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    Multiplex polymerase chain reaction (PCR) followed by hybridization for the diagnosis of Leishmania (Viannia) braziliensis infection in Lutzomyia neivai collected in Porto Alegre, Rio Grande do Sul State, Brazil. The phlebotomines were grouped into pools of 10 female specimens and PCR was performed with total DNA extracted from these pools. A, Ethidium bromide-stained 2% aga-rose gel revealing the 220 bp product from cacophony gene amplification (Lutzomyia genus) and the 120 bp fragment corresponding to the conserved kinetoplast minicircle region from Leishmania spp. M, molecular weight marker (100 bp DNA ladder). Lane 1, amplification reaction without added DNA (PCR negative control); lanes 2–5, negative controls for the DNA extraction step (male insect pools); lanes 6–19, female sand fly pools (lanes 15–17, Lu. neivai positive pools); lanes 20 and 21, PCR positive controls (DNA extracted from a mixture of male insect pool containing L. (V.) braziliensis promastigotes). B, Dot hybridization using a biotinylated probe specific for parasites from the Viannia subgenus.

  • 1

    Marzochi MCA, Marzochi KB, 1994. Tegumentary and visceral leishmaniasis in Brazil—emerging anthropozoonosis and possibilities for their control. Cad Saude Publica 10: 359–375 (in Portuguese).

  • 2

    Kawa H, Sabroza PC, 2002. Spatial distribution of tegumentary leishmaniasis in the city of Rio de Janeiro. Cad Saude Publica 18: 853–865 (in Portuguese).

  • 3

    Dias ES, Falcão AL, Silva JE, 1997. Notes on the sand fly fauna (Diptera: Psychodidae) in the State of Rio Grande do Sul. Mem Inst Oswaldo Cruz 92 :329–332.

    • Search Google Scholar
    • Export Citation
  • 4

    Silva OS, Grunewald J, 1999. Contribution to the sand fly fauna (Diptera: Phlebotominae) of Rio Grande do Sul, Brazil and Leishmania (Viannia ) infections. Mem Inst Oswaldo Cruz 94 :579–582.

    • Search Google Scholar
    • Export Citation
  • 5

    Gonçalves BRD, 2003. Identificação da fauna de flebotomíneos em função de casos autóctones de LTA. Boletim Epidemiológico, Secretaria Municipal de Saúde de Porto Alegre 21 :5 (in Portuguese).

    • Search Google Scholar
    • Export Citation
  • 6

    Young DG, Duncan MA, 1994. Guide to the identification and geographic distribution of Lutzomyia sandflies in Mexico, the West Indies, Central and South America (Diptera: Psychodidae). Mem Am Entomol Inst 54 :1–881.

    • Search Google Scholar
    • Export Citation
  • 7

    Marcondes CB, 1996. A redescription of Lutzomyia (Nyssomyia) intermedia (Lutz & Neiva, 1912), and resurrection of L. neivai (Pinto, 1926) (Diptera, Psychodidae, Phlebotominae). Mem Inst Oswaldo Cruz 91 :457–462.

    • Search Google Scholar
    • Export Citation
  • 8

    Andrade Filho JD, Galati EA, Falcão AL, 2003. Redescription of Nyssomyia intermedia (Lutz & Neiva, 1912) and Nyssomyia neivai (Pinto, 1926) (Diptera: Psychodidae). Mem Inst Oswaldo Cruz 98 :1059–1065.

    • Search Google Scholar
    • Export Citation
  • 9

    Pita-Pereira D, Alves CR, Souza MB, Brazil RP, Bertho A, Barbosa A, Britto C, 2005. Identification of naturally infected Lutzomyia intermedia and Lutzomyia migonei with Leishmania (Viannia) braziliensis in Rio de Janeiro (Brazil) revealed by a PCR multiplex non-isotopic hybridization assay. Trans R Soc Trop Med Hyg 99 :905–913.

    • Search Google Scholar
    • Export Citation
  • 10

    Pita-Pereira D, Cardoso MAB, Alves CR, Brazil RP, Britto C, 2008. Detection of natural infection in Lutzomyia cruzi and Lutzomyia forattinii (Diptera: Psychodidae: Phlebotominae) by Leishmania infantum chagasi in an endemic area of visceral leishmaniasis in Brazil using a PCR multiplex assay. Acta Trop 107 :66–69.

