in june 2003 , an outbreak of plague emerged in the oran area of algeria ( 4 ) . during the following weeks , a total of 11 confirmed and all cases were bubonic plague ; septicemia and coma later developed in 2 patients . according to national health records , the last outbreak in oran was in 1946 and the last human cases of plague occurred in algeria in 1950 . the aim of this study was , by using molecular methods , to investigate the presence of y. pestis in fleas collected from rodents . the sites of the original focus of reported plague cases were kehailia ( 3529n , 032e ) and benaouali ( 3533n , 021e ) , in the area of oran and mascara , 450 km west of the capital , algiers ( figure ) . fleas were collected from rodents trapped inside human residences and peridomestic areas within this area ( figure ) from september 2004 to may 2005 by using bts ( besancon technique service , inra , montpellier , france ) and sherman trap ( h.p . specimens were stored in absolute ethanol before being tested in marseille , france , in may 2005 . preliminary morphologic identification was performed ( by i.b . ) by using entomologic taxonomic keys ( 5 ) . identification was confirmed by sequencing regions of siphonapteran 18s rdna , as previously described ( 6 ) . sequences were compared with flea sequences deposited in the 18s rdna database of the whiting laboratory ( 6 ) . ethanol - preserved fleas were rinsed with distilled water for 10 minutes and dried on sterile filter paper in a laminar biosafety hood . fleas were crushed individually in sterile eppendorf tubes with the tips of a sterile pipette . dna was extracted by using the qiaamp tissue kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions . y. pestis dna was detected by real - time pcr with primers against the plasminogen activator ( pla ) gene of y. pestis ( eurogentec , angers , france ) as previously described ( 7 ) . for this assay , negative controls consisted of extracted dna of uninfected fleas from colonies of our laboratory . positive control consisted of a plasmid previously developed in our laboratory for detecting bioterrorism agents ; using this control permitted both control of cycling efficacy and detection of contamination during the pcr process ( 7 ) . to confirm positive results , extracted dna was amplified , and pcr products were sequenced by using 2 alternative spacer targets of y. pestis ( spacers yp8 and yp9 ) as previously described ( 8) . positive sample products were sequenced with an abi 3130xl genetic analyzer ( applied biosystems , coignieres , france ) . sequences were compared with those available in genbank by using the nucleotide - nucleotide blast ( blastn ) program ( available from http://www.ncbi.nlm.nih.gov/blast/ ) together with those of our local database ( 8) . map of the zone where fleas were collected and sites of epidemic plague reported , algeria , june 2003 . ninety five fleas were collected from rodents , including 21 rattus rattus , 13 r. norvegicus , 7 mus musculus , and 8 m. spretus trapped inside the houses and in the peridomestic areas of the cities of kehailia and benaouali ( figure ) . using taxonomic keys , we identified all 95 fleas morphologically as x. cheopis ( the rat flea ) ; fleas index was calculated according to rats or mice , respectively ( 3.333 and 3.125 ) . identity was confirmed by sequencing and comparison of an 1,867-bp informative region of siphonapteran 18s rdna ( 6 ) . using the lightcycler ( lc , roche diagnostics , mannheim , germany ) real - time - pcr assay previously developed for detecting bioterrorism agents targeting the plasminogen activator gene ( 7 ) , we found 20 ( 21.05% ) of 95 fleas positive with a cycle threshold ( ct ) value ranging from 27.2 to 33.91 . among these 20 positive fleas , 9 were also positive in multiple spacer typing ( mst ) assays by using primers targeting spacer yp8 and 8 with primers targeting the spacer yp9 ( table ) . the mean ct value obtained with the lc assay for the 9 fleas positive with the yp8 primers was significantly lower that the mean ct value for the remaining 11 fleas only positive with the lc assay ( 29.56  1.55 ; n = 9 vs. 31.98  1.13 ; n = 11 ; p = 0.0005 ) ( table ) . sequences of the pcr products obtained with yp8 and yp9 primers were 100% identical to sequences of y. pestis biovar orientalis ( genbank accession nos . * examined by lightcycler ( lc ) and multiple spacer typing ( mst ) assays . ct , cycle threshold ; sd , standard deviation ; nd , not done . p<0.05 between mean ct values of fleas positive with lc assay only and fleas positive with mst and lc assays . in this study we present molecular evidence of y. pestis in 20 x. cheopis fleas collected in the area of oran , algeria . the molecular methods used in our study have been previously validated ( 7,8 ) , and precautions were taken to reduce risks for contamination during processing . rieux , the hero of albert camus ( 9 ) in " la peste , " aimed to relate the events of the plague outbreak in oran in the 1940s with the highest objectivity . he stated that " the virus " of plague can come back 1 day and he asked to be aware when it did . apparently plague has retired but is waiting in numerous foci and could reemerge , as it did in india during the 1990s . the " comeback " of plague in the region of oran occurred in june 2003 . in this study , we detected y. pestis in rodent fleas collected from september 2004 to may 2005 in the same area as those plague cases occurred . the persistence of zoonotic foci of plague is worrying since persons living in these areas remain in close contact with rodents and fleas . despite the absence of new cases since june 2003 surveillance should be maintained to monitor this natural focus and potential spread resulting from climatic or habitat influences ( 10 ) . a strong case could be made to extend surveillance to adjacent countries such as libya and mauritania , which also have natural foci of plague , according to the world health organization . in conclusion we believe that detection of y. pestis in fleas can be a useful tool for epidemiologic surveillance of plague in specific settings and could thus serve to study the risk for reemergence of the disease .