BACKGROUND
candida spp.
is now the fourth most frequent cause of nosocomial blood-stream infections in critically ill patients and candida.
albicans is the most prevalent species  <cit> .
prevalence of other species such as candida tropicalis, candida glabrata, candida parapsilosis and candida krusei varies according to clinical conditions  <cit> .
rapid isolation and identification of pathogenic yeasts to species level is critical for treatment.
conventional diagnostic procedures, such as blood culture and biochemical tests lack the degree of sensitivity and specificity that would ensure reliable and early diagnosis of invasive candida infections  <cit> .
as a consequence, methods based on the amplification and detection of yeast dna have been developed.
conventional pcr techniques and now real-time pcr assays are performed for specific and rapid detection and identification of fungi from clinical isolates  <cit> .
however, as yeast load is often lower than  <dig> cfu per ml of blood, even in patients suffering from invasive yeast infection  <cit> , sensitivity is the major drawback with these techniques.
sensitivity can be improved by coupling pcr method with other methods, such as hybridization assay  <cit> , by using nested  <cit>  or by optimising dna extraction methods  <cit> .
however availability of pure dna without pcr inhibitors is essential.
another approach is to concentrate yeast cells before culture or dna extraction using techniques such as the immunomagnetic separation method  where magnetic beads coated with monoclonal antibodies are used to capture yeast cells present in clinical specimens.
the purpose of this study was to evaluate the usefulness of ims in improving culture yields.
the effectiveness of the ims was determined and the time to obtain colonies of candida species was compared to that of the bactec mycosis-ic/f automated blood culture system.
human blood artificially contaminated with c. albicans, c. tropicalis which are the most frequently strains isolated in candidemia was used.
c. glabrata and c. krusei were also included in the study because of their reduced susceptibility i.e.
higher resistance to fluconazole.
RESULTS
analytical sensitivity of ims
percentages of viable yeast cells captured by beads were calculated in relation to counts of viable cells initially present in inoculated blood and expressed for  <dig> ml of blood .
it was observed that yeast cells of all strains studied could be recovered by ims, but with variable recovery rates.
<dig> % and  <dig> % for the two strains of c. albicans , 38% for the two c. glabrata isolates  and about 15% for the two c. tropicalis isolates .
it was moreover noted that immunoseparation recovery rates were more variable for c. krusei with  <dig> % for isolate  <dig> and  <dig> % for isolate  <dig>  a possible explanation for the lower recovery rate of isolate  <dig> is that the antigen recognised by mab 6b <dig> might have been expressed at lower level in this strain.
nevertheless, ims appears as an easy method allowing yeast cells immunocapture in less than one hour and colonies of the four candida species tested after  <dig> h of culture on sda-c. if ims is coupled with a culture on chromogenic medium, it can allow the differentiation and presumptive identification of the pathogen within  <dig> hours.
c. parapsilosis was not tested because it is not well recognised by the mab 5b <dig>  the introduction of beads coated with other specific mabs  in the mix is expected to result in a technique for concentrating all major pathogenic candida species in blood
acalculation of mean and sd is derived from three different experiments
bpercentage of immunocaptured cells: number of cfu per  <dig> ml of blood after ims procedure/number of cfu per  <dig> ml of blood in initial contaminated blood
cfu: colony forming unit, ims : immunomagnetic separation, sd : standard deviation
ims method in comparison with conventional procedures
the ims method has several advantages over conventional methods: it is easy to use, no special equipment is required, processing time of ims is shorter.
in our study, with the automated blood culture method, microbial growth could be detected for all strains of candida after an incubation of about  <dig> h .
however, the viable colonies needed to perform identification were only obtained after subsequent sub-culturing and this required a further 24-h incubation.
despite efforts made by many investigators, early and rapid diagnosis of systemic yeast infections remains limited.
culture detection of candida species is often delayed or remains negative because of slow or absent growth of yeast isolates from clinical specimens.
blood cultures are positive for fewer than 50% of patients with invasive candidiasis  <cit> .
then, time to obtain colonies is shorter with ims, saving at least  <dig> h in comparison with blood culture.
of course, further optimization should seek for best ims recovery rates to increase its lower limit of detection.
in addition, as demonstrated by other authors, ims could increase the efficiency of culture or the yield of nucleic acid before a dna extraction.
pcr assays that amplify a highly conserved sequence of the multicopy rrna gene were developed using dna extracts from blood specimens.
