Molecular characterization of Trypanosoma cruzi and Leishmania spp. coinfection in mammals of Venezuelan coendemic areas.

BACKGROUND & OBJECTIVES: Trypanosoma cruzi and Leishmania spp. are protozoans that cause American trypanosomiasis and leishmaniasis, respectively. In endemic foci where both diseases coincide, coinfection can occur. The objective of this work was the characterization of the parasites involved in coinfection in several endemic areas of Venezuela. METHODS: Molecular characterization was done in 30 samples of several species of mammals (Didelphis marsupialis, Equus mulus, Rattus rattus, Canis familiaris, Felis catus, and Sciurus granatensis) from the states of Anzoategui, Cojedes and Capital District diagnosed with T. cruzi and Leishmania spp. coinfections. For the typing of T. cruzi DTUs, the markers of miniexon, 24Sa rDNA, 18Sa rDNA, and hsp60-PCR-RFLP (EcoRV) were used. Infection by Leishmania spp. was characterized by miniexon multiplex PCR for complexes of Leishmania and ITS1-PCR-RFLP (HaeIII, HhaI, and RsaI) for the identification of the species. RESULTS: The T. cruzi TcI was present in 100% of the coinfected mammals, which included 76.7% of triple infection by T. cruzi TcI-complex-L. (L) mexicana-L. infantum/chagasi, 13.3% of double infection by T. cruzi TcI-L. mexicana and 10% of double infection by T. cruzi Tcl-L. infantum/chagasi. INTERPRETATION & CONCLUSION: These results suggest that the double or triple infection is a phenomenon existing in almost all the coendemics areas and mammals studied, which might influence the mechanisms of adaptation and pathogenicity of these parasites.

Phlebotomine sandflies and the spreading of leishmaniases and other diseases of public health concern.

Phlebotomine sandflies transmit pathogens that affect humans and animals worldwide. We review the roles of phlebotomines in the spreading of leishmaniases, sandfly fever, summer meningitis, vesicular stomatitis, Chandipura virus encephalitis and Carrión's disease. Among over 800 species of sandfly recorded, 98 are proven or suspected vectors of human leishmaniases; these include 42 Phlebotomus species in the Old World and 56 Lutzomyia species in the New World (all: Diptera: Psychodidae). Based on incrimination criteria, we provide an updated list of proven or suspected vector species by endemic country where data are available. Increases in sandfly diffusion and density resulting from increases in breeding sites and blood sources, and the interruption of vector control activities contribute to the spreading of leishmaniasis in the settings of human migration, deforestation, urbanization and conflict. In addition, climatic changes can be expected to affect the density and dispersion of sandflies. Phlebovirus infections and diseases are present in large areas of the Old World, especially in the Mediterranean subregion, in which virus diversity has proven to be higher than initially suspected. Vesiculovirus diseases are important to livestock and humans in the southeastern U.S.A. and Latin America, and represent emerging human threats in parts of India. Carrión's disease, formerly restricted to regions of elevated altitude in Peru, Ecuador and Colombia, has shown recent expansion to non-endemic areas of the Amazon basin.

Isolation of Leishmania (Viannia) braziliensis from Lutzomyia spinicrassa (species group Verrucarum) Morales Osorno Mesa, Osorno and Hoyos 1969, in the Venezuelan Andean region.

Natural infection with Leishmania spp. in phlebotomine sandflies was searched for during a longitudinal study carried out from July 1997 to July 1998 in the village Catarnica, Municipality Independencia, Táchira State. This hamlet is an old endemic focus of cutaneous leishmaniasis in the Venezuelan Andean region, which lies close to the Colombian border at 1,300 m a.s.l., in an agricultural area mainly used for cultivating coffee. Phlebotomine sandflies were collected using Shannon traps placed in the peridomestic habitat from 19:00 to 21:00 hs. Males were stored in alcohol 70 % while females were kept in Nunc vials with 10 % DMSO and cryopreserved in liquid nitrogen for subsequent dissection and identification. The most abundant anthropophilic species was Lutzomyia spinicrassa with 3,032 males and 4,290 females (85.4%). Among 1,633 (38%) females of Lu. spinicrassa dissected, 26 11.6%) were infected with promastigotes, while no natural infection was found in 209 females of other species. The flagellates were identified as Leishmania braziliensis braziliensis using PCR with species specific primers derived from nuclear DNA and hybridization using species specific probe labelled with digoxigenin. This parasite had been previously isolated from patients with cutaneous leishmaniasis from the same area. These results show Lu. spinicrassa as a new proven vector of Leishmania braziliensis in the Andean region of Venezuela.