    • Search Google Scholar
    • Export Citation
  • 11

    Rangel EF, Lainson R, 2003. Ecologia das leishmanioses. Rangel EF, Lainson R, eds. Flebotomíneos do Brasil. (First edition). Fundação Oswaldo Cruz: Fiocruz, pp. 291–337 (in Portuguese).

  • 12

    Marcondes CB, Day JC, Ready PD, 1997. Introgression between Lutzomyia intermedia and both Lutzomyia neivai and Lutzomyia whitmani, and their roles as vectors of Leishmania braziliensis. Trans R Soc Trop Med Hyg 91 :725–726.

    • Search Google Scholar
    • Export Citation
  • 13

    Salomón OD, Wilson ML, Munstermann LE, Travi BL, 2004. Spatial and temporal patterns of phlebotomine sand flies (Diptera: Psychodidae) in a cutaneous leishmaniasis focus in northern Argentina. J Med Entomol 41 :33–39.

    • Search Google Scholar
    • Export Citation
  • 14

    Casanova C, Costa AI, Natal D, 2005. Dispersal pattern of sand fly Lutzomyia neivai (Diptera: Psychodidae) in a cutaneous leishmaniasis endemic rural area in southeastern Brazil. Mem Inst Oswaldo Cruz 100 :719–724.

    • Search Google Scholar
    • Export Citation
  • 15

    Córdoba-Lanús E, De Grosso ML, Piñero JE, Valladares B, Salomón OD, 2006. Natural infection of Lutzomyia neivai with Leishmania spp. in northwestern Argentina. Acta Trop 98 :1–5.

    • Search Google Scholar
    • Export Citation
  • 16

    Salomón OD, Quintana MG, Zaidenberg M, 2008. Urban distribution of Phlebotominae in a cutaneous leishmaniasis focus, Argentina. Mem Inst Oswaldo Cruz 103 :282–287.

    • Search Google Scholar
    • Export Citation

 

 

 

 

First Report of Lutzomyia (Nyssomyia) neivai (Diptera: Psychodidae: Phlebotominae) Naturally Infected by Leishmania (Viannia) braziliensis in a Periurban Area of South Brazil Using a Multiplex Polymerase Chain Reaction Assay

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  • 1 Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brasil; Seção de Reservatórios e Vetores, Instituto de Pesquisas Biológicas, Laboratório Central de Pública do Rio Grande do Sul, Fundação Estadual de Produção e Pesquisa em Saúde, Secretaria Estadual de Saúde do Rio Grande do Sul; Núcleo de Vigilância de Roedores e Vetores, Coordenadoria Geral de Vigilância em Saúde, Secretaria Municipal de Saúde de Porto Alegre; Laboratório de Transmissores de Leishmanioses, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brasil

To identify Lutzomyia (Nyssomyia) neivai naturally infected by Leishmania a multiplex polymerase chain reaction (PCR) was used for the analysis of 450 specimens (270 females, 180 males) collected in an endemic periurban area of cutaneous leishmaniasis in Porto Alegre, Brazil. Insects were grouped into pools of 10 and positive results were achieved in 3/27 Lu. (N.) neivai female pools. Infection by L. (Viannia) braziliensis was confirmed after hybridizing PCR products with a subgenus–specific biotinylated probe. Considering the detection of three positive pools with at least one infected insect in each, an infection rate of 1.1% was estimated. Our results associated with epidemiologic data suggest a potential ability of Lu. (N.) neivai in transmitting L. braziliensis in Porto Alegre, where the first notifications of autochthonous cutaneous leishmaniasis in humans occurred in 2002, with an increase in the number of cases in recent years possibly as a consequence of deforestation and agricultural activities in the area.

In Brazil, from 2003 to 2007 the Ministry of Health recorded 181,117 human cases of American cutaneous leishmaniasis (ACL) (Alves W, unpublished data). It has been reported that an increase of ACL incidence in all Brazilian states with dissemination of the disease to the periurban areas of some state capitals, such as Manaus, Rio de Janeiro, and Belo Horizonte, thus constituting a serious health problem. 1,2

There are few studies directed to the sand fly fauna in Rio Grande do Sul State, 3,4 mainly because of the inexistence of reports on autochthonous ACL cases in the last decades. More recently, in accordance with the Secretariat of Health of Rio Grande do Sul, the epidemiologic situation has changed. From 2002 to 2008, 20 confirmed autochthonous human cases resulting from Leishmania (Viannia) braziliensis were reported in the periphery of the city of Porto Alegre, in areas of residual vegetation close to creeks. The region presents rural zone characteristics, such as animal husbandry and agricultural activities in the proximity to residences. In previous entomologic studies, Lutzomyia (Nyssomyia) neivai (Pinto) was the most frequent species found in the area and was considered a potential ACL vector.5

