Abstract
BackgroundThe 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.MethodsA 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.ResultsDNA 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/µL blood) were not detected when thick blood smears were used as a DNA source.ConclusionsAlthough 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.
Highlights
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
It is well known that the success of the PCR technique depends on a variety of factors such as high quality DNA obtained from blood samples, good reagents and adequate conditions of amplification
Agreement of PCR results using DNA extracted from two different blood storage devices in comparison to microscopic examination The nested PCR using DNA from thick blood smears (TBS) was positive in 62 of 369 samples (16.8%), the same proportion observed by microscopy
Summary
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. The PCR method successfully detects parasites in mixed and low level infections, being more sensitive when compared to microscopic examination [36], validating its use in malaria epidemiological studies. It is well known that the success of the PCR technique depends on a variety of factors such as high quality DNA obtained from blood samples, good reagents and adequate conditions of amplification. Giemsa-stained or unstained TBS and, blood conserved on filter-papers have been used as a source of DNA in molecular and epidemiological studies [9,10,11,12,13,14]
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