Wing shape divergence between Rhodnius prolixus from Cojedes (Venezuela) and Rhodnius robustus from Mérida (Venezuela).

The existence of Rhodnius robustus as a species distinct from Rhodnius prolixus has long been the main epidemiological question about Chagas disease transmission in Venezuela and surrounding countries. These two taxa are morphologically and genetically very similar, but only R. prolixus is assumed to colonize houses and transmit Chagas disease to humans. R. robustus is assumed to be an exclusively sylvatic species, restricted to palm trees. If robustus and prolixus are actually the same species, the theoretical possibility exists of sylvatic specimens invading houses, even after insecticide application, and a control strategy similar to that of the successful Southern Cone Initiative against Triatoma infestans would be difficult to consider. Since no valid alternative control strategy exists, the answer to this biological question could be decisive about the future of vector control in this region. Although we believe genetic techniques are best suited to define species boundaries, we present here an example of the relevance of modern morphometrics in dealing with such an issue. Using both traditional and geometric morphometrics, we compared the wing size and shape in both sexes of these two taxa reared in the same laboratory for one generation. R. robustus specimens were collected from palm trees in the state of Mérida (Venezuela), and R. prolixus were collected from houses in the state of Cojedes (Venezuela). Our study provided no argument to question their specific status. Even after one generation of living in the same laboratory conditions, the two lines showed clear size differences, divergent allometric trends, and significant allometry-free differences in shape. These results suggest that R. robustus (Mérida, Venezuela) and R. prolixus (Cojedes, Venezuela) are distinct evolutionary units. Due to the epidemiological importance of this question, further studies in other geographic areas of Venezuela are required to accurately define the relationships of R. robustus and R. prolixus.

Phylogeography of the neotropical sand fly Lutzomyia longipalpis inferred from mitochondrial DNA sequences.

Sand flies in the Lutzomyia longipalpis species complex include the primary vector of Leishmania chagasi, the etiologic agent of visceral leishmaniasis in the Neotropics. Twelve L. longipalpis populations from South and Central America were compared using the cytochrome c oxidase I (COI) gene from the mitochondrial genome. The haplotype profiles for each population revealed that the majority of sequence variation was inter-population (98%) rather than intra-population, suggesting that sequence polymorphisms at the COI locus should provide excellent characters for the study of phylogenetic relationships among populations. Phylogenetic reconstruction using distance (neighbor-joining) and maximum parsimony analysis revealed the existence of four clades among the L. longipalpis populations studied: (1) Laran, (2) Brazilian, (3) cis-Andean and (4) trans-Andean. We suggest that these clades represent species. A biogeographical interpretation of the molecular phylogeny suggests that the process of speciation in the L. longipalpis complex began in the Pliocene, from a sub-Andean-Amazonian gene pool resulting from the Andean orogeny (formation of the East Andean Cordillera). The four clades probably diverged as a result of vicariance events that occurred throughout the late Pliocene and Pleistocene. We propose and discuss several historical scenarios, based on the biogeography and historical geology of Central and South America.

A possible role of bats as a blood source for the Leishmania vector Lutzomyia longipalpis (Diptera: Psychodidae).

Some evidence suggests that bats may provide an alternative blood source for Lutzomyia longipalpis, the main vector of American visceral leishmaniasis. Feeding trials were conducted to determine whether L. longipalpis feeds on captive bats. The high feeding success indicated that L. longipalpis is capable of feeding on at least four species of bats. Implications for the epidemiology of leishmaniases are discussed.

Epidemiologic aspects of American visceral leishmaniasis in an endemic focus in Eastern Venezuela.

