Seroprevalence of Human Hantavirus Infection in the Ribeirao Preto Region of Sao Paulo State, Brazil.

To the Editor: Hantavirus pulmonary syndrome (HPS) has been identified in the region of Ribeirao Preto, Sao Paulo State, Brazil, since 1993 (1-4). As of September 1999, 38 HPS cases had been reported in Brazil, 16 in the state of Sao Paulo (2). Between May 1998 and August 1999, the Adolfo Lutz Institute (ALI) in Sao Paulo city serologically confirmed five cases--three fatal--in the Ribeirao Preto region: two from Guariba, one from Jardinopolis, one from Cajuru, and one from Cassia dos Coqueiros (Luiza Teresinha Madia de Souza, ALI, pers. comm.).

Despite these reports and suspicions of additional cases, the prevalence of hantavirus infection and HPS in the region is not known. Laboratory confirmation has not been available locally, and sending serum samples to ALI for laboratory evaluation is not feasible in most cases. Thus, only presumptive diagnoses could be made until the Sin Nombre virus (SNV) enzyme-linked immunosorbent assay (ELISA) was developed.

To estimate the occurrence and distribution of human hantavirus infection, we used SNV ELISA to conduct a serologic survey of a sample of hospital patients requiring venipuncture for routine procedures. The patients came from three regional cities: Ribeirao Preto, Guariba, and Jardinopolis. Between February and June 1999, a total of 567 samples were collected: 257 from the public hospital of Guariba, 110 from the public hospital in Jardinopolis, and 200 from the General Hospital of the School of Medicine of Ribeirao Preto. When we compared our sample with the general population, the patients in the study sample were slightly older but similar in sex distribution.

Sixteen additional samples were evaluated to confirm the effectiveness of SNV ELISA in diagnosing hantavirus infection: 12 from patients in whom HPS was clinically suspected and 4 previously confirmed by ALI in the city of Sao Paulo between May 1998 and August 1999. Known HPS convalescent-phase plasma provided by the Centers for Disease Control and Prevention (CDC) was used as positive control. Negative controls were selected by simple random sampling from all previously negative samples.

Positive and negative recombinant SNV antigens provided by CDC were coated on microtiter plates at 1:2,000 dilution in phosphate-buffered saline overnight at 4 [degrees] C and used in a standard immunoglobulin G testing format. Reverse transcriptase-polymerase chain reaction analysis of serum from two fatal cases of HPS occurring in the cities of Franca and Araraquara suggested the presence of two genetically distinct hantaviruses in the area surrounding Ribeirao Preto. Antigen prepared from local virus is not considered to be necessary for immunoassays because the local virus is not sufficiently different from other isolates to require special antigen preparation (5).

All samples were screened in duplicate on both positive and negative antigens in the assay. A sample was considered positive if absorbance on the positive antigen was greater than absorbance on both the negative control antigen and the negative control of the plate. To confirm the diagnosis, samples satisfying these criteria were tested in duplicate along with 14 negative samples. Samples were considered positive when their subtractive absorbance was greater than the calculated mean subtractive absorbance of the 14 negative samples and three standard deviations.

From our serologic survey, the seroprevalence of human hantavirus infection was determined to be 1.23% (7/567) overall, 0.5% (1/200) in Ribeirao Preto, 0.4% (1/257) in Guariba, and 4.5% (5/110) in Jardinopolis. If one assumes the inhabitants sampled were representative, the seroprevalence provides an estimate of surviving past or recent hantavirus infections in the area. As the overall antibody prevalence of 1.23% is more than twice that observed in the U.S. populations at risk for hantavirus infection, such infections are not rare in the Ribeirao Preto region (6).

Three of the four HPS samples previously confirmed by the ALI in Sao Paulo tested positive by our ELISA. Of the remaining 12 suspected HPS cases assayed, three were positive. Two of these three were later confirmed as positive by the ALI (Luiza Teresinha Madia de Souza, ALI, pers. comm.) Thus, we report three previously unconfirmed HPS cases, one fatal, in the Ribeirao Preto area between May 1998 and August 1999.

Since the rodent reservoir is not known and the virus has not been isolated, rodent capture is currently being conducted in areas where human infections have been found. In addition, positive cases are being retrospectively investigated.

Thanks to Thomas Ksiazek for providing recombinant SNV antigen and positive serum and to Robert Shope for his assistance.

The study was partially funded by grants from the National Institutes of Health, Bethesda, Maryland, USA, and Fundcao de Amparo a Pesquisa do Estado de Sao Paulo, Brazil.

References

(1.) Figueiredo LTM, Moreli ML, Almeida VSO Felix PR, Bruno, JC, Ferreira IB, et al. Hantavirus pulmonary syndrome (HPS) in Guariba, SP, Brazil. A report of 2 cases. Rev Inst Med Trop Sao Paulo 1999; 41:131-7

(2.) Johnson AM, de Souza LTM, Ferreira IB, Pereira LE, Ksiazek TG, Rollin PE, et al. Genetic investigation of novel hantaviruses causing fatal HPS in Brazil. J Med Virol 1999;59:527-35.

(3.) Ferreira MS, Nishioka SA, Santos TL, Santos RP, Santos PS, Rocha A. Hantavirus pulmonary syndrome in Brazil: clinical aspects of three new cases. Rev Inst Med Trop Sao Paulo 2000;42:41-6.

(4.) Da Silva MV, Vasconcelos MJ, Hidalgo NT, Veiga AP, Canzian M, Marotto PC, et al. Hantavirus pulmonary syndrome: report of the first three cases in Sao Paulo, Brazil. Rev Inst Med Trop Sao Paulo 1997;39:231-4.

(5.) Peters CJ. Emerging infections: hantavirus pulmonary syndrome in the Americas. Washington: ASM Press; 1998.

(6.) Zeitz PS, Butler JC, Cheek JE, Samuel MC, Childs JE, Shands LA, et al. A case-control study of hantavirus pulmonary syndrome during an outbreak in the southwestern United States. J Infect Dis 1995;171:864-70.

Ryan Holmes,(*) Rodrigo Boccanera,([dagger]) Luiz Tadeu M. Figueiredo,([dagger]) Silvia Regina Mancano,([double dagger]) Clodoaldo Pane([sections])

(*) University of Texas Medical Branch at Galveston, University of Texas School of Public Health at Houston, Texas, USA; ([dagger]) Unidade Multidepartamental de Pesquisa em Virologia da Faculdade de Medicina de Ribeirao Preto-USP, Brazil; ([double dagger]) Centro de Saude de Guariba, Sao Paulo State, Brazil; and ([sections]) Centro de Saude de Jardinopolis, Sao Paulo State, Brazil.