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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

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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.


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Background of leishmaniasis to military forces deployed to Iraq and Afghanistan

  • 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 of leishmaniasis to military forces deployed to Iraq and Afghanistan

  • 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 of leishmaniasis to military forces deployed to Iraq and Afghanistan

  • 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) of leishmaniasis to military forces deployed to Iraq and Afghanistan

  • 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) of leishmaniasis to military forces deployed to Iraq and Afghanistan

Sorting

Sand flies

Collecting Sand flies

Testing Sand flies

Sorting Sand flies


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Sand fly Surveillance (3) of leishmaniasis to military forces deployed to Iraq and Afghanistan

Summary of Results to Date


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Sand fly Surveillance (4) of leishmaniasis to military forces deployed to Iraq and Afghanistan

  • 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


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Sand fly Surveillance (5) of leishmaniasis to military forces deployed to Iraq and Afghanistan

  • Sand fly Collection Sites

  • 5 sites in Afghanistan

  • Fair distribution throughout country

  • Relatively low numbers


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  • What are these? of leishmaniasis to military forces deployed to Iraq and Afghanistan

    • 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


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Sand fly Surveillance (7) of leishmaniasis to military forces deployed to Iraq and Afghanistan

  • 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) of leishmaniasis to military forces deployed to Iraq and Afghanistan


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Sand fly Surveillance (9) of leishmaniasis to military forces deployed to Iraq and Afghanistan

  • 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) of leishmaniasis to military forces deployed to Iraq and Afghanistan

  • 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) of leishmaniasis to military forces deployed to Iraq and Afghanistan

  • 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 of leishmaniasis to military forces deployed to Iraq and Afghanistan

  • 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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? of leishmaniasis to military forces deployed to Iraq and Afghanistan

Navy

Army

Air Force


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