Leishmaniasis in Brazil. XXII: Characterization of Leishmania from man, dogs and the sandfly Lutzomyia longipalpis (Lutz & Neiva, 1912) isolated during an outbreak of visceral leishmaniasis in Santarém, Pará State

During epidemiological studies on an outbreak of visceral leishmaniasis in Santarém, Pará State, north Brazil, isolates of Leishmania from two children, three dogs and six naturally infected specimens of the sandfly Lutzomyia longipalpis were compared, biochemically, by starch-gel enzyme electrophoresis. They have proved to be indistinguishable from each other, and from a reference strain of Leishmania chagasi Cunha & Chagas, 1937 from a case of human visceral leishmaniasis from Bahia State, north-east Brazil, on their enzyme profiles for ASAT, ALAT, PGM, GPI, MDH and MPI. Lu. longipalpis is the principal, and possibly the only vector to man in the Amazon Region of Brazil.     

Leishmaniasis in Brazil. XIX: visceral leishmaniasis in the Amazon Region, and the presence of Lutzomyia longipalpis on the Island of Marajó, Pará State

Sporadic cases of visceral leishmaniasis in Amazonian Brazil appear limited to Pará State, in the lower Amazon valley and principally near the Atlantic coast. The fox Cerdocyon thous (L.) has been incriminated as a natural host of the causative parasite, Leishmania donovani chagasi, but past doubts have existed over the identification of the most likely vector as Lutzomyia (Lutzomyia) longipalpis (Lutz & Neiva, 1912). Investigations on two of five recent cases of visceral leishmaniasis of man in the Districts of Cachoeira do Arari and Salvaterra, on the eastern part of the Island of Marajó, Pará showed undoubted Lu. longipalpis to be abundant in one house and in numerous chicken-houses. This is the first record of Lu. longipalpis on Marajó Island, and the finding supports previous implication of this sandfly in the epidemiology of visceral leishmaniasis in other parts of Pará. Morphological differences have been noted between this insect from Marajó and other specimens from more highly endemic regions in the States of Ceará and Minas Gerais, Brazil.     

Antibodies against Lutzomyia longipalpis saliva in the fox Cerdocyon thous and the sylvatic cycle of Leishmania chagasi

Sera of 11 wild Cerdocyon thous foxes from an endemic area for American visceral leishmaniasis were tested for the presence of antibodies against salivary gland homogenates (SGH) of Lutzomyia longipalpis. All foxes had higher levels of anti-Lu. longipalpis SGH antibodies than foxes from non-endemic areas, suggesting contact between foxes and the vector of visceral leishmaniasis. Sera of humans and dogs living in the same area were also tested for reactivity against Lu. longipalpis SGHs and had a lower proportion of reactivity than foxes. Antibodies against Leishmania chagasi were not detected in any of the foxes, but three foxes showed the presence of parasites in the bone marrow by direct examination, PCR or by infecting the vector. Both humans and dogs had higher levels of anti-Le. chagasi IgG antibodies than C. thous. The finding of an antibody response against saliva of Lu. longipalpis among C. thous together with the broad distribution of the vector in resting areas of infected foxes suggests that the natural foci of transmission of Le. chagasi exists independently of the transmission among dogs and humans.     

Cutaneous leishmaniasis due to Leishmania (Leishmania) amazonensis in Amazonian Brazil, and the significance of a negative Montenegro skin-test in human infections.

The clinical and epidemiological features of 62 cases of cutaneous leishmaniasis due to Leishmania (Leishmania) amazonensis, from Pará State, Amazonian Brazil, are discussed. The parasite, isolated in hamster skin and/or blood-agar culture medium, was in each case identified by both biological characteristics and a monoclonal antibody specific for promastigotes of L. (L.) amazonensis. Of the 62 patients, 46 (74.2%) presented with a single cutaneous lesion, and on no occasion was evidence found indicating metastatic spread to either the naso-pharyngeal mucosae or the viscera. Recent claims that this parasite may be responsible for both mucocutaneous leishmaniasis and typical visceral leishmaniasis are discussed. Meglumine antimoniate (Glucantime) proved highly efficient in the treatment of all patients. Of the 62 patients examined by the Montenegro skin test, only 32 (51.6%) gave a positive reaction. The significance of this finding is considered and the hypothesis made that the parasite itself may induce an immunoinhibition. Field studies amply confirmed the role of Lutzomyia flaviscutellata as the major sandfly vector of L. (L.) amazonensis in Amazonia.     

