LTC Russell E. Coleman, Ph.D.Director, Entomology Division

Walter Reed Army Institute of Research

Assessment of sand flies as a means of evaluating the threat of leishmaniasis to

military forces deployed to Iraq and Afghanistan

Opinions, interpretations, conclusions, & recommendations are those of the author and are not

necessarily endorsed by the U.S. Army.

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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;

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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.

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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

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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;

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Sand fly Surveillance (2)

Collecting Sand flies

Sorting Sand flies

Sorting Sand flies

Testing Sand flies

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Sand fly Surveillance (3)

Summary of Results to Date

Total All 162,890 6,857 58,973 737 1.25 4,544 3,297 156 6,250 14,247

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Sand fly Surveillance (4)

82,000

3000

500

14,0003,000

3,00010,000

10,000

500

6000

500

1,000250Sand fly Collection Sites • 27 sites in Iraq• Good distribution throughout

much of country except western desert

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Sand fly Surveillance (5)

Sand fly Collection Sites • 5 sites in Afghanistan• Fair distribution throughout

country• Relatively low numbers

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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 positive7/305 (2.3%) visceral positiveRemaining (~95%) negative

• What are these?False PositivesBelow detection limit of species-specific assaysOther species of Leishmania

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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

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Sand fly Surveillance (8)

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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!

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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;

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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

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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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?Air Force Army Navy