An endemic focus of American visceral leishmaniasis (AVL) in eastern Venezuela has been evaluated in terms of patients (n = 48), immunologic reactivity to Leishmania in household contacts (n = 187) and neighborhood controls (n = 170), detection of Leishmania (L. donovani complex) in dogs and wild animals by the polymerase chain reaction (PCR) and characteristics of the sandfly population. The male:female ratio of patients was 1.18:1; 89.6% were < or =12 years old. Serologic reactivity was significantly higher in household contacts than in controls (P = 0.0008), as was the size of leishmanin reactions in contacts < or =10 years of age (P = 0.0141). Leishmania donovani complex-specific PCRs were positive in dogs, an opossum (Didelphis marsupialis), and a black rat (Rattus rattus). Lutzomyia longipalpis and Lu. evansi, both implicated in the transmission of AVL, were identified among the 386 sand flies examined. These observations provide the bases for an active control program as well as further studies of reservoirs and vector-host relationships in this area.

Hourly activity of Lutzomyia ovallesi and L. gomezi (Diptera:Psychodidae), Vectors of cutaneous leishmaniasis in northcentral Venezuela.

The comparative hourly activity of Lutzomyia ovallesi (Ortiz) and L. gomezi (Nitzulescu), vectors of cutaneous leishmaniasis in Miranda State, Venezuela, was studied between November and March during 1991-1994 using a Shannon trap with a fluorescent light. Female abundance of L. ovallesi increased from 1800 to 2000 hours, plateaued from 2000 to 2400 hours, then decreased progressively. L. gomezi always exhibited maximum activity between 1900 and 2000 hours, then declined abruptly. The importance of these activity patterns in Leishmania transmission is discussed.

Lutzomyia pseudolongipalpis: the first new species within the longipalpis (Diptera: Psychodidae: Phlebotominae) complex from La Rinconada, Curarigua, Lara State, Venezuela.

Lutzomyia pseudolongipalpis, the first new sand fly species within the longipalpis complex, is described based on females and males from La Rinconada, Curarigua, Lara State, Venezuela. Similar to Lutzomyia longipalpis sensu lato, females of the new species show spermathecae with 8-10 annulations and cibarial armature with 8-12 horizontal teeth. However, L. pseudolongipalpis females show conspicuous vertical cibarial teeth, large stipites and wing vein sections, round cerci, and short and broad valvifers that separate them from L. longipalpis s. l. females. L. pseudolongipalpis and L. longipalpis s. l. males are isomorphic, both having parameres with two curved setae on a simple tubercle. The analysis of morphometric characters and their diagnostic value is in progress.

Phlebotomine sandflies in Venezuela: review of the verrucarum species group (in part) of Lutzomyia (Diptera: Psychodidae) with description of a new species from Lara.

The series townsendi and verrucarum of the verrucarum species group of the phlebotomine genus Lutzomyia França are reviewed. Lutzomyia nadiae, new species, from the mountains of Yacambú, Lara State, is described. Distribution maps, illustrated keys, notes on medical importance, taxonomy, and ecology are presented for species in these series known from Venezuela.

Egg surface ultrastructure of eight New World phlebotomine sand fly species (Diptera: Psychodidae).

The ultrastructure of the egg chorionic sculpturing of eight neotropical and fly species (Lutzomyia townsendi (Ortiz, 1959), L. youngi Feliciangeli & Murillo, 1987, L. spinicrassa Osorno, Morales, Osorno, & Hoyos, 1969, L. evansi Nuñez Tovar 1924, L. lichyi, Floch & Abonnenc, 1950, L. trinidadensis Newstead, 1922, L. venezuelensis Floch & Abonnenc, 1948, and L. olmeca bicolor, Fairchild & Theodor, 1971) was studied using scanning electron microscopy (SEM). A new pattern, ellipses bound by fine longitudinal ridges, was observed in L. venezuelensis. Moreover, the chorionic ultrastructure was useful for separating three isomorphic species (L. townsendi, L. youngi, and L. spinicrassa), of which L. youngi and L. spinicrassa are sympatric in some endemic foci of Táchira State, Venezuela.

Genetic divergence among Venezuelan populations of Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae).

Lutzomyia longipalpis (Lutz & Neiva) is the primary vector of visceral leishmaniasis in Venezuela. An analysis of alleles at seven enzyme-encoding loci among four populations from different geographic and epidemiological regions revealed strong genetic substructuring. Isozyme analysis indicated that L. longipalpis in Venezuela is a complex of at least two subspecies. Possible differences in population size during their evolutionary histories, varying colonization histories and geological events may explain discrepancies in the patterns of variation observed at genetic markers between these four populations.

