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.     

Establishment of a mass screening method of sand fly vectors for Leishmania infection by molecular biological methods

Surveillance of the prevalence of Leishmania and its vector, sand fly species, in endemic and surrounding areas is important for prediction of the risk and expansion of leishmaniasis. In this study, a method for the mass screening of sand flies for Leishmania infection was established. This method was applied to 319 field-captured specimens, and 5 positive sand flies were detected. Sand fly species were identified by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) of the18S rRNA gene, and all the positive flies were Lu. hartmanni. Furthermore, cytochrome b (Cyt b) gene sequence analyses identified all the parasites as Endotrypanum species including a probable novel species. Because the method requires minimum effort and can process a large number of samples at once, it will be a powerful tool for studying the epidemiology of leishmaniasis.     

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.     

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.     

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.     

热休克蛋白70（hsp70）基因对利什曼原虫中国分离株的系统发育分析

目的选用hsp70基因探讨我国各疫区不同宿主利什曼原虫的种系发育关系。方法采用PCR方法扩增分离自中国各疫区不同宿主的利什曼原虫之热休克蛋白70（hsp70）基因,将扩增的目的片段纯化后测序。使用MEGA5．0软件对测序产物进行多序列比对并构建系统发育树,同时与国外利什曼原虫分离株的hsp70序列共同分析,观察利什曼原虫中国分离株在世界范围利什曼原虫的系统发育和分子进化中的地位。结果中国各流行疫区利什曼原虫均获得约1300bp长度的hsp70基因片段,存在丰富的多态位点。中国23株利什曼原虫经hsp70基因分型聚集为四群,其中人来源的原虫分离株均在A群,为杜氏利什曼原虫（L．donovani）,并分为杜氏利什曼原虫指名亚种（L.donovani）和杜氏利什曼原虫婴儿亚种（L．infantum）两个亚支;分离自新疆沙鼠或白蛉的6株利什曼原虫聚在B群,为都兰利什曼原虫（L．turanica）;分离自新疆白蛉和内蒙古沙鼠的两株利什曼原虫为C群沙鼠利什曼原虫（L．gerbilli）;分离自内蒙古蜥蜴和新疆白蛉的两株原虫为D群蜥蜴利什曼原虫（Sauroleishmania）。结论利什曼原虫中国分离株属于利什曼原虫亚属的不同种和蜥蜴利什曼原虫亚属,我国利什曼原虫分离株的种系发育复杂多样。     

Asymptomatic human carriers of Leishmania chagasi

In Brazil, programs based on elimination of infected dogs have not curtailed the spread of visceral leishmaniasis (VL), suggesting that other reservoirs of infection exist. Persons with active VL can infect the sand fly vector, but in endemic areas, persons with asymptomatic infections, whose infectivity to sand flies is unknown, are far more numerous. In this study, a polymerase chain reaction-based assay detected kinetoplast DNA of Leishmania chagasi in the blood of eight of 108 asymptomatic persons living with patients with recently diagnosed VL. These eight persons had low or unmeasurable levels of IgG antibodies to Leishmania, demonstrating the insensitivity of serology for subclinical infection. All eight persons had positive leishmanin skin test results, as did 70% of persons living in households of persons with active VL. Even if a small proportion of such asymptomatic persons are infective to sand flies, they represent a formidable reservoir of infection in endemic areas.     

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.     

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.     

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.     

Phlebotomine sand fly (Diptera: Psychodidae) seasonal distribution and infection rates in a defined focus of Leishmania tropica.

A two-year study was conducted of phlebotomine sand fly fauna in a defined focus of Leishmania tropica. A total of 17,947 sand flies representing 10 species were collected from the location. Phlebotomus guggisbergi, a vector of L. tropica in Kenya, was the most prevalent species through the entire period, representing about 80% of the total catch. There was marked seasonal fluctuation in the populations of the three most common species, with highest population levels reached in December and lowest levels reached in July and August. Leishmania-like infections were encountered in 489 P. guggisbergi. No flagellate infections were observed in any other species of sand fly. Although infected P. guggisbergi were collected during each month of the year, the percent parous infected flies was highest (27.5%) during the November through January time period. These data show that the greatest risk of transmission to humans at this focus occurs during December, when the vector is prevalent and infections are common.     

Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection

Entomological monitoring of Leishmania infection in leishmaniasis endemic areas offers epidemiologic advantages for predicting the risk and expansion of the disease, as well as evaluation of the effectiveness of control programs. In this study, we developed a highly sensitive loop-mediated isothermal amplification (LAMP) method for the mass screening of sand flies for Leishmania infection based on the 18S rRNA gene. The LAMP technique could detect 0.01 parasites, which was more sensitive than classical PCR. The method was robust and could amplify the target DNA within 1 h from a crude sand fly template without DNA purification. Amplicon detection could be accomplished by the newly developed colorimetric malachite green (MG)—mediated naked eye visualization. Pre-addition of MG to the LAMP reaction solution did not inhibit amplification efficiency. The field applicability of the colorimetric MG-based LAMP assay was demonstrated with 397 field-caught samples from the endemic areas of Ecuador and eight positive sand flies were detected. The robustness, superior sensitivity, and ability to produce better visual discriminatory reaction products than existing LAMP fluorescence and turbidity assays indicated the field potential usefulness of this new method for surveillance and epidemiological studies of leishmaniasis in developing countries.     

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.     