In the present study, a multiplex polymerase chain reaction (PCR) assay associated to non-isotopic hybridization was used to evaluate the occurrence of Lu. (N.) neivai naturally infected by Leishmania parasites in an urban area of tourism activity located in Porto Alegre, where human ACL cases were recently described. Sand flies were collected monthly from October 2006 to May 2007 with Centers for Disease Control and Prevention (CDC) light traps in 10 monitoring stations distributed inside houses, in the peridomicile close to domestic animal shelters and in the forest. A sample constituted of 450 Lu. (N.) neivai phlebotomines (270 females, 180 males) was sent to the laboratory for PCR examination after taxonomic identification according to Young and Duncan6 for genus and subgenus, and Marcondes7 and Andrade-Filho and others8 for the species level taking into account the morphologic characteristics of males and females. The insects were grouped into pools of 10 specimens and submitted to molecular analysis for Leishmania infection. The multiplex PCR 9,10 was designed to simultaneously amplify the cacophony gene IVS6 region in sand flies of the neotropical genus Lutzomyia (as an internal control for the polymerase enzyme activity), and the conserved kinetoplast DNA minicircle region from Leishmania spp. The amplified products were further submitted to dot blot hybridization using a L. (Viannia)-specific biotinylated probe.9

The PCR assay showed positive results in 3 out of 27 female pools analyzed, and hybridization confirmed the infection with parasites from the subgenus Viannia (Figure 1). Considering the occurrence of at least one infected insect in each pool of 10 phlebotomines, we found that the minimal infection rate for Lu. (N.) neivai was 1.11%. The PCR approach was highly sensitive and able to reveal on agarose gel, a 120 bp fragment from Leishmania kDNA minicircles in all three positive sand fly pools before the hybridization step. All samples analyzed yielded a 220 bp amplified product corresponding to a constitutive gene (cacophony) from Lutzomyia spp., thus confirming the integrity of the insect DNA preparations and the absence of eventual PCR inhibitors (Figure 1).

In Brazil, ACL resulting from L. (V.) braziliensis has been reported in all states and involves a diversity of sand fly species, such as Lu. (Psychodopygus) wellcomei (Fraiha, Shaw, & Lainson, 1971); Lu. (P.) complexa (Mangabeira, 1941); Lu. (N.) whitmani (Antunes & Coutinho, 1939); Lu. Migonei (França, 1920), and Lu. (N.) intermedia (Lutz & Neiva, 1912), based on evidences regarding their anthropophily, natural infection by Leishmania parasites, and spatial distribution in accordance with human cases transmission sites. 11

Marcondes and others 12 discussed the epidemiologic role of Lu. (N.) neivai in the transmission of L. (V.) braziliensis in southeast and south of Brazil, in cold and dry areas. Afterwards, some aspects concerning the vectorial competence of this sand fly species were studied in Argentina 13 in a transmission area of ACL, where it was reported a high Lu. (N.) neivai density associated with human cases in the domiciliary environment, and in the secondary and primary forest. Studies on Lu. (N.) neivai in southeastern Brazil brought more insights about the biologic characteristics of this species and suggested that dispersion from the forest to peridomicile areas could be important for the transmission of ACL inside houses. 14 In 2006, Córdoba-Lanús and others 15 reported for the first time natural infection of Lu. (N.) neivai with Leishmania from an area in Argentina where Leishmania (Viannia ) was the main circulating parasite. More recently, also in Argentina, in an area considered as hyper-endemic for ACL, the transmission was associated to Lu. (N.) neivai in periurban vegetation and ecotone modified regions. 16

Taking into account the first reports of ACL in Porto Alegre, south Brazil, studies concerning the sand fly fauna were performed. Captures carried out inside houses and in peridomicile areas, with the occurrence of human ACL cases, revealed the predominance of Lu. (N.) neivai (94.9%) without the identification of Lu. (N.) intermedia (Azevedo A and others, unpublished data). Epidemiologic data from the Secretariat of Health of Porto Alegre5 suggested Lu. (N.) neivai as the main vector of ACL, because of its anthropophylic characteristic and spatial and seasonal abundance in anthropic environments. This information associated with the evidence of natural infection by Leishmania indicate the importance of Lu. (N.) neivai in the epidemiologic cycle of ACL in Porto Alegre, and suggests this species as another L. (V.) braziliensis vector in the southeast and south regions of Brazil.

