Incidence Rate for Hantavirus Infections without Pulmonary Syndrome, Panama

During 2001–2007, to determine incidence of all hantavirus infections, including those without pulmonary syndrome, in western Panama, we conducted 11 communitywide surveys. Among 1,129 persons, antibody prevalence was 16.5%–60.4%. Repeat surveys of 476 found that patients who seroconverted outnumbered patients with hantavirus pulmonary syndrome by 14 to 1.

I n the Americas, hantavirus species that occur at low frequency are associated with the severe disease hantavirus pulmonary syndrome (HPS) (1,2), and species that occur at higher frequency are associated with milder disease (3)(4)(5). In Panama, HPS is caused by the Choclo virus, for which a common rodent, the fulvous pygmy rice rat (6), is host. Serum antibody prevalence against this virus is 3%-33% in neighborhoods where HPS cases have occurred (7) and 16%-45% according to selected communitywide surveys (8). Neutralization-inhibition assays of antibody-positive serum indicated past infections caused by Choclo virus (9). To obtain a more accurate incidence of hantavirus infections in Panama, we conducted repeat surveys to identify hantavirus seroconversions during 1-to 3-year intervals between surveys. Our goal was to compare incidence of seroconversion with that of concurrent HPS in the same communities.

The Study
During 2001-2007, a total of 4 communities (3 in Los Santos Province and 1 in Veraguas Province) within hantavirus-endemic agroecosystems in western Panama were sampled 2-4 times at 1-to 3-year intervals ( Table  1). Informed written consent was obtained from all adult participants and from parents or legal guardians of minors. Consent and assent forms were reviewed and approved by institutional ethics review boards at the University of New Mexico, the Gorgas Memorial Institute in Panama City, and the protocol review committee of the International Centers for Infectious Diseases Research program of the National Institute of Allergy and Infectious Diseases. Eligible participants were all adults and children >2 years of age who permanently resided in each community according to the 2000 national census. The reasons for noninclusion in the fi rst and subsequent surveys were absence during the week of the survey and refusal to participate.
A questionnaire administered to the head of household asked for a history of respiratory-related illnesses and hospitalizations within the past 3 years. Venous blood was collected from all family members for serologic testing. Results of the surveys were provided to each participating community through community meetings. Surveillance for HPS was conducted in the same communities as the serosurvey and nationally through reports to the Ministry of Health, and cases of HPS were confi rmed by questionnaire. The diagnosis of HPS required fi nding immunoglobulin (Ig) M in acute-phase serum, detection of Choclo virus RNA in serum by reverse transcription PCR, typical respiratory signs and symptoms, and chest radiographic fi ndings compatible with pulmonary edema.
Heparinized whole blood collected by arm venipuncture was separated by centrifugation; plasma was stored at −20°C until analysis. In binding assays, antibody to all known hantaviruses indigenous to the Americas cross-react with the N protein of Sin Nombre virus (10). A strip immunoblot assay for IgG containing recombinant N protein of the 3H226 genotype of Sin Nombre virus was used as described (10); the criterion for positivity was a dark band for Sin Nombre N protein at a serum dilution of 1:200. An enzyme immunoassay used recombinant nucleocapsid protein from Sin Nombre virus (11); the cutoff value was established at 3 SD above reactivity to a panel of known positive serum. All samples were tested by both assays; the concordance of the enzyme immunoassay and strip immunoblot assay in this study was 97%, and the criterion for seropositivity was a positive reaction in both assays. Loss of antibody in persons with previously positive serum was determined by 2 independent tests with both assays. IgM against hantavirus was not tested. In Panama, all HPS patients tested have had positive reverse transcription PCR results for Choclo genomic RNA in acute-phase blood samples (9), and antibody has been detected by both assays.
Data were transferred from fi eld collection forms to a database (Epi Info version 6.04d, Centers for Disease Control and Prevention, Atlanta, GA, USA) for statistical analyses using Epi Info software. Changes in seroprevalence within each community were tested by Fisher exact test for each interval and by longitudinal analysis for all intervals and communities by a generalized estimating equation (12). Increases in seroprevalence according to community and year of survey were tested by using analysis of covariance.
The 11 surveys repeatedly sampled 60%-85% of the total population of each community, for a total of 1,838 samples from 1,129 persons. Overall antibody prevalence was 32.9%, varying from 16.5% to 60.4% in individual surveys (Table 1). In each of the 3 Los Santos communities (Agua Buena, Isla Cañas, San Jose), seroprevalence increased annually by ≈5% (Table 1); the overall seroprevalence increases for the combined Los  Santos communities were signifi cant (Fisher exact test, p = 0.0014). The changes in seroprevalence were community specifi c (analysis of covariance F = 5.24, p = 0.0043), but increases in seroprevalence in the 4 communities combined was not (general estimating equation). Among the study population, seroconversion was documented for 70 persons, and HPS was diagnosed for 5 other persons in the same communities during the intervals studied (Table 2). In the cohort of 476 persons in all 3 Los Santos communities sampled in 2 back-to-back surveys ( Table 2), the 70 seroconversions occurred in persons in all age cohorts and equally among persons of both sexes (data not shown). No person who seroconverted gave a history of HPS-like illness or hospitalization for an acute respiratory illness. A separate study of outpatients with febrile illnesses was conducted in 4 clinics in the hantavirusendemic area. This study found 48 adults and children with the typical febrile prodrome, unremarkable chest radiographs, and either serum IgM specifi c for hantavirus nucleoprotein or Choclo virus genomic RNA in the acutephase blood sample (B. Armien and J.M. Pascale, unpub. data). These fi ndings of symptomatic hantavirus infections confi rm previous observations derived from neutralizationinhibition antibody assays (9).
The incidence of 70 seroconversions in 857 personyears of observation (Table 2) was equivalent to 8 infections per 100 person-years. The ratio of infection detected by seroconversion to infection resulting in HPS was 14:1. The mean ages of persons who seroconverted (43 years) and those with HPS (43 years) were the same. Undercounting of HPS cases was not likely because HPS is a highly publicized illness throughout Panama, and diagnostic serologic testing is readily available through the Ministry of Health.
A total of 16 seroreversions, compared with 70 seroconversions, occurred among persons in most age cohorts, mean age 48 years. For HPS caused by Sin Nombre and Andes viruses, serum antibody typically persists for years (13). Serum antibodies after mild or asymptomatic infections may not persist for many years.

Conclusions
Antibody prevalence surveys are useful for identifying populations and locations at risk, monitoring changes in incidence, and focusing limited public health resources. Determining whether the observed increases in seroprevalence will be sustained requires additional surveys, but this information will be useful as the new agroeconomy increasingly emphasizes the monoculture of products (rice and sugar cane) favorable to rodents (14). Nonetheless, the documentation of large numbers of mild or asymptomatic hantavirus infections not progressing to HPS has identifi ed a larger effect of this zoonotic disease.