Lutzomyia longipalpis naturally infected by Leishmania (L.) chagasi in Várzea Grande, Mato Grosso State, Brazil, an area of intense transmission of visceral leishmaniasis

The American visceral leishmaniasis (AVL) is caused by parasites belonging to the genus Leishmania (Trypanosomatidae) and is transmitted to humans through the bite of certain species of infected phlebotomine sand flies. In this study, we investigated the natural infection ratio of Lutzomyia longipalpis, the main vector species of AVL in Brazil, in Várzea Grande, Mato Grosso State. Between July 2004 and June 2006, phlebotomine sand flies were captured in peridomestic areas using CDC light-traps. Four hundred and twenty (420) specimens of Lu. longipalpis were captured. 42 pools, containing 10 specimens of Lu. longipalpis each, were used for genomic DNA extraction and PCR (polymerase chain reaction) amplification. Leishmania spp. DNA was detected in three out of the 42 pools tested, resulting in a minimal infection ratio of 0.71%. Restriction fragment length polymorphism (RFLP) analysis indicated that Leishmania (L.) chagasi was the infective agent in the positive pools.     

Molecular analysis of natural infection of Lutzomyia longipalpis in an endemic area for visceral leishmaniasis in Brazil

The main purpose of this study was to investigate natural infection by Leishmania chagasi in female sand flies in a visceral leishmaniasis (VL) focus on S?o Luís Island, Maranh?o State, Brazil. Molecular analysis by polymerase chain reaction (PCR) was applied to determine the rate of natural infection of Lutzomyia longipalpis by L. chagasi in areas of old and recent human settlement on S?o Luís Island. Based on a sample of 800 female specimens captured from March to August 2005, the natural infection rate was 1.25% in an area of old settlement and 0.25% in two recently settled areas. Infection of L. longipalpis was detected in both areas, regardless of the number of reported human VL cases, indicating that other factors modulating infection in the wild need to be investigated. The results confirm PCR as a specific technique and an important tool for epidemiological surveillance.     

Natural infection of Lutzomyia longipalpis by Leishmania sp. in Teresina, Piauí State, Brazil

In Brazil, control of the vector Lutzomyia longipalpis is one of the main strategies used to limit the expansion of American visceral leishmaniasis. However, studies on the ecology of this sand fly are rare, especially regarding its natural infection with species of Leishmania. A study of the natural infection of Lu. longipalpis by Leishmania sp. was carried out in the Bela Vista neighborhood in the city of Teresina, Piauí State, Brazil, an important area of American visceral leishmaniasis transmission. From February 2004 to January 2005, sand flies were captured with CDC light traps. Approximately 10 female sand flies in each capture were dissected and examined for the presence of evolutionary forms of Leishmania sp. Two sand fly species were identified: 1,832 were Lu. longipalpis and six Lu. whitmani. Twenty female sand flies (1.1%), all Lu. longipalpis, were infected with procyclic and nectomonad forms of Leishmania sp., found mostly in the hindgut. Higher proportions of infected sand flies were found four months after the rainy season, suggesting that environmental factors may predict not only vector abundance (as already known) but also their level of infection.     

