006801:Humans; D008117:Loa / Q000235:genetics* / Q000302:isolation & purification; D008118:Loiasis / Q000175:diagnosis*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D010274:Parasitology / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers