Detection and identification of Leishmania species within naturally infected sand flies in the andean areas of ecuador by a polymerase chain reaction

The surveillance of prevalent Leishmania and sand fly species in endemic areas is important for prediction of the risk and expansion of leishmaniasis. In this study, we developed a polymerase chain reaction (PCR)-based method for detection of Leishmania minicircle DNA within individual sand flies. Using this method, we detected minicircle DNA in 6 (3.3%) of 183 sand flies, while 5 (3.5%) of 143 were positive for Leishmania promastigotes in the same areas by microscopic examination. The species were identified as Leishmania (Leishmania) mexicana by nucleotide sequencing of the cytochrome b gene. Additionally, all the Leishmania-positive sand flies were identified as Lutzomyia ayacuchensis by the restriction enzyme digestion of the PCR-amplified 18S ribosomal RNA gene fragments. Since this combined method is relatively easy and can process a large number of samples, it will be a powerful tool for the rapid identification of prevalent sand fly and Leishmania species as well as monitoring the infection rate in sand fly populations in endemic areas.     

Natural infection of Lutzomyia tortura with Leishmania (Viannia) naiffi in an Amazonian area of Ecuador

Natural infection of sand flies with Leishmania parasites was surveyed in an Amazonian area in Ecuador where leishmaniasis is endemic. Seventy-one female sand flies were dissected and one was positive for Leishmania protozoa. The species of this sand fly was identified as Lutzomyia (Lu.) tortura on the basis of morphologic characteristics. Analysis of the cytochrome b gene sequence identified the parasite as L. (Viannia) naiffi. We report the distribution of L. (V.) naiffi in Ecuador and detection of a naturally infected sand fly in the Ecuadorian Amazon and natural infection of Lu. tortura with Leishmania parasites in the New World.     

Molecular mass screening to incriminate sand fly vectors of Andean-type cutaneous leishmaniasis in Ecuador and Peru

Sand flies from the Andean areas of Ecuador and Peru were examined for Leishmania infections by using our recently established molecular mass screening method. Leishmanial minicircle DNA-positive sand flies were detected in 3 of 192 and 1 of 462 samples from Ecuador and Peru, respectively. Sand fly species were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the 18S ribosomal RNA (rRNA) gene, and the positive flies were Lutzomyia (Lu.) ayacuchensis and Lu. peruensis, respectively. Furthermore, cytochrome b and mannose-phosphate isomerase gene sequence analyses identified the parasites from Ecuador and Peru as Leishmania (Leishmania) mexicana and L. (Viannia) peruviana, respectively. Thus, the mass screening method was confirmed to be a powerful tool for sand fly research.     

First report on natural infection of Phlebotomus sergenti with Leishmania promastigotes in the cutaneous leishmaniasis focus in southeastern Tunisia

During September 2010, 133 female sand flies were caught inside houses of patients with cutaneous leishmaniasis in the focus for this disease in southeastern Tunisia and subsequently dissected. One specimen was positive for Leishmania protozoa. This sand fly species was identified as Phlebotomus sergenti, and the parasite was identified as L. tropica. This is the first report of P. sergenti involvement in transmission of L. tropica in Tunisia.     

Prevalence of sand flies and Leishmania donovani infection in a natural population of female Phlebotomus argentipes in Bihar State, India

Leishmaniasis is a vector-borne disease, and in the Indian subcontinent the female Phlebotomus argentipes is the vector for Leishmania donovani. However, data on the extent of sand fly infection rates in natural settings using molecular methods have not been extensively reported in India. In this study a PCR technique was applied targeting the 18S rRNA encoding region to determine the prevalence of Leishmania infection in female P. argentipes captured in the field. For this study, sand flies were collected from 897 houses selected from 50 villages endemic for visceral leishmaniasis (VL) in Muzaffarpur district, Bihar state, using CDC miniature light traps and mouth aspirators. A total of 14,585 sand flies were collected of which 449 were female P. argentipes divided into 132 pools. Molecular detection using PCR targeting the 18S rRNA gene was carried out for the identification of P. argentipes and Leishmania. The overall prevalence of infection was 4.90-17.37% for L. donovani in female P. argentipes in endemic regions of Bihar state. In this study no correlation was found between the presence of infected sand flies and the occurrence of clinical VL. This study provides the first report evaluating the prevalence of Leishmania infection in sand flies in a region endemic for VL in India. Sergentomyia species are the most common species of sand fly. Knowledge of the infection rate in female P. argentipes may help in predicting severity of disease and in vector elimination programs.     

Diversity and ecology of sand flies (Diptera: Psychodidae: Phlebotominae) in coastal French Guiana

In French Guiana, at least five Leishmania species are known to be sympatically transmitted in sylvatic ecotopes. However, the previous surveys on the phlebotomine sand fly fauna were published 20 years ago. During that period, many ecological changes have occurred. Sand fly collections were conducted with CDC light traps in five stations representing the main ecotopes of French Guiana. A total of 817 sand flies belonging to 2 genera, 18 sub-genera, and 46 different species were identified. The species Lutzomyia umbratilis (16.6% of the collected specimens), Lu. infraspinosa (12.7%), Lu. ininii (8.0%), and Lu. flaviscutellata (6.1%) were the most common species. The stratification by height, activity period, and resting site preferences of the most abundant sand flies were analyzed. Population abundance and diversity were compared for each ecotope. The potential of certain sand fly species in leishmaniasis transmission is discussed.     

DNA barcoding for identification of sand fly species (Diptera: Psychodidae) from leishmaniasis-endemic areas of Peru

Phlebotomine sand flies are the only proven vectors of leishmaniases, a group of human and animal diseases. Accurate knowledge of sand fly species identification is essential in understanding the epidemiology of leishmaniasis and vector control in endemic areas. Classical identification of sand fly species based on morphological characteristics often remains difficult and requires taxonomic expertise. Here, we generated DNA barcodes of the cytochrome c oxidase subunit 1 (COI) gene using 159 adult specimens morphologically identified to be 19 species of sand flies, belonging to 6 subgenera/species groups circulating in Peru, including the vector species. Neighbor-joining (NJ) analysis based on Kimura 2-Parameter genetic distances formed non-overlapping clusters for all species. The levels of intraspecific genetic divergence ranged from 0 to 5.96%, whereas interspecific genetic divergence among different species ranged from 8.39 to 19.08%. The generated COI barcodes could discriminate between all the sand fly taxa. Besides its success in separating known species, we found that DNA barcoding is useful in revealing population differentiation and cryptic diversity, and thus promises to be a valuable tool for epidemiological studies of leishmaniasis.     

Phlebotomus guggisbergi (Diptera: Psychodidae), a vector of Leishmania tropica in Kenya

Sand flies were collected in light traps and on oiled papers at four active case sites of human cutaneous leishmaniasis due to Leishmania tropica at Muruku Sublocation, Laikipia District, Kenya. Nearly 5,200 females of five species, including Phlebotomus guggisbergi, were dissected and examined for flagellates. Of 3,867 P. guggisbergi females collected at a multiple case site, 168 (4.3%) harbored mature infections (to include metacyclic promastigotes) of flagellates morphologically identical to Leishmania, while all other flies were negative. Of the infected flies, 164 were collected in a cave near the patients' home, three from crevices on an escarpment immediately behind the house, and one from the bedroom of one of the patients. One hundred sixty-four of the isolates were successfully grown in Schneider's Drosophila medium and harvested for typing by cellulose-acetate electrophoresis. Isoenzyme profiles of the first 22 of these were compared with those of WHO reference strains and well characterized local strains using 12 enzyme loci. The isolates yielded isoenzyme migration patterns that were indistinguishable from those of two L. tropica reference strains and of six L. tropica patient isolates from the same locality. This is the first reported isolation of L. tropica from a sand fly in Kenya, the first reported isolation of Leishmania parasites from P. guggisbergi, and the first confirmed isolation of this Leishmania from a sand fly other than P. sergenti. The finding of such a large number of P. guggisbergi naturally harboring mature infections of L. tropica at an active case site of cutaneous leishmaniasis due to this agent strongly implicates this fly as a vector.     

Short report: surveillance of Leishmania sp. among sand flies in Sicily (Italy) using a fluorogenic real-time polymerase chain reaction

Leishmaniasis caused by Leishmania infantum is a complex zoonotic disease, resulting in cutaneous and visceral manifestations in both dogs and humans. The present study involved a published Taqman fluorogenic real-time polymerase chain reaction (PCR) assay for surveillance of Leishmania sp. parasites among sand flies trapped in two provinces in Sicily, Catania and Agrigento, during the summer and fall of 2003. Only male specimens were identified to species level, while females were used to evaluate Leishmania sp. infection by PCR testing. The two most prevalent sand fly species found were Phlebotomus perfiliewi and P. perniciosus. Of the female sand flies tested, 2.9% were positive for Leishmania sp. DNA by the PCR.     