Leishmaniasis in Brazil: XVIII. Further evidence incriminating the fox Cerdocyon thous (L) as a reservoir of Amazonian visceral leishmaniasis

Major endemic areas of visceral leishmaniasis in Brazil are located in the drier, poorly forested regions, principally in the northeastern States such as Ceará and Bahia. Cases of the human disease in the Amazon Region are rare, very sporadic, and seldom present opportunities for epidemiological study. Following the report of a fatal case near Salvaterra, the Island of Marajó, Pará State, a preliminary investigation has resulted in the isolation of a parasite regarded as Leishmania donovani chagasi from the viscera and skin of an apparently healthy fox, Cerdocyon thous, captured in the same locality. This represents the third recorded isolation of the parasite from this species of fox in the Amazon Region. The inapparent nature of the infections supports the suggestion that this canid may represent the primitive natural host of L. d. chagasi. C. thous is commonly associated with forested or wooded areas, and enzymic profiles for the enzymes ASAT, ALAT, PGM, GPI, MDH, MPI, G6PD, PEP and ACP failed to distinguish an isolate of L. d. chagasi from this animal in Pará from others obtained from cases of human visceral leishmaniasis in the neighbouring States of Maranhão, Ceará and Bahia. This suggests that the major, present-day endemics may have originated from a primary silvatic enzootic.     

American visceral leishmaniasis: wild animal hosts

In Colombia it has been suggested that the rodent Proechimys canicollis may be a reservoir-host of Leishmania (L.) chagasi, the cause of American visceral leishmaniasis, based on polymerase chain reaction/hybridization tests. We have detected no infection in laboratory-bred specimens of another species, P. guyannensis, after their inoculation with promastigotes or amastigotes of this parasite from Amazonian Brazil.     

Phlebotomine sandfly species from an American visceral leishmaniasis area in the Northern Rainforest region of Pernambuco State, Brazil

The aim of this study was to identify the fauna of phlebotomine sandflies in an area with incidence of American visceral leishmaniasis (AVL) in S?o Vicente Férrer, a municipality (county) located in the northern Zona da Mata in Pernambuco State, Brazil. Sandfly captures were conducted monthly for four nights/month from December 2002 to November 2003, by means of manual captures (with a Shannon trap) and by CDC light traps. A total of 23,156 specimens of phlebotomines sandflies were collected in a remnant of the Atlantic rainforest, houses, and animal shelters. Lutzomyia complexa was the most prevalent species, with a frequency of 62.5% (14,445/23,156), followed by Lutzomyia migonei, with 33.2% (7,677/23,156). No Lutzomyia longipalpis (the main vector of Leishmania chagasi in Brazil and the Americas) was found in the target area. Some specimens were dissected, but no Leishmania parasites were found.     

Sandfly fauna of endemic leishmaniasis foci in Anzoátegui State, Venezuela

A census of the sandfly fauna was undertaken in 1993-98 in 5 endemic leishmaniasis foci situated at different altitudes in Anzoátegui State, Venezuela. From the 17 species of Lutzomyia identified, we believe that Lu. ovallesi, Lu. panamensis and Lu. gomezi are the probable vectors of cutaneous leishmaniasis, while Lu. evansi might transmit visceral leishmaniasis.     

A real-time PCR assay to estimate Leishmania chagasi load in its natural sand fly vector Lutzomyia longipalpis

Leishmania chagasi, transmitted mainly by Lutzomyia longipalpis sand flies, causes visceral leishmaniasis and atypical cutaneous leishmaniasis in Latin America. Successful vector control depends upon determining vectorial capacity and understanding Leishmania transmission by sand flies. As microscopic detection of Leishmania in dissected sand fly guts is laborious and time-consuming, highly specific, sensitive, rapid and robust Leishmania PCR assays have attracted epidemiologists' attention. Real-time PCR is faster than qualitative PCR and yields quantitative data amenable to statistical analyses. A highly reproducible Leishmania DNA polymerase gene-based TaqMan real-time PCR assay was adapted to quantify Leishmania in sand flies, showing intra-assay and inter-assay coefficient variations lower than 1 and 1.7%, respectively, and sensitivity to 10 pg Leishmania DNA ( approximately 120 parasites) in as much as 100 ng sand fly DNA. Data obtained for experimentally infected sand flies yielded parasite loads within the range of counts obtained by microscopy for the same sand fly cohort or that were around five times higher than microscopy counts, depending on the method used for data analysis. These results highlight the potential of quantitative PCR for Leishmania transmission studies, and the need to understand factors affecting its sensitivity and specificity.     

