Although the microscopic examination of TBS remains the method of choice for the diagnosis of human malaria, in recent years, considerable attention has been given to molecular methods, including the PCR technique. In malaria studies, this method is considered to have a promising future, especially due to the identification of parasites in areas where four Plasmodium species occur simultaneously . Nevertheless, it has been recognized that the success of the technique depends on the quality of DNA. It has been observed that intrinsic (as DNA amount or a high content of human DNA or haemoglobin) and extrinsic (use of heparin or inadequate conditions of blood collecting, storage and amplification of samples) factors can inhibit the PCR assay [3, 18, 19]. This retrospective study has evaluated the sensitivity and specificity of PCR to detect malaria parasites according to the blood conservation devices used for DNA extraction. Considering the two alternatives for DNA preparation, the best results (44.7% of prevalence) were obtained for nested PCR analysis of the 18 SSU rRNA genes when target DNA was isolated from blood on filter-papers. The study showed that the sensitivity and specificity of nested PCR were 73% and 61%, respectively, when target DNA was extracted from filter-paper and 65% and 93% when DNA was obtained from thick smears. This is probably due to the conservation of the biological material used as a source of DNA, which directly affects the quality of DNA and, consequently, the sensitivity and specificity of the PCR assay. The sensitivity rates below 80% observed for both DNA preparation methods could be explained by the low level of circulating parasites in the blood of the individuals who were part of our study. The number of false negative results observed with PCR using DNA from thick smears could be an effect of a reduced number of parasites present in the samples, as some could have been lost during the process of scraping the slides. Furthermore, factors involved in preparing slides for microscopic examination may contribute to the stability of DNA template. Classical methods for fixing and storage of cell may be crucial factors in determining the rate of DNA degradation. For example, methanol fixation may affect the dissociation of protein-nucleic acid complexes within the cell. Thus, Giemsa-stain and excessive manipulation of samples could act negatively on DNA integrity. Thus, TBS should be used as a DNA source mainly when a large number of parasites is present on the slides, which will reduce the risk of false negative results enabling the success of the technique.
In contrast to the results of the present study, the use of DNA from thick or thin smears has produced good results by PCR assay in the different studies involving Plasmodium [9, 12–15, 20]. All these studies demonstrated that Plasmodium DNA might be successfully isolated from TBS indicating that this method of DNA preservation could be considered adequate and convenient for epidemiological studies. A possible explanation for the differences between our results and the above mentioned is the relatively reduced number of parasites present in our samples. On the other hand, the results obtained by PCR using isolated DNA from filter-papers indicate its great usefulness in field studies. Although false negative results have occurred in our study, the use of isolated DNA from filter-paper allowed the detection of Plasmodium in several samples previously negative by microscopic examination. Plasmodium falciparum is the most prevalent species in the study area. A higher number of mixed infections involving this species was observed when PCR was performed using DNA from filter-paper. In this case, delayed or missed diagnosis increases the possibility of complicated or severe malaria. Moreover, the results of this study demonstrated that the P. malariae (11%) and the prevalence of mixed infections (10%) may be substantially higher than previously reported in Brazil. The false negative results observed using DNA from filter-paper may reflect trapping of parasite DNA in the filter-paper, as previosuly reported . The present study corroborates previous results [10, 22], which showed that material on filter-paper appears to keep its characteristics and that this method is a simple, low cost way for preserving blood, with less risk of contamination and/or DNA degradation due to handling, transport and storage.
An important point to be discussed is that routine microscopy failed to detect very low parasite densities in Apiacás population studied. However, malaria prevalence as diagnosed by PCR using filter-paper DNA showed a high number of subclinical parasitaemia (32.5%) mainly among symptomless subjects. In the Apiacás epidemiological setting, the detection of low-level parasitaemia may not be clinically relevant or represent an indication of treatment since the development of clinical immunity has been reported for this population [6, 23, 24] and others  living in the Brazilian Amazon. Whether those individuals with negative thick blood smears but positive PCR may act as reservoirs of the parasite remains unclear. Although in a malaria endemic area it is most probable that the PCR actually detects infection, a prospective study performed in the symptomless individuals would be advisable to confirm the infection.