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 .
 after an outbreak of human plague , 95 xenopsylla cheopis fleas from algeria were tested for yersinia pestis with pcr methods . 
 nine fleas were definitively confirmed to be infected with y. pestis biovar orientalis . 
 our results demonstrate the persistence of a zoonotic focus of y. pestis in algeria . 
The Study
Conclusions