Influence of vertebrate blood meals on the development of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis in the sand fly Lutzomyia migonei (Diptera: Psychodidae)

The effect of blood meals from humans and seven domestic, wild, or laboratory animals (dogs, horses, chickens, rats, opossums, mice, and hamsters) on the development of Leishmania braziliensis and L. amazonensis was studied in the sand fly Lutzomyia migonei. The development of L. braziliensis and L. amazonensis exhibited peripylarian and suprapylarian patterns of development, respectively, in the sand fly gut with all blood meals tested. The blood meal sources influenced the infection rate of the sand flies. In both the Leishmania species, the highest parasite density was obtained with blood from wild rats followed by skunk, human, and horse. The epidemiological significance of these observations may be related to the distribution of leishmaniasis and needs to be evaluated further.     

First report on natural Leishmania infection of Phlebotomus sergenti due Leishmania tropica by high resolution melting curve method in Southeastern Iran

Objective:To identify the Leishmania species in infected sand flies by Real-time PCR coupled with HRM analysis.Methods:Real-time PCR coupled with HRM analysis targeting the first internal transcribed spacer(ITS1)of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish Lelthmania species in sand flies specimens.Results:Three out of 115females of Phlebotomus sergenti(P.sergenti)(2.6%)were positive to Leishmania tropica(L.tropica).Conclusions:This is the first report on P.sergenti as the main and proven vector of anthroponitic cutaneous leishmaniasis in Dehbakri County using Real-time PCR coupled with HUM analysis.This method is rapid,sensitive and specific for diagnosing of parasites in infected Sand flies and ideal for large scale genotyping projects.     

Sand fly feeding on noxious plants: a potential method for the control of leishmaniasis.

The sand fly Phlebotomus papatasi transmits Leishmania major, which causes cutaneous leishmaniasis, in vast regions of the Old World. In addition to blood, the sand flies feed on plants. In a study of this diet, we observed that one night of feeding on branches of Solanum jasminoides, Ricinus communis, or Bougainvillea glabra drastically shortened the life span of the sand flies. Flowering B. glabra attracted P. papatasi in the field. Nevertheless, in the region endemic for L. major in yards abounding with vector sand flies, the number of P. papatasi trapped near hedges of B. glabra was eight times less (62 versus 502 flies trapped) than in the control sites. The results imply that B. glabra affords local protection against sand fly bites and decreases the risk of leishmaniasis. We suggest that this and other ornamental plants that are harmful to sand flies can be used as a tool for this purpose.     

Ecology of sand flies in a low-density residential rural area,with mixed forest/agricultural exploitation,in north-eastern Brazil

Cutaneous leishmaniasis caused by Leishmania braziliensis is endemic in Brazil, where Lutzomyia whitmani is the most important vector involved in the transmission to humans, particularly in the peridomestic environment. Herein, we assessed the ecology of sand flies, including Lu. whitmani, in a low-density residential rural area with mixed forest/agricultural exploitation in north-eastern Brazil, where cutaneous leishmaniasis is endemic. Particularly, we hypothesized that sand fly abundance was correlated with climatic variables. Sand fly collections were carried out monthly from August 2013 to August 2014, using seven CDC light traps, for three consecutive nights, in three kinds of environments: indoor, peridomicile and forest. Collected sand flies were identified based on morphology and females of Lu. whitmani (n = 169), Lu. amazonensis (n = 134) and Lu. complexa (n = 21) were selected and tested by PCR for Leishmania (Viannia) spp. In total, 5167 sand flies belonging to 19 species were identified, being that Lu. choti (43.2%) was the most frequent species, followed by Lu. amazonensis (16.6%), Lu. whitmani (15.8%), Lu. sordellii (10.7%) and Lu. quinquefer (5.8%), which together represented over 90% of the collected sand flies. All females tested by PCR were negative. The number of sand flies collected daily was positively correlated with temperature and negatively correlated with rainfall and relative humidity. Furthermore, there was a positive correlation between daily number of sand flies and daily average saturation deficit. This study points out that the number of sand flies captured daily is correlated to climatic variables, including saturation deficit, which may represent a useful parameter for monitoring sand fly populations in leishmaniasis-endemic areas.     