Figure 1.
Figure 1.

Multiplex polymerase chain reaction (PCR) followed by hybridization for the diagnosis of Leishmania (Viannia) braziliensis infection in Lutzomyia neivai collected in Porto Alegre, Rio Grande do Sul State, Brazil. The phlebotomines were grouped into pools of 10 female specimens and PCR was performed with total DNA extracted from these pools. A, Ethidium bromide-stained 2% aga-rose gel revealing the 220 bp product from cacophony gene amplification (Lutzomyia genus) and the 120 bp fragment corresponding to the conserved kinetoplast minicircle region from Leishmania spp. M, molecular weight marker (100 bp DNA ladder). Lane 1, amplification reaction without added DNA (PCR negative control); lanes 2–5, negative controls for the DNA extraction step (male insect pools); lanes 6–19, female sand fly pools (lanes 15–17, Lu. neivai positive pools); lanes 20 and 21, PCR positive controls (DNA extracted from a mixture of male insect pool containing L. (V.) braziliensis promastigotes). B, Dot hybridization using a biotinylated probe specific for parasites from the Viannia subgenus.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 4; 10.4269/ajtmh.2009.80.593

*

Address correspondence to Elizabeth Rangel, Laboratório de Transmissores de Leishmanioses, Instituto Oswaldo Cruz, FIOCRUZ, Pavilhão Carlos Chagas, Avenida Brasil 4365, Manguinhos, 21045-900, Rio de Janeiro, RJ, Brasil. E-mail: elizabethrangel@fiocruz.br

Authors’ addresses: Daniela Pita-Pereira, Carlos R. Alves, and Constança Britto, Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, FIOCRUZ, Pavilhão Leônidas Deane sala 209, Avenida Brasil 4365, Manguinhos, 21045-900, Rio de Janeiro, RJ, Brasil. Getúlio D. Souza, Seção de Reservatórios e Vetores, Instituto de Pesquisas Biológicas, Laboratório Central de Pública do Rio Grande do Sul, Fundação Estadual de Produção e Pesquisa em Saúde, Secretaria Estadual de Saúde do Rio Grande do Sul, Avenida Ipiranga 5400, Bairro Jardim Botânico, 90610-000, Porto Alegre, RS, Brasil; Núcleo de Vigilância de Roedores e Vetores, Coordenadoria Geral de Vigilância em Saúde, Secretaria Municipal de Saúde de Porto Alegre, Avenida Padre Cacique 372, Bairro Menino Deus, 90810-240, Porto Alegre, RS, Brasil. Adriana Zwetsch and Elizabeth F. Rangel, Laboratório de Transmissores de Leishmanioses, Instituto Oswaldo Cruz, FIOCRUZ, Pavilhão Carlos Chagas, Avenida Brasil 4365, Manguinhos, 21045-900, Rio de Janeiro, RJ, Brasil, Tel: +55-21-2590-9590, Fax: +55-21-2560-7011, E-mail: elizabethrangel@fiocruz.br.

Acknowledgments: We thank J. A. Ferreira, A. N. Martínez, and B. S. Pereira for the English revision.

Financial support: This investigation was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico–CNPq, Brasil (476380/2004-9).

Disclosure: Daniela Pita-Pereira is a fellow PhD student from FIOCRUZ/CAPES, Carlos R. Alves, Constança Britto, and Elizabeth F. Rangel are fellows of the CNPq Institution, and Adriana Zwetsch is a fellow of the FAPERJ Institution.

REFERENCES

  • 1

    Marzochi MCA, Marzochi KB, 1994. Tegumentary and visceral leishmaniasis in Brazil—emerging anthropozoonosis and possibilities for their control. Cad Saude Publica 10: 359–375 (in Portuguese).

  • 2

    Kawa H, Sabroza PC, 2002. Spatial distribution of tegumentary leishmaniasis in the city of Rio de Janeiro. Cad Saude Publica 18: 853–865 (in Portuguese).

  • 3

    Dias ES, Falcão AL, Silva JE, 1997. Notes on the sand fly fauna (Diptera: Psychodidae) in the State of Rio Grande do Sul. Mem Inst Oswaldo Cruz 92 :329–332.

    • Search Google Scholar
    • Export Citation
  • 4

    Silva OS, Grunewald J, 1999. Contribution to the sand fly fauna (Diptera: Phlebotominae) of Rio Grande do Sul, Brazil and Leishmania (Viannia ) infections. Mem Inst Oswaldo Cruz 94 :579–582.