Multivariate analysis to discriminate species of phlebotomine sand flies (Diptera:Psychodidae): Lutzomyia townsendi, L. spinicrassa, and L. youngi.

Multivariate discriminant analysis was employed to discriminate on a morphological basis females of 3 closely related sand fly species, Lutzomyia townsendi (Ortiz), L. spinicrassa Morales, Osorno-Mesa, Osorno & Hoyos, and L. youngi Feliciangeli & Murillo. Principal component and canonical discriminant analysis compared a set of 31 morphological characters measured from known specimens to detect differences among the 3 species. A subset of 6 characters separated the 3 species with a high level of confidence. A simple method is presented to identify an unknown specimen as L. townsendi, L. spinicrassa, or L. youngi using these 6 morphological characters.

Vectors of leishmaniases in Venezuela.

Information on the vectors of leishmaniases in Venezuela is reviewed and updated. Lutzomyia longipalpis is considered the vector of visceral leishmaniasis. Lutzomyia ovallesi of the northcentral area is the only human feeding species from which parasites have been isolated and typed as Leishmania braziliensis. In the Andean Region there is strong evidence that Lu. youngi is the vector of cutaneous leishmaniasis but the parasites require further identification. Lutzomyia spinicrassa may be responsible for transmission throughout areas bordering the endemic foci in Columbia. New methods for identification of parasites and vectors are needed in areas where more than one parasite species and isomorphic Lutzomyia are sympatric.

Man-vector contact of phlebotomine sand flies (Diptera: Psychodidae) in north-central Venezuela as assessed by blood meal identification using dot-ELISA.

Human bait is traditionally used to assess man-vector contact, which is a key point in the study of the epidemiology of vector-borne diseases. However, in highly endemic foci, where this method should be avoided, this information could be obtained by blood meal analysis of engorged insects. In the village El Ingenio, Miranda State, Venezuela, Lutzomyia ovallesi and Lutzomyia gomezi are vectors of cutaneous leishmaniasis (CL). From June 1994 to March 1995, sand flies were collected inside houses on 974 CDC trap nights from 1900 to 0700 h. A total of 7,281 female sand flies were caught: 68.7% of them were identified as L. ovallesi, and 3.3% were identified as L. gomezi. Almost all of the blood-engorged flies (233 of 237) were dissected and identified, and gut contents were examined by dot enzyme-linked immunosorbant assay (dot-ELISA) using antisera against humans and common household animals including the domestic mouse. The Human Blood Index was 0.817 for L. ovallesi. These results suggest that intradomiciliary transmission may occur and account for the cases of CL frequently observed in newborn children in El Ingenio.

Correlated morphologic and genetic diversity among Lutzomyia longipalpis (Diptera: Psychodidae) collections in Venezuela.

Diversity among Lutzomyia longipalpis populations in Venezuela was characterized using 2 methods: larval mouthpart morphology-morphometry and isoenzyme electrophoresis. Analysis of the results suggested the presence of 2 morpho-genotypes. The mentum, maxillary comb, mandibular ventral teeth, and adenylate kinase and hexokinase enzyme-encoding loci suggested that a population from the northwestern Coriano System (Curarigua) is a distinct lineage within the L. longipalpis complex. Three widely separated populations from the Llanos (savanna), Andes, and northcentral Coastal Cordillera showed no significant substructure. These studies provide morphologic markers that are congruent with genetic data and suggest that the morphologic markers may be used to characterize and differentiate populations within this species complex.

Susceptibility of Lutzomyia longipalpis (Diptera: Psychodidae) to selected insecticides in an endemic focus of visceral leishmaniasis in Venezuela.