Natural Leishmania Infection of Lutzomyia auraensis in Madre de Dios, Peru, Detected by a Fluorescence Resonance Energy Transfer-Based Real-Time Polymerase Chain Reaction

Abstract. Leishmania species of the Viannia subgenus are responsible for most cases of New World tegumentary leishmaniasis. However, little is known about the vectors involved in disease transmission in the Amazon regions of Peru. We used a novel real-time polymerase chain reaction (PCR) to assess Leishmania infections in phlebotomines collected in rural areas of Madre de Dios, Peru. A total of 1,299 non-blood fed female sand flies from 33 species were captured by using miniature CDC light traps. Lutzomyia auraensis was the most abundant species (63%) in this area. Seven of 164 pools were positive by PCR for Leishmania by kinetoplast DNA. The real-time PCR identified four Lu. auraensis pools as positive for L. (Viannia) lainsoni and L. (V.) braziliensis. The minimum infection prevalence for Lu. auraensis was estimated to be 0.6% (95% confidence interval = 0.20-1.42%). Further studies are needed to assess the importance of Lu. auraensis in the transmission of New World tegumentary leishmaniasis in hyperendemic areas of Peru.     

Distribution of cave-dwelling phlebotomine sand flies and their nocturnal and diurnal activity in Phitsanulok Province, Thailand

An entomological survey of sand flies was conducted in Naresuan Cave in Noen Maprang District, Phitsanulok Province, during November 2009 to December 2010. A total of 10,115 cave-dwelling sand flies were collected with CDC light traps nocturnally (06:00 AM and 06:00 PM) and diurnally (06:00 PM and 06:00 AM). The ratio between male and female sand flies was 1:1.3 (4,363:5,752). The ratio between the number of sand flies caught nocturnally and diurnally was 2.6:1 (7,268:2,847). In this study, 13 species belonging to 4 genera were identified, of which 4 belonged to the genus Phlebotomus, 7 to Sergentomyia, 1 to Nemopalpus and 1 to Chinius. An abundance of species were observed: Nemopalpus vietnamensis (49.15%), P. argentipes (20.15%), C. barbazani (15.79%), P. teshi (9.53%), and S. anodontis (3.21%). Less common species (<1%) were S. barraudi (0.63%), P. stantoni (0.57%), S. dentata (0.49%), S.quatei (0.17%), P. philippinensis gouldi (0.12%), S.silvatica (0.10%), S. gemmea (0.05%), and S. iyengari (0.04%). The predominant species in the Naresuan Cave was Nemopalpus vietnamensis (49.15%). The data demonstrates variability in sand fly prevalence, species composition, and relative abundance in caves. P. argentipes was found throughout the day in the caves, which is important because it is believed to be the Leishmania spp vector. This study highlights the diurnal activity of the sand fly and the day-time risk of leishmaniasis. In conclusion, although leishmaniasis has not been reported in Phitsanulok, there should be heightened awareness of infection in these areas with vectors of the protozoa.     

Synthetic glycovaccine protects against the bite of leishmania-infected sand flies

Leishmaniasis is a vectorborne disease transmitted to human and other mammalian hosts by sand fly bite. In the present study, we show that immunization with Leishmania mexicana promastigote secretory gel (PSG) or with a chemically defined synthetic glycovaccine containing the glycans found in L. mexicana PSG can provide significant protection against challenge by the bite of infected sand flies. Only the glycan from L. mexicana was protective; those from other species did not protect against L. mexicana infection. Furthermore, neither PSG nor the glycovaccine protected against artificial needle challenge, which is traditionally used in antileishmanial vaccine development. Conversely, an antigen preparation that was effective against needle challenge offered no protection against sand fly bite. These findings provide a new target for Leishmania vaccine development and demonstrate the critical role that the vector plays in the evaluation of candidate vaccines for leishmaniasis and other vectorborne diseases.     

