The performance of a single-tube nested-PCR (STNPCR) technique was evaluated for plague diagnosis in comparison to conventional (one step) and two step nested PCR (NPCR). Assays were carried out with primers targeting the gene caf1 that encodes the Yersinia pestis F1 antigen. For STNPCR inner primers were immobilized onto the inside of the microtube caps and after the first amplification they were eluted by inversion of the tube. This procedure avoids opening the tube, reducing the risks of false-positive results by cross-contamination. The immobilized primers are stable for several months at -20 degrees C, thus, the tubes can be prepared beforehand and stored until use. STNPCR was more sensitive than conventional PCR, and less sensitive than NPCR. This drawback is compensated by a lower risk of cross-contamination. The experiments with infected animals showed that NPCR and STNPCR were able to produce positive results in all samples tested, despite contamination with other organisms. In contrast, conventional PCR yielded positive results in a smaller number of samples. Three out of 62 culture-negative rodents from plague areas, were positive by STNPCR. In conclusion, the PCR approaches evaluated, particularly NPCR and STNPCR have potential to be used as alternative tools in epidemiological surveys of plague. Furthermore, as the results can be obtained quickly (less than 24 hour), these techniques could be useful in emergency situations in which the rapidity in diagnosis is essential for adoption of immediate measures of control.

Sera from 269 rodents obtained during the routine surveillance operations in plague areas of Rio de Janeiro and Pernambuco states, Brazil were tested by ELISA for specific IgG antibodies against a recombinant nucleocapsid (N) protein of Araraquara hantavirus. ELISA-positive sera were submitted to reverse transcriptase-polymerase chain reaction (RT-PCR) for amplification of the virus genome and later sequencing for identification of the viral variant. The samples from the state of Pernambuco were antibody negative, and although four from Rio de Janeiro were ELISA-positive, they failed to yield viral cDNA by RT-PCR. This is the first report of the presence of antibodies to a hantavirus among rodents from Rio de Janeiro and suggests the possibility of human cases of hantavirus pulmonary syndrome (HPS) in that state, although no case has yet been reported.

RESUMOA peste, infecção pela Yersinia pestis, mantém-se em vários focos naturais na África, Ásia e Américas e atualmente é considerada uma doença reemergente pela Organização Mundial da Saúde 15 24 , constituindo um problema de Saúde Pública. A sua epidemiologia é bastante complexa e a erradicação ainda não é exeqüível, apesar dos avanços científicos e tecnológicos 13 . O seu potencial de epidemização e elevada letalidade justificam a sua inclusão na Classe I do Regulamento Sanitário Internacional (RSI) vigente, que exige notificação compulsória de toda atividade pestosa e manutenção de vigilância permanente nos focos e locais por onde a infecção possa ser introduzida a partir de focos ativos.Vários fatores de virulência da Y. pestis são codificados em genes localizados no cromossomo e nos três plasmídeos prototípicos: pPst, pFra e pYV. O plasmídeo pPst, específico da Y. pestis, codifica uma protease (Pla) ou ativador do plasminogênio. No plasmídeo pFra, também específico, estão localizados os genes que codificam uma proteína de envoltório, a fração antigênica F1,

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