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. 2012 Sep;87(3):511-7.
doi: 10.4269/ajtmh.2012.11-0708. Epub 2012 Jul 16.

Natural Leishmania infection of Lutzomyia auraensis in Madre de Dios, Peru, detected by a fluorescence resonance energy transfer-based real-time polymerase chain reaction

Hugo O Valdivia  1 , Maxy B De Los Santos, Roberto Fernandez, G Christian Baldeviano, Victor O Zorrilla, Hubert Vera, Carmen M Lucas, Kimberly A Edgel, Andrés G Lescano, Kirk D Mundal, Paul C F Graf
Affiliations

Natural Leishmania infection of Lutzomyia auraensis in Madre de Dios, Peru, detected by a fluorescence resonance energy transfer-based real-time polymerase chain reaction

Hugo O Valdivia et al. Am J Trop Med Hyg. 2012 Sep.

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.

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Conflict of interest statement

Disclosure: Several authors of this manuscript are military service members or employees of the U.S. Government. This work was prepared as part of their duties. Title 17 U.S.C. § 105 provides that Copyright protection under this title is not available for any work of the United States Government. Title 17 U.S.C. § 101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person's official duties.

Figures

Figure 1.
Figure 1.
Flor de Acre, Iberia in Madre de Dios, Peru. Inset shows satellite images of 10 houses around where sand flies were collected. Source: “Flor de Acre” S11° 23′ 09″ and W69° 30′ 55″ Google Earth.
Figure 2.
Figure 2.
Detection of Lutzomyia 12S ribosomal DNA and of Leishmania kinetoplast DNA by polymerase chain reaction (PCR) of pools of field-collected Lutzomyia sand flies. A, Agarose gel electrophoresis of 12S ribosomal DNA PCR products in a representative subset of pools. Lane 1, 100-basepair (bp) ladder; lanes 2–8, DNA from Lutzomyia pools; lane 9, positive control of male Lutzomyia spp.; lane 10, blank. B, Agarose gel electrophoresis of kinetoplast DNA PCR products. Lane 1, 100-bp ladder; lanes 2–8, positive Lutzomyia pools; lane 9, positive control of L. (V.) braziliensis strain MHOM/BR/84/LTB300; lane 10, negative control of male Lutzomyia spp.; lane 11, blank.
Figure 3.
Figure 3.
Melting curve analysis of real-time polymerase chain reaction of 6-phosphogluconate dehydrogenase (6PGD) and mannose phosphate isomerase (MPI). A, Melting curves corresponding to 6PGD-based amplification reactions of seven sand fly pools (I–VII) were run and compared with a battery of six standard strains of the New World Leishmania. B, Melting curves of MPI-based real-time amplification reactions of seven sand fly pools (I–VII) were run and compared with a battery of six standard strains of New World Leishmania. Species were identified on the basis of melting curves that had the same or similar peaks. pan = panamensis; guy = guyanensis; lain = lainsoni; bra = braziliensis; per = peruviana; ama = amazonensis.
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