title: Low sensitivity of nested PCR using Plasmodium DNA extracted from stained thick blood smears: an epidemiological retrospective study among subjects with low parasitaemia in an endemic area of the Brazilian Amazon region.
abstract: BACKGROUND
  The success of PCR technique depends on many factors, such as high quality DNA pellets obtained from blood samples, good reagents and adequate conditions of amplification. Taking these limitations into account, a retrospective epidemiological study for malaria diagnosis was conducted in a mesoendemic area in the Brazilian Amazon.
  
  
  METHODS
  A nested PCR protocol with DNA extracted from two blood storage devices obtained from Giemsa-stained thick blood smears and filter-papers was used for malaria diagnosis. The extracted DNA was used as a template to amplify approximately 100 bp species-specific sequences of the small subunit of the ribosomal RNA (18S SSU rRNA) of Plasmodium sp. The prevalence of single and mixed infections was examined in a cross-sectional survey carried out among 369 miners living in the district of Apiacás, Mato Grosso State. The parasitemia levels detected by microscopic examination were compared to the PCR results.
  
  
  RESULTS
  DNA samples isolated from blood on filter-paper allowed the detection and identification of Plasmodium in 165 (44.7%) of the 369 individuals evaluated, while only 62 (16.8%) had positive results using DNA obtained from thick smears, a similar rate observed by microscopic examination. The sensitivities of PCR using DNA from blood smears and filter-papers were 65% and 73.0%, respectively. Low parasite infections (below 20 parasites/ micro L blood) were not detected when thick blood smears were used as a DNA source.
  
  
  CONCLUSIONS
  Although the blood preserved as thick blood smears provides an alternative and useful tool for malaria molecular diagnosis, its relatively poor performance at low level parasitemias impairs the correct determination of malaria prevalence in epidemiological studies. However, the results obtained in the present study confirm that the use of filter-paper to collect blood is useful for field studies.
authors.affiliation: Departamento de Parasitologia, Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Belo Horizonte, Minas Gerais, Brazil. keziascopel@bol.com.br
authors.forename: Kézia K G
authors.initials: KK
authors.lastname: Scopel
authors.forename: Cor J F
authors.initials: CJ
authors.lastname: Fontes
authors.forename: Alvaro C
authors.initials: AC
authors.lastname: Nunes
authors.forename: M F
authors.initials: MF
authors.lastname: Horta
authors.forename: Erika M
authors.initials: EM
authors.lastname: Braga
mesh_terms: D000818:Animals; D001800:Blood Specimen Collection / Q000379:methods; D001938:Brazil / Q000453:epidemiology; D003430:Cross-Sectional Studies; D016054:DNA, Protozoan / Q000097:blood*; D004275:DNA, Ribosomal / Q000097:blood / Q000145:classification; D006801:Humans; D008288:Malaria / Q000175:diagnosis / Q000453:epidemiology*; D018512:Parasitemia / Q000175:diagnosis / Q000453:epidemiology* / Q000469:parasitology; D010961:Plasmodium / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000592:standards*; D015995:Prevalence; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012189:Retrospective Studies; D012680:Sensitivity and Specificity
chemical_list: D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D012337:RNA, Ribosomal, 18S