A multiplex PCR test based on the simultaneous amplification of two lipoprotein genes, oprI and oprL, was designed and evaluated for its ability to directly detect fluorescent pseudomonads (amplification of oprI open reading frame, 249 bp) and Pseudomonas aeruginosa (amplification of oprL open reading frame, 504 bp) in clinical material. A collection of reference strains including 20 different species of fluorescent pseudomonads was tested. Positive PCR results for both genes were observed only for P. aeruginosa isolates (n ‫؍‬ 150), including strains of clinical and environmental origin, while only one gene, oprI, was amplified from the other fluorescent pseudomonads. All other bacteria tested (n ‫؍‬ 15) were negative by the amplification test. The lower detection level for P. aeruginosa was estimated to be 10 2 cells/ml. Preliminary evaluation on testing skin biopsy specimens from patients with burns (n ‫؍‬ 14) and sputum samples from cystic fibrosis patients (n ‫؍‬ 49) and other patients (n ‫؍‬ 19) showed 100% sensitivity and 74% specificity in comparison with culture. This multiplex PCR assay appears promising for the rapid and sensitive detection of P. aeruginosa in clinical specimens. Further evaluation of its specificity in longitudinal clinical studies is warranted.

We evaluated minimal residual disease (MRD) in 23 CD5 + B-chronic lymphocytic leukemia (CLL) patients who achieved clinico-hematological remission confirmed by bone-marrow biopsy. MRD was evaluated by dual marker analysis flow-cytometry using CD5 and CD19 markers, and by the study of Ig heavy chain gene rearrangements using the fast polymerase chain reaction (PCR). According to our laboratory conditions patients were considered to be in complete phenotypic remission when total CD19+ cells were < 25% and the ratio of CD5 + CD19 + /CD19 + cells was < 25%. According to these strict criteria only 9 of the 23 patients were in complete phenotypic remission. In order to evaluate the sensitivity of the above method, PCR analysis of the configuration of the Ig heavy chain gene region was performed in 12 of these patients. Five of 7 patients in complete phenotypic remission retained a detectable monoclonal rearrangement of the Ig heavy chain gene. For the remaining 5 patients in partial phenotypic remission, only one failed to show a monoclonal band and this is probably explained by the presence of an unusual gene rearrangement. In conclusion, this study suggests that PCR is more sensitive than dual marker flow-cytometry for evaluation of residual disease and that it is indeed possible to achieve complete remission at the molecular level, in B-CLL. Nevertheless, we suggest a word of caution as this was a retrospective study, and samples were not assessed before treatment. Thus the possibility that apparent molecular remission might correspond to unusual gene rearrangements cannot be completely excluded in these cases.

Seoul hantavirus (SEOV), carried by Rattus rattus (black rat) and R. norvegicus (Norway, brown rat), was reported to circulate as well as cause HFRS cases in Asia. As Rattus sp. are present worldwide, SEOV has the potential to cause human disease worldwide. In Europe however, only SEOV prevalence in rats from France was reported and no confirmed cases of SEOV infection were published. We here report genetic and serological evidence for the presence of SEOV virus in brown rat populations in Belgium. We also serologically screened an at-risk group that was in contact with R. norvegicus on a daily basis and found no evidence for SEOV infection.

Statement of findingsWe developed a real-time detection (RTD) polymerase chain reaction (PCR) with rapid thermal cycling to detect and quantify Pseudomonas aeruginosa in wound biopsy samples. This method produced a linear quantitative detection range of 7 logs, with a lower detection limit of 10 3 colony-forming units (CFU)/g tissue or a few copies per reaction. The time from sample collection to result was less than 1 h. RTD-PCR has potential for rapid quantitative detection of pathogens in critical care patients, enabling early and individualized treatment. Introduction: Early diagnosis of wound colonisation or prediction of wound sepsis provides an opportunity for therapeutic intervention. There is need for qualitative and quantitative tests that are more rapid than bacterial culture. Pseudomonas aeruginosa results in high morbidity and mortality rates, is inherently resistant to common antibiotics, and is increasingly being isolated as a nosocomial pathogen. We developed three PCR-based methods to detect and quantify P aeruginosa in wound biopsy samples: conventional PCR, enzyme-linked immunosorbent assay (ELISA)-PCR, and RTD-PCR with rapid thermal cycling (LightCycler™ technology), all based on the amplification of the outer membrane lipoprotein gene oprL. We compared the efficacy of these methods to bacterial culture by quantitatively measuring levels of P aeruginosa in serial dilutions, in reconstituted skin samples and 21 burn wound biopsy samples. Materials and methods: Serial 10-fold dilutions were made from an overnight P aeruginosa culture and plated out onto Luria-Bertani and cetrimide agar plates. The agar plates were incubated overnight at 37°C, and the colonies were counted in order to estimate the number of CFU per dilution tube. A sample was taken from each dilution tube as a template for the three PCR-based methods. Serial P aeruginosa dilutions (see above) were added to uninfected cadaveric skin. The reconstituted biopsy samples were homogenized using a tissue tearer and DNA was extracted using XTRAX DNA buffer. The DNA was resuspended in distilled water. A sample was taken as a template for the PCR-based methods. Twenty-one burn wound biopsy samples were taken from nine patients with suspected P aeruginosa burn wound infection. The biopsy samples were longitudinally divided into two pieces. From one piece, DNA was extracted (using XTRAX DNA buffer) and used as a template for PCR-based techniques (see above). The other piece was homogenized, in physiological water, using a tissue tearer. Serial 10-fold dilutions of the suspension were spread on Luria-Bertani and cetrimide agar plates. Colony counts were performed after overnight incubation at 37°C. The PCR mixture contained sterile distilled water, PCR buffer, deoxynucleotide mixture or digoxigenin labelling mix, MgCl 2 , diluted template, primers PAL1 and PAL2, and AmpliTaQ DNA polymerase. The amplification was performed in a GeneAmp ® http://ccforum.com/content/4/4/255 PCR System 2400. An aliquot of the reaction mixture was put on an agaros...

In order to determine the habitat preferred by Myodes (before Clethrionomys) glareolus and the corresponding Puumala hantavirus seroprevalence in those habitats, we captured rodents simultaneously in three significantly different habitats. We compared trapping success and presence of virus per habitat during an ongoing epidemic in order to test the hypothesis of a density-dependent seroprevalence. Our study showed that bank vole population density, as well as Puumala virus seroprevalence, were habitat dependent. Apodemus sylvaticus was found more vulnerable for deteriorating habitat conditions than M. glareolus and could play a role as vehicle for Puumala virus and as mediator for inter- and conspecific virus transmission.