Leishmaniasis in Brazil: IX. Considerations on the Leishmania braziliensis complex:—Importance of sandflies of the genus Psychodopygus (mangabeira) in the transmission of L. braziliensis braziliensis in North Brazil

At least 3 different parasites may produce cutaneous leishmaniasis in man in Brazil. Leishmania mexicana amazonensis is widely distributed and very common in wild animals, but rarely infects man because the vector is not anthropophilic. L. braziliensis guyanensis and L. braziliensis braziliensis have highly anthropophilic vectors and are thus the major causes of the disease in man.Infections with Leishmania b. braziliensis are described in 17 persons from an endemic area in the Serra dos Carajás, Pará, north Brazil. A nearby Indian tribe (Xikrín) was examined and leishmanin skin-tests carried out on 116 individuals. No evidence of past or present infection was found, but strong positive reactions were noted in 83·9% adult males, 38·8% adult females, 14·3% male children and 4·8% female children.A total of 2,701 man-biting and rodent-biting sandflies were dissected and the guts examined for flagellates. Promastigotes were found in 3 out of 1,656 Psychodopygus wellcomei, 2 out of 175 Ps. paraensis and 1 out of 127 Ps. amazonensis. Hamsters inoculated with promastigotes from one Ps. wellcomei developed a typical L. b. braziliensis infection. The importance of sandflies of the genus Psychodopygus is stressed: Ps. wellcomei is of particular importance due to its tendency to bite man during the day and the fact that it is attracted to both man and rodents in large numbers.Nearly all the sandflies were caught at ground level. The absence of a large, arboreal population of man-biters suggests that the reservoir-hosts of L. b. braziliensis are likely to be terrestrial or frequently coming to the ground.     

Detection of circulating Leishmania chagasi DNA for the non-invasive diagnosis of human infection

A polymerase chain reaction (PCR) assay for the detection of Leishmania spp. DNA in peripheral blood was optimized and evaluated for the diagnosis of human visceral leishmaniasis (VL) in Brazil during May 2001 to December 2002. Optimization of the technique resulted in a detection limit of 1.65 fg of purified L. (L.) chagasi DNA, equivalent to 1.65 x 10(-2) parasites. Leishmania DNA was detected in the blood of 48 of 53 patients with parasitologically-confirmed VL, which corresponds to a sensitivity of 91%. No DNA was detected in the peripheral blood of 15 healthy, non-exposed volunteers, giving a specificity of 100%. We conclude that detection of parasite DNA in peripheral blood offers a non-invasive, sensitive and rapid method for the detection of VL caused by L. (L.) chagasi.     

Current epidemiological status of visceral leishmaniasis in Northeastern Brazil

The objective was to describe the current epidemiological status of visceral leishmaniasis in the State of Pernambuco, Northeastern Brazil. For that, a search for scientific production on epidemiology of visceral leishmaniasis in Pernambuco was carried out in electronic databases MEDLINE, SciELO and LILACS. Additionally, relevant papers that have not been retrieved from the searches were analyzed. The 18 studies selected for this review indicate that: visceral leishmaniasis is widely spread in Pernambuco; human cases are often associated to anthropic pressure on the environment; and children have been more frequently affected by the disease. These results showed the need for further studies on risk factors for visceral leishmaniasis in humans, the role of Leishmania chagasi hosts in the zoonotic transmission cycle, and the vector behavior in different geographical regions.     

