Epidemiologic aspects of the malaria transmission cycle in an area of very low incidence in Brazil

Abstract

Background: Extra-Amazonian autochthonous Plasmodium vivax infections have been reported in mountainous regions surrounded by the Atlantic Forest in Espírito Santo state, Brazil.

Methods: Sixty-five patients and 1,777 residents were surveyed between April 2001 and March 2004. Laboratory methods included thin and thick smears, multiplex-PCR, immunofluorescent assay (IFA) against P. vivax and Plasmodium malariae crude blood-stage antigens and enzyme-linked immunosorbent assay (ELISA) for antibodies against the P. vivax-complex (P. vivax and variants) and P. malariae/Plasmodium brasilianum circumsporozoite-protein (CSP) antigens.

Results: Average patient age was 35.1 years. Most (78.5%) were males; 64.6% lived in rural areas; 35.4% were farmers; and 12.3% students. There was no relevant history of travel. Ninety-five per cent of the patients were experiencing their first episode of malaria. Laboratory data from 51 patients were consistent with P. vivax infection, which was determined by thin smear. Of these samples, 48 were assayed by multiplex-PCR. Forty-five were positive for P. vivax, confirming the parasitological results, while P. malariae was detected in one sample and two gave negative results. Fifty percent of the 50 patients tested had IgG antibodies against the P. vivax-complex or P. malariae CSP as determined by ELISA. The percentages of residents with IgM and IgG antibodies detected by IFA for P. malariae, P. vivax and Plasmodium falciparum who did not complain of malaria symptoms at the time blood was collected were 30.1% and 56.5%, 6.2% and 37.7%, and 13.5% and 13%, respectively. The same sera that reacted to P. vivax also reacted to P. malariae. The following numbers of samples were positive in multiplex-PCR: 23 for P. vivax; 15 for P. malariae; 9 for P. falciparum and only one for P. falciparum and P. malariae. All thin and thick smears were negative. ELISA against CSP antigens was positive in 25.4%, 6.3%, 10.7% and 15.1% of the samples tested for "classical" P. vivax (VK210), VK247, P. vivax-like and P. malariae, respectively. Anopheline captures in the transmission area revealed only zoophilic and exophilic species.

Conclusion: The low incidence of malaria cases, the finding of asymptomatic inhabitants and the geographic separation of patients allied to serological and molecular results raise the possibility of the existence of a simian reservoir in these areas.

Figure 1

Location of the state of Espírito Santo in Brazil and the study area.

Figure 2

Distribution of positive IFA results for antibodies against P. malariae (A) and P. vivax (B) crude-blood-form antigens by municipality in the endemic area of the state of Espírito Santo.

Figure 3

DNA extracted from acute malaria patients and amplified by multiplex PCR. 1: Molecular weight (100 bp, Invitrogen); 2: negative control; 3 and 10: negative results from positive patients; 4–9, 11–13 and 15–16: P. vivax fragment (499 bp); 14: P. malariae fragment (269 bp).

Figure 4

DNA extracted from blood samples collected from the residents of the endemic area in the state of Espírito Santo and amplified by multiplex PCR. Numbers in the columns represent numbers of the samples. MW: (100 bp, Invitrogen). Upside: 48 = P. vivax; 56 = P. malariae and P. falciparum; 65 to 230 = P. malariae. Downside: 68 = P. vivax; 95 = P. malariae; 69, 86, 87, 92, 94, 96 and 97 = negative samples.