A field population of Lutzomyia longipalpis from La Rinconada, Lara State, an endemic focus of visceral leishmaniasis in Venezuela, was tested for susceptibility to organochlorine (DDT 2%), carbamate (propoxur 0.01%), organophosphate (malathion 2%, fenitrothion 1%, and pirimiphos methyl 1%), and pyrethroid (deltamethrin 0.06%, lambdacyhalothrin 0.06%, and permethrin 0.2%) insecticides. Susceptibility to the insecticides tested was evaluated in the field population of L. longipalpis and compared with a laboratory reference strain. The (LT95) to propoxur and malathion insecticides for the field population was lower than the LT95 for the laboratory reference strain, demonstrating high susceptibility to these compounds. A low level of resistance at LT50 (< 3-fold) was found for fenitrothion, pirimiphos methyl, and permethrin insecticides, but no resistance was detected at LT95. No significant resistance at the LT50 and LT95 was detected for the pyrethroids deltamethrin and lambdacyhalothrin. The susceptibility levels of L. longipalpis to the insecticides tested are discussed in view of a future control program against endophilic vectors of leishmaniases based on the use of pesticides.

Activity of Lutzomyia pseudolongipalpis and L. longipalpis s.l. (Diptera: Psychodidae) in Venezuela.

The nocturnal activity of the phlebotomine sandfly Lutzomyia pseudolongipalpis and two populations of L. longipalpis s.l. from different American visceral leishmaniasis foci in Venezuela was studied using collection bottle rotator traps. The activity of L. pseudolongipalpis from Lara State was continuous throughout the night, while that of L. longipalpis s.l. from El Layero, Guárico State and from Santa Ana del Valle, Margarita Island, was greatest before 23:00 h. The activity of sandflies of both populations and sexes steadily decreased thereafter. These different patterns seem to correlate with genetic data that indicate the presence in Venezuela of at least two sibling species in the L. longipalpis complex. The advantages of the bottle rotator trap for this type of study are discussed.

The re-emergence of American visceral leishmaniasis in an old focus in Venezuela. II. Vectors and parasites.

As part of an epidemiological study in an old focus of American Visceral Leishmaniasis (AVL) in Venezuela (Guayabita, Aragua State), a longitudinal entomological survey (January 1993-June 1994) was carried out. A total of 3,239 males and 6,043 females belonging to 11 phlebotomine sandfly species were collected. The two recognised vectors of AVL in the New World, Lutzomyia evansi and Lu. longipalpis were found to be sympatric. Lutzomyia evansi was the dominant species (86.4%), almost ten fold times more abundant than Lu. longipalpis (10.6%). The two species alternated seasonally: Lu evansi peaked at the end of the rainy season while Lu. longipalpis, almost virtually absent during such period, increased in the dry season. This species seems more greatly influenced by the temperature. Seven of 4,559 Lutzomyia evansi (0.15%) and one of 353 Lu. longipalpis (0.28%) were found positive for suprapyloric promastigotes. Using the polymerase chain reaction (PCR) with universal primers, all isolates were identified as Leishmania spp. Two cultures from Lu. evansi, IEVA/VE/93/UCNA-2 and IEVA/VE/93/UCNA-3, were established. k-DNA restriction analysis showed high homologies between these isolates and Leishmania chagasi. High hybridization signal with L. chagasi specific kDNA confirmed these results. These findings suggest that Lu. evansi may play a role as vector of visceral leishmaniasis in this area. The identity of the parasite carried by Lu. longipalpis needs to be confirmed.

The re-emergence of American visceral leishmaniasis in an old focus in Venezuela: present situation of human and canine infections.

The report of a new autochthonous case of human American Visceral Leishmaniasis (AVL) in 1992 in the village of Guayabita, Aragua State, Venezuela (10 degrees 16'N, 67 degrees 28'W; 500 m asl), led us to undertake an epidemiological study in this locality. A demographic survey was conducted using a structured questionnaire in which data was collected regarding sex, age, occupation, length of residence in the area and migratory history. A leishmanin skin test (LST) was applied and samples of venous blood for counter immunoelectrophoresis (CIEP) and immunofluorescent antibodies (IFAT) tests were drawn. The prevalence of positive LST was 11.4%. The positivity was highest among males and increased with age. Young males seemed to be more exposed to infection than females. Since occupation per se did not seem to account for this association, it may be explained as a gender-associated behavior, leading to different degrees of exposure to sand flies. The canine population was also screened for leishmanial infection. One dog was shown to be parasitologically infected with Leishmania sp. Four out of 71 dogs (5.6%) were positive for FG, CIEP, IFAT and Western Blot and 11 (15.5%) were positive for CIEP. These results suggest that after almost 30 years of epidemiological silence, American visceral leishmaniasis has re-emerged in this focus.