Despite control and prevention measures, malaria has not been eliminated from non-endemic areas in southeastern Brazil. Autochthonous cases have been identified in several regions [33–35], and human malaria is predominantly caused by P. vivax and its variants [36, 37]. Subclinical Plasmodium infections have been considered rare in Brazil; however, they have been detected and identified in endemic regions . A retrospective analysis of human malaria in the State of São Paulo from 1985–2006 found 83 cases, four of which were subclinical infections . Other studies have shown a low parasitaemia rate in humans who inhabit areas within the Atlantic Forest, with cases caused by P. vivax and P. malariae.
Recently, molecular tools have been employed to detect Plasmodium infection in human blood. Although PCR techniques do not represent a rapid test for malaria diagnosis, they allow detection of as little as 0.5–1 parasite/μL blood, making them 20 times more sensitive than thick blood smear microscopic examination . PCR methods are also more sensitive in verifying mixed infections . Additionally, real-time PCR allows pathogen quantification, reduces the risk of cross-contamination, provides accurate results , and identifies subclinical infections in humans  that might otherwise remain undetected by routine tests for malaria. Consequently, the new technology reveals a prevalence of subclinical infections in humans that is higher than previously thought .
Human reservoirs can maintain transmission in areas of low, moderate, or high endemicity because they can infect mosquitoes . Despite the role of subclinical infection in maintaining malaria transmission, little is known about the factors that determine its occurrence and whether it is associated with human protective immune responses .
Blood transfusion is an important intervention in hospitals and health centres, but it also represents a route for transmission of parasites and pathogens that circulate in the blood, such as HIV, human Plasmodium, viruses, and others . Consequently, several measures are being taken to improve blood recipient safety, including high-sensitivity screening tests. Considering only Plasmodium, after the collection of the blood, the parasite can survive for at least one week at 4°C, as well as in frozen erythrocytes . Plasmodium transmission by transfusion usually occurs through whole blood and red blood cells and may occur less frequently through platelet concentrates, white blood cells, cryoprecipitate, and fresh frozen plasma [18, 45]. The blood bank is therefore an important target for controlling undesirable Plasmodium transmission in endemic [38, 46] and non-endemic regions [43, 47].
Here, subclinical malarial infections were detected from blood donors in the most important centre of public healthcare in the State of São Paulo. The presence of P. falciparum among blood donors and the high prevalence of these infections were not expected. In the present study, each blood donor was interviewed and denied having been in malaria endemic regions. Consequently, it is plausible to assume that all subclinical infections were autochthonous. It is noteworthy that all individuals reported here would be accepted for blood donation using the current protocol adopted in blood transfusion centres in the State of São Paulo, which includes a questionnaire and an interview to detect history of exposure to malaria, both applied by a qualified professional. Therefore, although the clinical importance of the findings described here remains to be investigated, the presence of asymptomatic Plasmodium infection in blood donors in non-endemic areas should be an important concern for medical services, health authorities, and blood recipient safety .
Plasmodium vivax and its variants as well as P. falciparum and P. malariae circulate in non-endemic areas, where they are responsible for only a few, mostly oligosymptomatic cases of malaria annually . Because Plasmodium species can circulate in cycles that involve humans with low parasitaemia and sylvatic mosquitoes, and likely in non-human primate reservoirs [48, 49], malaria can be maintained indefinitely in silent cycles without the consequences of high-level epidemics. The estimated PR of subclinical infection found in blood donors in São Paulo clearly shows that P. falciparum is positively associated with forested areas. Juquitiba (30,239 inhabitants, 55.03 inhabitants/km2) and São Lourenço da Serra (14,874 inhabitants, 75 inhabitants/km2) are primarily rural locations embedded in a well-preserved and extensively forested area on the slope of Serra do Mar, where the vectors are mainly sylvatic mosquitoes from the subgenus Kerteszia of Anopheles. The human population from these areas was here defined as the exposed population whereas inhabitants of urban areas intercalated by forest fragments formed the non-exposed population. In the non-exposed population, the PR of P. vivax revealed a positive (PR = 0.47; 95% CI, 0.2, 1.12) between the parasite infection and urban inhabitants. However, this result may arise from the fact that the residences of the blood donors are situated in urban areas at the edge of forest fragments. Consequently, the presence of P. vivax is likely to be associated with forest fragments and involves both Kerteszia and Nyssorhynchus species, or even other Anopheles subgenera [48, 50]. The evolution of transmission of Plasmodium species that cause malaria in humans is a dynamic process that involves ecological, environmental, and climate factors as well as social, political, and economic determinants. The high prevalence of Plasmodium spp. subclinical infections in blood donors living either in forested areas or urban regions intermixed with forest fragments suggests that some evolutionary mechanisms are modulating the virulence of P. falciparum and P. vivax in a way that does not negatively affect survival of the parasite, humans, mosquitoes, and non-human primates that seem to be involved in malaria transmission in the Atlantic Forest. The dynamics of human malaria transmission in the Brazilian Amazon forest seems to involve similar evolutionary mechanisms that lead to subclinical infections in humans , and transmission cycles that involve humans and other mosquito species as vectors (in addition to An. darlingi) , and non-human primates, which were recently found to be infected with P. falciparum. In the Atlantic Forest, Alouatta monkeys have been found to be infected by P. vivax and P. malariae, along with P. falciparum.
The scientific novelty and translational importance of the present results are two-fold: 1) They could represent a serious problem for the blood transfusion programme in the State of São Paulo; and 2) they could be evidence of evolutionary mechanisms of attenuation of malarial symptoms in humans.
The translational importance of the first aspect is obvious. Because some infected recipients may become asymptomatic carriers and constitute a reservoir for parasites, maintaining their transmission, further investigations are needed to better document the risk of transfusion-transmitted malaria in these regions and to establish whether a specific malarial infection test should be considered for blood donors who live in or near the Atlantic Forest. The second aspect is more subtle and needs more data before being established as a paradigmatic cornerstone for the evolution of malaria transmission dynamics. In terms of clinical and economic viewpoints, the Atlantic Forest’s dynamics of malaria transmission could be a real-world example of symptomatic malaria elimination.