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Leishmaniasis is a vector-borne zoonotic disease occurring in three distinct manifestations: cutaneous, mucocutaneous, and visceral. This disease is endemic in approximately 90 countries in tropical and subtropical regions of the world.1 Transmission can occur in rain forests and desert areas, rural and periurban habitats, and sylvatic and domestic areas in Asia, Africa, Europe, and South America.2,3
The disease is transmitted by the bite of infected female phlebotomine sand flies, with two species considered the main vectors of human leishmaniasis in Italy: Phlebotomus perniciosus4–6 and P. perfiliewi.7 Of the two species, P. perniciosus is the main anthropophilic vector,8 while P. perfiliewi is considered mainly zoophilic, although it has been implicated in transmission of leishmaniasis to humans.9 Moreover, in some Italian territories, P. neglectus is also suspected to play a role in human transmission and this species is a proven vector of L. infantum in Greece.10,11 The etiologic agents are several obligate intra-macrophage parasitic protozoan species within the genus Leishmania that are widespread in the Mediterranean basin, with L. infantum being the most common species in Italy,12 and include the island of Sicily.8 Several studies have found higher sensitivity and specificity in the detection of Leishmania sp. parasites using the polymerase chain reaction (PCR) rather than standard methods such as culture and microscopy.13–21 The present study involved a published Taqman fluorogenic real-time PCR assay22 to determine the prevalence of Leishmania sp. parasites among sand flies trapped during the summer and fall of 2003 in two provinces in Sicily.
Two types of trapping devices were used to collect sand flies: U.S. Army miniature light trap23 and Malaise traps.24 The entomologic survey was carried out in Catania Province in housing areas of the Sigonella plain (Gli Ulivi, Marinai, and Mineo), urban areas (Trecastagni and Motta), semi-urban areas (Pennisi of the Acireale commune), and one agricultural habitat in a semi-rural environment (Valcorrente of the Belpasso commune). Additional sand flies were collected in Agrigento Province in four areas considered to have a relatively high prevalence of leishmaniasis: Palma di Montechiaro, Favara, Licata, and Ribera, in camping grounds within semi-urban environments. Traps were set up in the evening and collected the next morning during the period July–October 2003. After collection, the captured insects were placed in a freezer for at least one hour and then observed with a dissecting microscope to separate the sand flies from other insects and to determine sex. Immediately after segregation, the sand flies were stored in 1.5-mL Nunc (Roskilde, Denmark) vials containing 95% ethanol. Female sand flies were used for PCR testing and male specimens were identified to the species level by their morphologic characteristics.25,26
Female sand flies were homogenized in phosphate-buffered saline using a metal probe and DNA was then extracted using the DNeasy Tissue DNA extraction Kit (Qiagen SPA, Milan, Italy) following the manufacturer’s instructions. Extracts of DNA were stored refrigerated (2–8°C).
Leishmania infantum zymodeme MON1, international code MHOM/TN/80/IPT1, promastigotes were kindly provided by the Istituto Superiore di Sanità and DNA was extracted using the DNeasy kit as described for the sand flies. Control DNA from L. braziliensis WR U1150 was provided by the Walter Reed Army Institute of Research (Silver Spring, MD).
A 2-µL aliquot of DNA extract was used as template for the PCR using published22 genus-specific primers that flank a 67-basepair region of the Leishmania small subunit ribosomal RNA gene in a 20-µL reaction volume. The master mixture contained Taqman
Universal PCR Master Mix (PE Applied Biosystems, Foster City, CA), oligonucleotide primers LEISU1 and LEISL1 (both at concentrations of at 0.5 µM), and the fluorogenic probe LEISP1using a 5'-reporter dye 6-carboxy-fluorescein monophosphate (6FAM) and a 3'-quencher dye 6-carboxy-tetramethyl-rhodamine (TAMRA) at a concentration of 0.1 µM. Thermus aquaticus DNA polymerase was used at a concentration of one unit per 20-µL reaction. Oligonucleotide primers and probes were synthesized by PE Applied Biosystems.