Environmental factors underlying spatial patterns of sand flies (Diptera: Psychodidae) associated with leishmaniasis in southern Sinai, Egypt

Although Leishmania major is endemic in parts of the Sinai of Egypt, the ecology and distribution of Leishmania sand fly vectors in southern Sinai has not been well characterized. Accordingly, additional sand fly samples were obtained at 41 sites in the southern Sinai region during 1996-1997, and analyzed to improve the characterization of risk of sand fly-borne pathogens. Using a Geographic Information System (GIS), species-specific spatial distributions that might suggest zoonotic cutaneous leishmaniasis (ZCL) risk areas were determined in relation to contextual environmental factors, including geology, hydrogeology, climate variables and elevation. Southern Sinai was characterized by a diverse sand fly fauna (eight Phlebotomus species), probably attributable to highly variable landscape and environmental factors. Phlebotomus alexandri, Phlebotomus kazeruni and Phlebotomus sergenti were widespread and abundant, Phlebotomus papatasi and Phlebotomus bergeroti were less frequent, and Phlebotomus arabicus, Phlebotomus major and Phlebotomus orientalis had highly restricted distributions. Logistic regression models indicated that elevation and climatic conditions were limiting determinants for the distributions of sand flies in southern Sinai. Based on the predicted distribution of P. papatasi, a recognized vector of L. major, about one-quarter of southern Sinai may be at high risk of ZCL. Risk areas for the suspected ZCL vector P. bergeroti had a more patchy distribution. Results suggest that future studies should include other factors related to vector abundance, vector competence, human population, and parasite and reservoir host(s) to produce more comprehensive ZCL transmission risk maps, thus helping in planning effective prevention and control strategies.     

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.     

Molecular detection of the blood meal source of sand flies (Diptera: Psychodidae) in a transmission area of American cutaneous leishmaniasis,Paraná State,Brazil

The feeding behavior of sand flies provides valuable information about the vector/host interactions and elucidates the epidemiological patterns of American cutaneous leishmaniasis (ACL) transmission. The aim of this study was to identify the blood meal sources of sand flies in endemic areas of leishmaniasis in Paraná State through polymerase chain reaction (PCR) amplification of a prepronociceptin (PNOC) gene fragment and its subsequent DNA sequencing. Moreover, molecular assays were conducted to evaluate the sensitivity and reproducibility of the PNOC gene amplification. Besides that, a time-course digestion test of the blood using sand flies that fed artificially on BALB/c mice was performed. Of 1263 female sand flies collected in the field, 93 (3.6%) specimens were engorged and 27 allowed efficient amplification of the PNOC gene. These flies had fed on equine (Equus caballus), porcine (Sus scrofa) and canine (Canis lupus familiaris) species. The results also showed that the identification of the blood meal sources of the sand flies using the molecular method was directly linked to the level of digestion of the blood (time-course) and not to the amount of blood that had been ingested or to the presence of inhibitors in the blood.     

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.     

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.     

Exploring the utility of phylogenetic analysis of cytochrome oxidase gene subunit I as a complementary tool to classical taxonomical identification of phlebotomine sand fly species (Diptera,Psychodidae) from southern Europe

Phlebotomine sand flies (Diptera, Psychodidae) are known to be vectors of several pathogens such as Leishmania and Phlebovirus genera. The identification of phlebotomine sand fly species is currently based on morphological characters, and requires considerable taxonomic expertise and skilfulness, but may be complemented by DNA-based analyses for (i) accurate species identification and (ii) for estimating sand fly diversity. The aim of this study was to evaluate the utility of mitochondrial cytochrome oxidase gene subunit I (cox1) sequence analysis as a complementary tool to classical taxonomical for the identification of the most prevalent phlebotomine sand fly species from southern Europe (i.e. Phlebotomus ariasi, P. perniciosus, P. sergenti and Sergentomyia minuta).     

Leishmania and sand flies: proximity to woodland as a risk factor for infection in a rural focus of visceral leishmaniasis in west central Venezuela

OBJECTIVE: To relate entomological, epidemiological and geographical data to understand the transmission dynamics of visceral leishmaniasis (VL) in a closed focus in western Venezuela. METHODS: Spatial and temporal patterns of Lutzomyia pseudolongipalpis, the most prevalent phlebotomine sand fly species (99.7%), were studied in El Brasilar, Curarigua, Lara State, Venezuela, a small rural community of 20 dwellings and 118 inhabitants. The sand fly population was monitored using Centers for Disease Control light traps monthly throughout 1 year in the domestic and sylvatic habitats and for 3 months in all inhabited houses. RESULTS: Temporal variation followed the yearly bimodal pattern of precipitation with the highest population densities in April and December. Infection with flagellates suggestive of Leishmania spp. was detected in 0.01% of 10,026 dissected females of L. pseudolongipalpis, which proved to be highly endophilic. Prevalence of Leishmania infection in people, as measured by the leishmanin skin test, was correlated with distance of the houses from the woodland and with sand fly abundance. A logistic regression model showed that for people who live in the village, the proximity to the woodland (linear) should be considered a risk factor for Leishmania infection (binary) (z = -2.02, P = 0.04, OR = 0.98, 95% CI = 0.97-0.99). This was consistent with the association between the proportion of VL infection and the log of sand fly abundance, which was negatively correlated with distance from the woodland. CONCLUSION: We discuss strategies that might be useful in controlling VL transmission in this endemic focus.