<dig> to  <dig> cfu/ml has been reported as a lower limit for dna detection by pcr.
it was shown that ims combined with real-time pcr to detect plasmodium falciparum in blood samples  <cit> , or bacteria such as escherichia coli 0157:h <dig> and listeria monocytogenes in food  <cit> , or some viruses  from environmental samples or food  <cit>  is particularly attractive because of the potential for concentrating micro-organisms.
therefore ims, in conjunction with pcr, considered as one of the most sensitive methods to detect low levels of dna from pathogens in clinical samples, could be a helpful tool in the diagnosis of candidemia.
CONCLUSIONS
in our study, ims has been used to capture yeast cells from artificially contaminated human blood using magnetic beads coated with monoclonal antibodies  specific to surface antigens of candida.
when numbers of cfu ranged from  <dig> to  <dig> cfu per ml of blood, ims allowed immunocapture for the four candida species studied.
we are fully aware that it is a study using artificially contaminated blood and another study is now in progress to evaluate what really happens in natural candidemia.
ims should be useful in order to facilitate the isolation of yeasts from blood saving at least  <dig> h to obtain colonies before classic identification.

 candida spp. is now the fourth most frequent cause of nosocomial blood-stream infections in critically ill patients and candida. albicans is the most prevalent species. prevalence of other species such as candida tropicalis, candida glabrata, candida parapsilosis and candida krusei varies according to clinical conditions. rapid isolation and identification of pathogenic yeasts to species level is critical for treatment. conventional diagnostic procedures, such as blood culture and biochemical tests lack the degree of sensitivity and specificity that would ensure reliable and early diagnosis of invasive candida infections. as a consequence, methods based on the amplification and detection of yeast dna have been developed. conventional pcr techniques and now real-time pcr assays are performed for specific and rapid detection and identification of fungi from clinical isolates. however, as yeast load is often lower than cfu per ml of blood, even in patients suffering from invasive yeast infection, sensitivity is the major drawback with these techniques. sensitivity can be improved by coupling pcr method with other methods, such as hybridization assay, by using nested or by optimising dna extraction methods. however availability of pure dna without pcr inhibitors is essential. another approach is to concentrate yeast cells before culture or dna extraction using techniques such as the immunomagnetic separation method where magnetic beads coated with monoclonal antibodies are used to capture yeast cells present in clinical specimens. the purpose of this study was to evaluate the usefulness of ims in improving culture yields. the effectiveness of the ims was determined and the time to obtain colonies of candida species was compared to that of the bactec mycosis-ic/f automated blood culture system. human blood artificially contaminated with c. albicans, c. tropicalis which are the most frequently strains isolated in candidemia was used. c. glabrata and c. krusei were also included in the study because of their reduced susceptibility i.e. higher resistance to fluconazole.  analytical sensitivity of ims percentages of viable yeast cells captured by beads were calculated in relation to counts of viable cells initially present in inoculated blood and expressed for ml of blood. it was observed that yeast cells of all strains studied could be recovered by ims, but with variable recovery rates. % and % for the two strains of c. albicans, 38% for the two c. glabrata isolates and about 15% for the two c. tropicalis isolates. it was moreover noted that immunoseparation recovery rates were more variable for c. krusei with % for isolate and % for isolate a possible explanation for the lower recovery rate of isolate is that the antigen recognised by mab 6b might have been expressed at lower level in this strain. nevertheless, ims appears as an easy method allowing yeast cells immunocapture in less than one hour and colonies of the four candida species tested after h of culture on sda-c. if ims is coupled with a culture on chromogenic medium, it can allow the differentiation and presumptive identification of the pathogen within hours. c. parapsilosis was not tested because it is not well recognised by the mab 5b the introduction of beads coated with other specific mabs in the mix is expected to result in a technique for concentrating all major pathogenic candida species in blood acalculation of mean and sd is