Specimens were incubated at 95°C for 30 seconds followed by 45 two-step cycles of denaturation for 0 seconds at 95°C and primer annealing/extension for 20 seconds at 60°C in a Ruggedized Advanced Pathogen Identification Device (RAPID) (Idaho Technologies, Salt Lake City, UT). The fluorescent detector of the RAPID feeds data acquisition directly to a laptop computer running LightCycler software (Idaho Technologies). Fluorescence is measured after every cycle repeat and plotted as a function of time or cycle number.
We collected 351 sand flies (234 females and 117 males) from July to October 2003 (Table 1
). Based on the gross morphology of male specimens, the most prevalent sand fly species identified during our study was P. perfiliewi (48.1%), followed by P. perniciosus (41.5%), Sergentomyia minuta (6.6%), P. neglectus, (1.9%), and P. papatasi (1.9%) (Table 2). In Catania Province, 180 (54%) sand flies were caught using Malaise traps (8.57 sand flies per trap night), whereas 154 (46%) were caught with light traps (4.97 sand flies per trap night). Eleven male sand flies were not accounted for and were probably lost in transit.
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). The majority (86.3%) of the male sand flies trapped in Mineo were identified as P. perfiliewi, whereas the majority (87.8%) of those trapped in Belpasso/Valcorrente were P. perniciosus (Table 2).
Of 234 female sand flies tested, seven were found to be positive for Leishmania sp. by the fluorogenic PCR (Figure 1). The overall positive rate was 2.9% using our genus-specific assay. Three of 10 sand flies captured in Gli Ulivi were positive, as were one of seven in Marinai, two of 99 in Mineo, and one of 90 in Valcorrente (Table 3). All positive sand flies were trapped in August, which was also the month when the greatest number of sand flies was captured.
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Of the areas surveyed, the Gli Ulivi site is of great interest for future studies since three of 10 sand flies trapped there were positive for Leishmania sp. DNA, and many of the human cases of cutaneous and visceral leishmaniasis among the Sigonella military community occurred in residents of a nearby town. However, the sample size at this location was very small, and further analysis will be performed when we have more data. Other collection sites, such as Mineo and Valcorrente, were more abundant in sand flies, although they showed a lower prevalence of Leishmania sp. The most prevalent species in Valcorrente was P. perniciosus, which suggests a higher potential risk for human leishmaniasis transmission at this location. Conversely, the most prevalent species in Mineo was P. perfiliewi. Both P. perniciosus and P. perfiliewi are considered to be competent vectors of L. infantum, although P. perniciosus is the most significant due to its anthropophilic and zoophilic behavior.8 The high prevalence of P. perfiliewi in Mineo might suggest a higher risk for canine leishmaniasis transmission because of its mainly zoophilic behavior.28
Detection of parasite DNA by a PCR using this protocol does not prove that a specific sand fly was acting as a competent vector of leishmaniasis, only that there were parasites present at the time of capture. Nevertheless, this study shows the prevalence of Leishmania sp. parasites in sand flies in the Sigonella area in the Catania Province of Sicily that involves either a competent vector, infected hosts/reservoirs, or both.
Received June 7, 2004. Accepted for publication August 1, 2004.
Acknowledgments: We thank Dr. Marina Gramiccia for providing reference L. infantum promastigotes and Colonel Glenn Wortmann for providing L. braziliensis DNA. We also thank Drs. Vera D’Urso, Antonio Cascio, and Rosario Russo for helpful epidemiologic information. Several U.S. Navy Environmental and Preventive Medicine Unit 7 staff members donated their time to set up traps in their homes and neighborhoods.
Financial support: This work was supported by the U.S. Navy Bureau of Medicine and Surgery.
Disclaimer: The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, or the U.S. Government.
Author’s addresses: Eduardo Gómez-Saladín and Carl W. Doud. U.S. Navy Environmental and Preventive Medicine Unit 7, PSC 812, Box 3540, FPO AE 09627–3540, Telephone: 39–095–86–9251 Fax: 39–095–86–9252, E-mails: egomez{at}nepmu7.sicily.navy.mil and cdoud{at}nepmu7.sicily.navy.mil. Michele Maroli, Section of Vector-Borne Diseases and International Health, Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy, Telephone: 39–06–4990–2302, Fax: 39–06–4938–7065, email: maroli{at}iss.it.
Reprint requests: Eduardo Gómez-Saladín, PSC 812, Box 3540, FPO AE 09627–3540.
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