Genotyping of sand fly species in Peruvian Andes where leishmaniasis is endemic

Genotyping of sand fly species circulating in Peru was established on the basis of PCR-restriction fragment length polymorphisms (RFLPs) of the 18S ribosomal RNA (rRNA) gene. The sequences of 18S rRNA gene fragments from 12 Lutzomyia and 1 Warileya species were determined and their RFLP-patterns were analyzed. Consequently, RFLP analysis with the restriction enzyme AfaI and then HapII or KpnI, followed by XspI successfully differentiated them. Intraspecific genetic diversity affecting RFLP-patterns was not detected in the specimens collected from 24 areas of 8 departments. The genotyping was applied to the surveillance of sand flies collected from Andean areas where leishmaniasis is endemic, and its usability was verified. The present method promises to be a powerful tool for the classification and surveillance of sand flies circulating in Peru.     

Possible determination of the vector and reservoir of leishmaniasis in the Dominican Republic.

Two species of sand flies were collected by various methods from sites in the Dominican Republic. Lutzomyia cayennensis hispaniolae was the more common of the two. It was found in wooded habitats from sea level to an elevation of 442 m. This species was observed feeding on lizards (Anolis sp.) in the wild. In the laboratory, it fed only on lizards and only under lighted conditions. The other species, Lu. christophei was only found in the vicinity of seven leishmaniasis case sites. It readily fed on or probed rodents and humans. Although no naturally infected sand flies were collected, in the laboratory Lu. christophei was readily capable of transmitting the Dominican Leishmania parasite to uninfected BALB/c mice. We collected 167 specimens of three species of rodents and three Herpestes auropunctatus (mongoose) from the vicinity of two case sites. All four species are non-endemics introduced in post-Columbian times. Although we were unable to isolate parasites from any of these specimens, four of 44 Rattus rattus from one case site were seropositive for antibodies against Leishmania by indirect fluorescent antibody testing. This represents the first report of transmission of the Dominican Leishmania parasite by a sympatric species of sand fly and suggests that commensal rodents may play a role in the epidemiologic cycle.     

Evidence for leishmania (viannia) parasites in the skin and blood of patients before and after treatment

BACKGROUND: American cutaneous leishmaniasis is considered to be a zoonotic disease transmitted by sand flies that feed on infected sylvatic mammals. However, the "domestication" of transmission and the increase in treatment failure with antimonial drugs have raised the suspicion of anthroponotic transmission of American cutaneous leishmaniasis. METHODS: The objective of the present study was to explore the potential of humans as a source of infection for sand flies. Biological (xenodiagnosis and culture) and molecular (polymerase chain reaction/Southern blot) detection methods were used to evaluate peripheral-blood monocytes and tissue fluids from sites accessible to sand flies from 59 adult patients with parasitologically confirmed American cutaneous leishmaniasis. RESULTS: Overall, 44.1% of patients (26/59) presented biological and/or molecular evidence of Leishmania parasites in normal skin, peripheral-blood monocytes, lesion scars, or lesion border (by xenodiagnosis) before (18/59 [30.5%]) or after (10/27 [37.0%]) treatment. Leishmania parasites were cultured from the unaffected skin of 2 (3.6%) of 55 patients, and xenodiagnosis gave positive results for 5 (8.8%) of 57 patients before treatment. CONCLUSIONS: The presence of Leishmania parasites in the unaffected skin and peripheral-blood monocytes of a high proportion of patients even after treatment and the acquisition of infection by sand flies support the plausibility of anthroponotic transmission of American cutaneous leishmaniasis.     

Natural infections with promastigotes in man-biting species of sand flies in leishmaniasis-endemic areas of Ecuador

In order to determine the vectors of leishmaniasis in Ecuador, 1,054 man biting sand flies from the Department of Ca?ar were dissected and examined for promastigotes. There were 2 man-biting species, Lu. trapidoi and Lu. hartmanni in this endemic area of the disease. The infection rates were 7.7% in the former and 3.9% in the latter species, demonstrating the different rates in various localities and altitudes of the study areas. There was an association between infection rates and the time of day, suggesting some connection with biting activity of sand fly species. In collections using human bait at 7 study areas in 5 Departments, 6 man-biting species were recognized, indicating different dominant species in each area. It was assumed that the dominant species would play an important role as the principal vector of leishmaniasis in each endemic area. As to species determination of the present Leishmania promastigotes, suffice it to say that the parasites are Leishmania sp., presumably L. braziliensis s.l., until the isolates have been typed.