ltc russell e. coleman, ph.d. director, entomology division walter reed army institute of research

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