The FML-vaccine (Leishmune) against canine visceral leishmaniasis: a transmission blocking vaccine

Transmission blocking vaccines are one of the control strategies for vector-transmitted protozoan diseases. Antibodies raised in the vaccinated host prevent the development of the parasite in the insect vector, interrupting the epidemiological cycle. The FML antigen of Leishmania donovani in combination with saponin (FML-vaccine and Leishmune) induced 92-97% of protections against zoonotic visceral leishmaniasis. We assayed the ability of FML to inhibit Leishmania donovani and Leishmania chagasi procyclic promastigote-binding to dissected Lutzomyia longipalpis midguts. We found a dose-dependent inhibition, more pronounced on L. donovani (80%) than on L. chagasi promastigotes (p<0.001). On the other hand, the Fab-IgG serum fraction of Leishmune vaccinated dogs (IgG2 predominant), also inhibited parasite binding in a dose-response (p<0.0001) with an equally potent effect against L. donovani or L. chagasi (p = 0.061). The transmission blocking properties of the Leishmune vaccine was also assessed by an in vivo membrane assay, with sand flies fed with 1.5 x 10(7) amastigotes, human blood and, vaccinated or normal control dog sera. Significantly higher values were found in rate of infection (p<0.025) and intensity of infection (number of parasites/insect) (p<0.05) of control sand flies, making a very reduced infection index (20.7%) in the vaccine group. Our results disclosed that the Leishmune vaccine is a TBV, and that the dog antibodies present in sera, even 12 months after vaccination, lead to a significant effective protection of 79.3%.     

Leishmaniasis in Bolivia. I. Lutzomyia longipalpis (Lutz & Neiva, 1912) as the vector of visceral leishmaniasis in Los Yungas

A relatively high leishmanial infection rate was found in the phlebotomine sandfly Lutzomyia longipalpis collected from three villages of the Los Yungas region (Department of La Paz, Bolivia). 2,578 female sandflies were dissected. In three houses surveyed in Santa Barbara promastigote infection rates of Lu. longipalpis were 4.2, 2.2 and 3.2% respectively. Anatomical localization of the infection in the insect, and biochemical characterization of the strains indicate that the parasite belongs to the Leishmania donovani complex. The geographical area and the biotopes of Lu. longipalpis are discussed in relation to the vector-parasite relationship.     

Sandflies (Diptera, Psychodidae) from an endemic leishmaniasis area in the cerrado region of the State of Maranh?o, Brazil]

This article presents a list of ten sandfly species from the genus Lutzomyia Fran?a, 1924 found in the counties of Aldeias Altas, Capinzal do Norte, Caxias, Codó, Coelho Neto, Timbiras, Timon and Tuntum in northeastern Maranh?o, Brazil. Presence of sandflies was associated with cases of visceral and cutaneous leishmaniasis. Some 377 specimens were captured indoors with CDC light traps and 1491 specimens in the peridomicile. The species were: Lutzomyia cortelezii, Lutzomyia evandroi, Lutzomyia goiana, Lutzomyia intermedia, Lutzomyia lenti, Lutzomyia longipalpis, Lutzomyia longipennis, Lutzomyia squamiventris, Lutzomyia termitophila and Lutzomyia whitmani. The most abundant species was L. longipalpis (67. 4% and 70.2%) followed by L. whitmani (31.0% and 24.7%). L. longipalpis was captured both indoors and outdoors in all the months studied.     

Leishmaniasis in Brazil. 8. Observations on the phlebotomine fauna of an area highly endemic for cutaneous leishmaniasis, in the Serra dos Carajas, Pará State

A study has been made of the phlebotomine fauna in an area highly endemic for Leishmania braziliensis braziliensis in forest of the Serra dos Carajás, Pará State, Brazil, using human and rodent bait.A total of 23 different species were encountered, including 2 new sandflies, to be described at a later date. Over 92% of the total catch off man was made up by species of the genus Psychodopygus. Of these, Ps. wellcomei formed 65% of the total caught off man and 24·5% of the total off rodents. This species is of particular interest in view of its tendency to bite man throughout daylight hours.Once more we have confirmed that although Lutzomyia flaviscutellata may be the predominent species feeding on rodents, it is rarely taken biting man.Very few sandflies were captured above ground-level; and attempts to find the daytime resting places for the major man-biting species were unsuccessful.The relationship of rainfall to sandfly density is discussed. It is concluded that the population density of certain forest species may depend not so much on the quantity of rainfall, but more on the amount of water which persists in the topsoil in a given region.     

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.