LeishmaniaDetection in Sand Flies Using a Field-Deployable Real-Time Analytic System

We describe here the development and evaluation of advanced vector surveillance analytic technologies for real-time leishmaniasis risk assessment. Leishmania genus and visceral leishmaniasis causative agent–specific dual fluorogenic-probe hydrolysis (TaqMan), thermally stable (freeze-dried) polymerase chain reaction assays were developed using field-durable analytic instrumentation. In laboratory testing with a panel of diverse Leishmania species from culture and infected sand flies, the sensitivity and specificity of both assays were 100% concordant with DNA sequencing. In specificity testing with Leishmania genetic near neighbors, clinically significant organisms, and human genomic DNA, no detectable fluorescence above background was observed. Field evaluation was conducted in southern Iraq using wild sand flies. In field testing, Leishmania genus assay was 100% sensitive and 96% specific with a single false-positive result. The visceral leishmaniasis genotype assay was 100% sensitive and 100% specific compared to DNA sequencing. Thermally stable polymerase chain reaction assays vastly simplified transportation and storage. Assay preparation and analysis required less than 2 hours. INTRODUCTION Currently, more than 350 million people are at risk for leishmaniasis and 12 million people in 90 countries are infected. Leishmaniasis is ranked among the top 40 diseases in the U.S. Department of Defense global risk-severity index. Leishmaniasis is a zoonotic disease caused by obligate intracellular parasites of the genus Leishmania transmitted to humans through infected sand flies. Primary reservoirs are canids and rodents. The potentially fatal form of the disease, visceral leishmaniasis (VL), is caused by Leishmania donovani complex spp L. donovani and Leishmania infantum in the Old World and Leishmania chagasi in the New World. The nonfatal form of the disease, cutaneous leishmaniasis, is caused primarily by Leishmania tropica and Leishmania major complex spp in the Old World and Leishmania mexicana complex spp and subgenus Viannia complex Leishmania brazilliensis and Leishmania guyanensis spp in the New World. In the absence of a vaccine or prophylactic drug, the only effective means of protecting deployed military personnel from leishmaniasis is to prevent infected sand flies from biting them. Prevention of bites from infected sand flies can be achieved through effective use of personal protective measures (PPM) or by reduction in vector populations. A key tenet of military vector control operations is that the objective is not to merely kill potential vectors but to reduce transmission of the pathogen to deployed military personnel. To effectively reduce transmission, it is critical that insecticides be applied in areas where the risk is greatest (i.e., in those areas where infected vectors are found). Clearly, many factors affect the distribution of leishmaniasis and the associated risk posed to deployed military forces. Likewise, many different methods can be used to assess potential risk. The presence or absence of a key vector, abundance of the vector, and presence of pathogen-specific antibodies in animal reservoirs or in people living in the area of operations can all provide useful information that can help to assess the risk and facilitate the development of targeted control programs. Isoenzyme analysis is normally used to identify Leishmania parasites; however, polymerase chain reaction (PCR) is finding a place as accepted methodology as well. However, these methodologies are designed for instrumentation and reagents intended for use within traditional laboratory and logistical infrastructures and thus are unsuitable in terms of portability, durability, and thermal stability for use in field environments. With the emergence of field-deployable diagnostic assays, early detection of Leishmania parasites in sand fly populations has emerged as an effective method of rapidly assessing risk. The analytic system used in this study was designed specifically for vector surveillance conducted under challenging environmental conditions. The Vector Surveillance Analytic *Division of Entomology, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910. †Molecular Epidemiology, Brooks City-Base, 2730 Louis Bauer Drive, San Antonio, TX 78235. ‡407th Expeditionary Medical Support Hospital, United States Air Force, Tallil Air Base, Iraq. §United States Army Center for Health Promotion and Preventive Medicine-West, 5th & Blaine Street, Fort Lewis, WA 98433-9500. k520th Theater Army Medical Laboratory, United States Army, Tallil