Am. J. Trop. Med. Hyg., 80(4), 2009, pp. 593–595 Copyright © 2009 by The American Society of Tropical Medicine and Hygiene 593 Short Report: 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 Daniela Pita-Pereira, Getúlio D. Souza, Adriana Zwetsch, Carlos Roberto Alves, Constança Britto, and Elizabeth F. Rangel* 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 Abstract. 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 autochtho- nous 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2003 to 2007 the Ministry of Health recorded 181,117 human cases of American cutaneous leishmaniasis (ACL) (Alves W, unpublished data).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 dis- semination 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 result- ing 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 char- acteristics, such as animal husbandry and agricultural activities in the proximity to residences. In previous entomologic stud- ies, Lutzomyia ( Nyssomyia) neivai (Pinto) was the most fre- quent species found in the area and was considered a potential ACL vector. 5 In the present study, a multiplex polymerase chain reac- tion (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 Duncan 6 for genus and subgenus, and Marcondes 7 and Andrade-Filho and others 8 for the species level taking into account the morphologic charac- teristics 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 hybrid- ization 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 consti- tutive 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 spe- cies, 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 * Address correspondence to Elizabeth Rangel, Laboratório de Trans- missores 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