title: Plasmodium falciparum in the southeastern Atlantic forest: a challenge to the bromeliad-malaria paradigm?
abstract: BACKGROUND
  Recently an unexpectedly high prevalence of Plasmodium falciparum was found in asymptomatic blood donors living in the southeastern Brazilian Atlantic forest. The bromeliad-malaria paradigm assumes that transmission of Plasmodium vivax and Plasmodium malariae involves species of the subgenus Kerteszia of Anopheles and only a few cases of P. vivax malaria are reported annually in this region. The expectations of this paradigm are a low prevalence of P. vivax and a null prevalence of P. falciparum. Therefore, the aim of this study was to verify if P. falciparum is actively circulating in the southeastern Brazilian Atlantic forest remains.
  
  
  METHODS
  In this study, anophelines were collected with Shannon and CDC-light traps in seven distinct Atlantic forest landscapes over a 4-month period. Field-collected Anopheles mosquitoes were tested by real-time PCR assay in pools of ten, and then each mosquito from every positive pool, separately for P. falciparum and P. vivax. Genomic DNA of P. falciparum or P. vivax from positive anophelines was then amplified by traditional PCR for sequencing of the 18S ribosomal DNA to confirm Plasmodium species. Binomial probabilities were calculated to identify non-random results of the P. falciparum-infected anopheline findings.
  
  
  RESULTS
  The overall proportion of anophelines naturally infected with P. falciparum was 4.4% (21/480) and only 0.8% (4/480) with P. vivax. All of the infected mosquitoes were found in intermixed natural and human-modified environments and most were Anopheles cruzii (22/25 = 88%, 18 P. falciparum plus 4 P. vivax). Plasmodium falciparum was confirmed by sequencing in 76% (16/21) of positive mosquitoes, whereas P. vivax was confirmed in only 25% (1/4). Binomial probabilities suggest that P. falciparum actively circulates throughout the region and that there may be a threshold of the forested over human-modified environment ratio upon which the proportion of P. falciparum-infected anophelines increases significantly.
  
  
  CONCLUSIONS
  These results show that P. falciparum actively circulates, in higher proportion than P. vivax, among Anopheles mosquitoes of fragments of the southeastern Brazilian Atlantic forest. This finding challenges the classical bromeliad-malaria paradigm, which considers P. vivax circulation as the driver for the dynamics of residual malaria transmission in this region.
authors.affiliation: Departamento de Epidemiologia, Faculdade de Saúde Pública da Universidade de São Paulo, São Paulo, SP, 01246-904, Brazil. gabrielzorelo@usp.br.|Laboratório de Informática Médica (LIM 01), Faculdade de Medicina da Universidade de São Paulo, São Paulo, SP, 05405-000, Brazil. gabrielzorelo@usp.br.
authors.forename: Gabriel Zorello
authors.initials: GZ
authors.lastname: Laporta
authors.affiliation: Laboratório de Informática Médica (LIM 01), Faculdade de Medicina da Universidade de São Paulo, São Paulo, SP, 05405-000, Brazil. mnburatt@usp.br.|Divisão de Doenças Infecciosas, Hospital São Paulo, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, 04024-002, Brazil. mnburatt@usp.br.
authors.forename: Marcelo Nascimento
authors.initials: MN
authors.lastname: Burattini
authors.affiliation: Laboratório de Genética e Hematologia Molecular (LIM 31), Faculdade de Medicina da Universidade de São Paulo, São Paulo, SP, 05403-000, Brazil. deb.levy@lim31.fm.usp.br.
authors.forename: Debora
authors.initials: D
authors.lastname: Levy
authors.affiliation: Divisão de Pesquisa, Fundação Pró-Sangue Hemocentro de São Paulo, Faculdade de Medicina da Universidade de São Paulo, São Paulo, SP, 05403-000, Brazil. linah@lim31.fm.usp.br.
authors.forename: Linah Akemi
authors.initials: LA
authors.lastname: Fukuya
authors.affiliation: Departamento de Epidemiologia, Faculdade de Saúde Pública da Universidade de São Paulo, São Paulo, SP, 01246-904, Brazil. porangaba@usp.br.
authors.forename: Tatiane Marques Porangaba
authors.initials: TM
authors.lastname: de Oliveira
authors.affiliation: Laboratório de Genética e Hematologia Molecular (LIM 31), Faculdade de Medicina da Universidade de São Paulo, São Paulo, SP, 05403-000, Brazil. lu.morganti@lim31.fm.usp.br.|Divisão de Pesquisa, Fundação Pró-Sangue Hemocentro de São Paulo, Faculdade de Medicina da Universidade de São Paulo, São Paulo, SP, 05403-000, Brazil. lu.morganti@lim31.fm.usp.br.
authors.forename: Luciana Morganti Ferreira
authors.initials: LM
authors.lastname: Maselli
authors.affiliation: Department of Health, Wadsworth Center, Slingerlands, NY, 12159, USA. jan.conn@health.ny.gov.|Department of Biomedical Sciences, School of Public Health, State University of New York-Albany, Albany, NY, 12222, USA. jan.conn@health.ny.gov.
authors.forename: Jan Evelyn
authors.initials: JE
authors.lastname: Conn
authors.affiliation: Laboratório de Informática Médica (LIM 01), Faculdade de Medicina da Universidade de São Paulo, São Paulo, SP, 05405-000, Brazil. edmassad@usp.br.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Massad
authors.affiliation: Laboratório de Genética e Hematologia Molecular (LIM 31), Faculdade de Medicina da Universidade de São Paulo, São Paulo, SP, 05403-000, Brazil. spbydlow@usp.br.
authors.forename: Sergio Paulo
authors.initials: SP
authors.lastname: Bydlowski
authors.affiliation: Departamento de Epidemiologia, Faculdade de Saúde Pública da Universidade de São Paulo, São Paulo, SP, 01246-904, Brazil. masallum@usp.br.
authors.forename: Maria Anice Mureb
authors.initials: MA
authors.lastname: Sallum
mesh_terms: D000818:Animals; D000852:Anopheles / Q000469:parasitology*; D001938:Brazil; D031229:Bromeliaceae / Q000502:physiology*; D065928:Forests; D006801:Humans; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000235:genetics / Q000302:isolation & purification*; D010966:Plasmodium vivax / Q000235:genetics / Q000302:isolation & purification*; D015800:Protozoan Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D015800:Protozoan Proteins