    • Search Google Scholar
    • Export Citation
  • 5

    Gonçalves BRD, 2003. Identificação da fauna de flebotomíneos em função de casos autóctones de LTA. Boletim Epidemiológico, Secretaria Municipal de Saúde de Porto Alegre 21 :5 (in Portuguese).

    • Search Google Scholar
    • Export Citation
  • 6

    Young DG, Duncan MA, 1994. Guide to the identification and geographic distribution of Lutzomyia sandflies in Mexico, the West Indies, Central and South America (Diptera: Psychodidae). Mem Am Entomol Inst 54 :1–881.

    • Search Google Scholar
    • Export Citation
  • 7

    Marcondes CB, 1996. A redescription of Lutzomyia (Nyssomyia) intermedia (Lutz & Neiva, 1912), and resurrection of L. neivai (Pinto, 1926) (Diptera, Psychodidae, Phlebotominae). Mem Inst Oswaldo Cruz 91 :457–462.

    • Search Google Scholar
    • Export Citation
  • 8

    Andrade Filho JD, Galati EA, Falcão AL, 2003. Redescription of Nyssomyia intermedia (Lutz & Neiva, 1912) and Nyssomyia neivai (Pinto, 1926) (Diptera: Psychodidae). Mem Inst Oswaldo Cruz 98 :1059–1065.

    • Search Google Scholar
    • Export Citation
  • 9

    Pita-Pereira D, Alves CR, Souza MB, Brazil RP, Bertho A, Barbosa A, Britto C, 2005. Identification of naturally infected Lutzomyia intermedia and Lutzomyia migonei with Leishmania (Viannia) braziliensis in Rio de Janeiro (Brazil) revealed by a PCR multiplex non-isotopic hybridization assay. Trans R Soc Trop Med Hyg 99 :905–913.

    • Search Google Scholar
    • Export Citation
  • 10

    Pita-Pereira D, Cardoso MAB, Alves CR, Brazil RP, Britto C, 2008. Detection of natural infection in Lutzomyia cruzi and Lutzomyia forattinii (Diptera: Psychodidae: Phlebotominae) by Leishmania infantum chagasi in an endemic area of visceral leishmaniasis in Brazil using a PCR multiplex assay. Acta Trop 107 :66–69.

    • Search Google Scholar
    • Export Citation
  • 11

    Rangel EF, Lainson R, 2003. Ecologia das leishmanioses. Rangel EF, Lainson R, eds. Flebotomíneos do Brasil. (First edition). Fundação Oswaldo Cruz: Fiocruz, pp. 291–337 (in Portuguese).

  • 12

    Marcondes CB, Day JC, Ready PD, 1997. Introgression between Lutzomyia intermedia and both Lutzomyia neivai and Lutzomyia whitmani, and their roles as vectors of Leishmania braziliensis. Trans R Soc Trop Med Hyg 91 :725–726.

    • Search Google Scholar
    • Export Citation
  • 13

    Salomón OD, Wilson ML, Munstermann LE, Travi BL, 2004. Spatial and temporal patterns of phlebotomine sand flies (Diptera: Psychodidae) in a cutaneous leishmaniasis focus in northern Argentina. J Med Entomol 41 :33–39.

    • Search Google Scholar
    • Export Citation
  • 14

    Casanova C, Costa AI, Natal D, 2005. Dispersal pattern of sand fly Lutzomyia neivai (Diptera: Psychodidae) in a cutaneous leishmaniasis endemic rural area in southeastern Brazil. Mem Inst Oswaldo Cruz 100 :719–724.

    • Search Google Scholar
    • Export Citation
  • 15

    Córdoba-Lanús E, De Grosso ML, Piñero JE, Valladares B, Salomón OD, 2006. Natural infection of Lutzomyia neivai with Leishmania spp. in northwestern Argentina. Acta Trop 98 :1–5.

    • Search Google Scholar
    • Export Citation
  • 16

    Salomón OD, Quintana MG, Zaidenberg M, 2008. Urban distribution of Phlebotominae in a cutaneous leishmaniasis focus, Argentina. Mem Inst Oswaldo Cruz 103 :282–287.

    • Search Google Scholar
    • Export Citation

Author Notes

Reprint requests: Elizabeth Rangel, Laboratório de Transmissores de Leishmanioses, Instituto Oswaldo Cruz, FIOCRUZ, Pavilhão Carlos Chagas, Avenida Brasil 4365, Manguinhos, 21045-900, Rio de Janeiro, RJ, Brasil.
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