Probable dengue virus infection among Italian troops, East Timor, 1999-2000 - Dispatches

Emerging Infectious Diseases, July, 2003 by Mario Stefano Peragallo, Loredana Nicolletti, Florigio Lista, Raffaele D'Amelio

To investigate the attack rate and risk factors for probable dengue fever, a cross-sectional study was conducted of an Italian military unit after its deployment to East Timor. Probable dengue was contracted by 16 (6.6%) of 241 army troops and caused half of all medical evacuations (12/24); no cases were detected among navy and air force personnel.

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Dengue fever (DF), caused by dengue virus (DENV) serotypes 1 to 4, is an emerging public health problem in many tropical countries (1). Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), the severe manifestations of DENV infection, were first recognized in the 1950s in Southeast Asia and are today a leading cause of childhood illness and death in many tropical countries. More recently, DHF and DSS have emerged in Central and South America and in the Pacific region (2,3). DF is also recognized as an emerging health problem for international travelers (4,5) and for troops deployed to tropical countries (6,7).

In 1999, following a United Nations Security Council recommendation, the International Force for East Timor (INTERFET) was formed to restore peace on the island. In November 1999, INTERFET troops totaled 11,000 from 17 countries. The Italian Armed Forces contributed 640 soldiers.

DF is endemic in East Timor. The peak transmission periods for DF are July-August and December-January, corresponding to the rainy months (8). In 1998, at least 11% of hospital inpatient deaths in East Timor were attributed to DHF (9). In October 1999, a localized outbreak of DF in a western district was attributed to serotype 3 (9) and serotype 2 was isolated in December 1999 (10). Serotypes 2 and 3 were also responsible for DF cases among Australian troops returning from East Timor in January--February 2000 (11).

During deployment, a high attack rate of febrile illness consistent with DF was reported among Italian troops. A seroepidemiologic survey was therefore conducted in February 2000 among soldiers returning home, in an attempt to determine the cause of this outbreak and to define infection rates and risk factors for infection.

The Study

All Italian troops eligible for deployment are routinely vaccinated against diphtheria/tetanus, tetravalent meningococcal meningitis, measles/mumps/rubella, hepatitis A and B, polio (with inactivated virus), typhoid fever (orally), and yellow fever (YF). In this situation, troops were also vaccinated against Japanese encephalitis (JE) (Nakajama strain, 3 doses on days 0, 7, and 14)just before landing in East Timor.

DF prevention consisted of the use of personal protection measures against mosquitoes (repellents applied to the skin; permethrin-treated bed nets and uniforms) along with environmental mosquito control. Adulticide spraying was conducted weekly by pesticide-dispersal units but only within the campsite and in its nearest surroundings, which were also inspected daily to reduce or eliminate breeding sites of vectors.

Italian troops were deployed in East Timor from late September 1999 to mid-February 2000, and all 640 participating military personnel were eligible for inclusion in the study. Army soldiers were permanently based on the ground and operated in Dili and surrounding areas, while air force and navy personnel had only logistical tasks and their presence in Dili was episodic, since they were mainly aboard ship or based in Darwin (Australia).

A seroepidemiologic survey was conducted February 15-28, 2000, among troops returning to Italy after their 3-month period of duty in East Timor. After informed consent was obtained, peripheral blood specimens were drawn and a written questionnaire administered. The questionnaire asked for personal health data, including all symptoms experienced during deployment and information about compliance with personal protection measures. Immunization status and clinical data concerning febrile illness cases consistent with DF were obtained from standardized records kept by medical personnel. Soldiers and navy/air force personnel were studied according to their serologic status and disease status during deployment.

All specimens were screened for antibodies to dengue virus serotype 2 (DEN-2), yellow fever virus (YVF), and West Nile virus (WNV) by hemagglutination-inhibition test (HI). All serum specimens positive for DEN-2 were tested by neutralization test (NT) for DEN-2. Additionally, serum samples from participants who had experienced an acute clinical syndrome suggestive of DF were directly tested by NT for antibodies to DEN-2. Serum specimens negative for DEN-2 were then tested for neutralizing antibodies to dengue virus serotypes 1, 3, and 4 (DEN-1 DEN-3 and DEN-4).

The HI test was performed by the method of Clarke and Casals (12) and NT as 90% plaque reduction neutralization test (PRNT) on Vero cells. Briefly, serum specimens (twofold dilutions) and virus ([10.sup.2] PFU) were incubated overnight at 4[degrees]C, injected onto monolayers of Vero cells, and overlaid with 1% Tragacanth gum (Sigma-Aldrich S.r.I., Milan, Italy). Seven days postinfection, cells were washed with saline and stained with 1% crystal violet in 20% ethanol (DEN-2 and DEN-3) or by immunodetection assay (DEN-1 and DEN-4) as described (13). Vero cells were propagated in minimum essential medium with Earle's salts (EMEM), supplemented with nonessential amino acids, 10% fetal calf serum, 100 IE/mL of penicillin G, and 100 IE/mL of streptomycin.