LTC Russell E. Coleman, Ph.D. Director, Entomology Division Walter Reed Army Institute of Research

1. Assessment of sand flies as a means of evaluating the threat of leishmaniasis to military forces deployed to Iraq and Afghanistan LTC Russell E. Coleman, Ph.D. Director, Entomology Division Walter Reed Army Institute of Research Opinions, interpretations, conclusions, & recommendations are those of the author and are not necessarily endorsed by the U.S. Army.

2. Background • Leishmaniasis is transmitted by phlebotomine sand flies; • There are no vaccines or prophylactic drugs that can protect deployed military personnel from the disease; • Best protection is to prevent infected sand flies from biting susceptible personnel;

3. U.S. Military in OIF/OEF • To date, >2,000 cases of leishmaniasis in U.S. military personnel deployed to Iraq and Afghanistan. All but 5 were cutaneous leishmaniasis due to L. major; 5 were visceral leishmaniasis. • In 2003/2004, most cases sent to WRAMC/BAMC for treatment. More recently, many cases not definitively diagnosed and/or not treated.

4. Preventive Medicine Support • Protection from biting sand flies is the responsibility of: • Individual Soldier: Must use Personal Protective Measures (use of DEET-containing insect repellent on the skin, wear of permethrin-treated uniforms, and sleeping under a permethrin-treated insect net); • Preventive Medicine Personnel: Ability to conduct surveillance for sand flies and to control them using pesticides; Level of PVNTMED Support Soldier: Uses PPM to protect self Company: 2-person Field Sanitation Team (additional duty) BDE/DIV: Full-time PVNTMED Section with 1 ESO, 1 (BDE) or 4-5 (Div) technicians, limited control capabilities PvntMed Unit: 11-12 Personnel, with 1 ESO, 1 entomologist, 10 technicians, excellent surveillance/control capabilities

5. Sand fly Surveillance (1) • In 2003 established a Sand-fly Surveillance Program: • Assess threat of leishmaniasis in Iraq and Afghanistan; • Provide PVNTMED Personnel with information on threat in their AO so that they can implement control measures as necessary; • Program Elements: • PVNTMED Units collect sand flies and ship to WRAIR • WRAIR test sand flies using Real-time PCR and conducts sequencing to determine species; • Threat maps developed and feedback provided to PVNTMED Units;

6. Sand fly Surveillance (2) Sorting Sand flies Collecting Sand flies Testing Sand flies Sorting Sand flies

7. Sand fly Surveillance (3) Summary of Results to Date

8. Sand fly Surveillance (4) • Sand fly Collection Sites • 27 sites in Iraq • Good distribution throughout • much of country except western desert 1,000 250 10,000 500 500 10,000 3,000 3,000 14,000 500 6000 3000 82,000

9. Sand fly Surveillance (5) • Sand fly Collection Sites • 5 sites in Afghanistan • Fair distribution throughout country • Relatively low numbers

10. What are these? • False Positives • Below detection limit of species-specific assays • Other species of Leishmania Sand fly Surveillance (6) • Total of 737 sand fly pools positive using Leishmania-generic assay • Retested using L. major and visceral (L. infantum/donovani) specific assays. • 14/569 (2.5%) L. major positive • 7/305 (2.3%) visceral positive • Remaining (~95%) negative

11. Sand fly Surveillance (7) • Sequencing of PCR-positive samples to determine species; • 360 BP region of Glucose 6-Phosphate Isomerase (GPI) gene; • 606 samples evaluated: • 1 (0.2%) L. major • 2 (0.3%) L. major/tropica • 24 (4.0%) L. infantum/donovani (visceral – Southern Iraq) • 252 (42%) L. tarentolae (“Lizard Leishmania”) • 258 (43%) Anopheles-like GPI • 69 (11%) no sequence

12. Sand fly Surveillance (8)

13. Sand fly Surveillance (9) • Because so many positive samples were “Lizard Leishmania” we decided to determine if sorting sand flies to genus (Phlebotomus versus Sergentomyia) prior to testing would be useful; More testing needed!

14. What does this Mean? (1) • L. major appears rare in sand flies; however, is the causative agent in >99% (>2,000) of U.S. military personnel. L. major- specific Real-time PCR assay good for sand fly testing; • L. infantum is rare in U.S. military personnel (only 5 cases from Iraq/Afghanistan); however, is relatively common in sand flies from Southern Iraq. Suggests we may have a significant number of asymptomatic cases. Visceral Real-time PCR assay appears good for sand flies; • L. tarentolae (“Lizard Leishmania“) is common in sand flies. Must develop a method of rapidly differentiating medically-important Leishmania sp. from unimportant species;

15. What does this Mean? (2) • High % of PCR-positive samples that yield a “Anopheles-like” sequence is a concern. Testing of un-infected sand flies yields same results, suggesting this is a sand fly related sequence. We believe that Leishmania were present in these samples; however, at levels that did not yield a good sequence

16. Bottom-Line • Require the following improvements in ability to rapidly test sand flies for Leishmania parasites: • Better species-specific assays (L. tropica, L. tarentolae) • Assay that can separate medically-important from un-important species • Confirmatory assays • Better understanding of parasite loads in sand flies (so we can relate PCR results to whether the sand fly can transmit the parasite). • Field-deployable assays similar to the Malaria VecTEST assay

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