derived from three different experiments bpercentage of immunocaptured cells: number of cfu per ml of blood after ims procedure/number of cfu per ml of blood in initial contaminated blood cfu: colony forming unit, ims: immunomagnetic separation, sd: standard deviation ims method in comparison with conventional procedures the ims method has several advantages over conventional methods: it is easy to use, no special equipment is required, processing time of ims is shorter. in our study, with the automated blood culture method, microbial growth could be detected for all strains of candida after an incubation of about h. however, the viable colonies needed to perform identification were only obtained after subsequent sub-culturing and this required a further 24-h incubation. despite efforts made by many investigators, early and rapid diagnosis of systemic yeast infections remains limited. culture detection of candida species is often delayed or remains negative because of slow or absent growth of yeast isolates from clinical specimens. blood cultures are positive for fewer than 50% of patients with invasive candidiasis. then, time to obtain colonies is shorter with ims, saving at least h in comparison with blood culture. of course, further optimization should seek for best ims recovery rates to increase its lower limit of detection. in addition, as demonstrated by other authors, ims could increase the efficiency of culture or the yield of nucleic acid before a dna extraction. pcr assays that amplify a highly conserved sequence of the multicopy rrna gene were developed using dna extracts from blood specimens. to cfu/ml has been reported as a lower limit for dna detection by pcr. it was shown that ims combined with real-time pcr to detect plasmodium falciparum in blood samples, or bacteria such as escherichia coli 0157:h and listeria monocytogenes in food, or some viruses from environmental samples or food is particularly attractive because of the potential for concentrating micro-organisms. therefore ims, in conjunction with pcr, considered as one of the most sensitive methods to detect low levels of dna from pathogens in clinical samples, could be a helpful tool in the diagnosis of candidemia.  in our study, ims has been used to capture yeast cells from artificially contaminated human blood using magnetic beads coated with monoclonal antibodies specific to surface antigens of candida. when numbers of cfu ranged from to cfu per ml of blood, ims allowed immunocapture for the four candida species studied. we are fully aware that it is a study using artificially contaminated blood and another study is now in progress to evaluate what really happens in natural candidemia. ims should be useful in order to facilitate the isolation of yeasts from blood saving at least h to obtain colonies before classic identification. 
candida species have become the fourth most-frequent cause of nosocomial bloodstream infections in immunocompromised patients. thus, this easy to use method provides a promising basis for concentrating all candida species in blood to improve sensitivity before identification. conventional diagnostic procedures such as blood culture or biochemical tests are lacking both sensitivity and species specificity, so development of rapid diagnostic is essential. in comparison with an automated blood culture, processing time of immunomagnetic separation is shorter, saving at least hours to obtain colonies before identification. therefore, rapid identification of pathogenic fungi to species level has been considered critical for treatment. an immunomagnetic method involving anti-candida monoclonal antibodies was developed to capture and concentrate in human blood four different species of candida cells responsible for invasive yeast infections.
 candida species have become the fourth most-frequent cause of nosocomial bloodstream infections in immunocompromised patients. therefore, rapid identification of pathogenic fungi to species level has been considered critical for treatment. conventional diagnostic procedures such as blood culture or biochemical tests are lacking both sensitivity and species specificity, so development of rapid diagnostic is essential.  an immunomagnetic method involving anti-candida monoclonal antibodies was developed to capture and concentrate in human blood four different species of candida cells responsible for invasive yeast infections. in comparison with an automated blood culture, processing time of immunomagnetic separation is shorter, saving at least hours to obtain colonies before identification.  thus, this easy to use method provides a promising basis for concentrating all candida species in blood to improve sensitivity before identification. 