A new molecular method for rapid etiological diagnosis of sepsis with improved performance.

The value of blood cultures for confirming the clinical diagnosis of sepsis is suboptimal. There is growing interest in the potential of real-time PCR technology by detection of minute amounts of pathogen DNA in patient blood samples with results available within 4-6 h. Adopting a two-step approach, we evaluated the compliance of two versions of the MicrobScan assay on a total of 748 patients with suspected bloodstream infections. The results obtained with a second version of the MicrobScan assay are characterized by increased specificity (from 95.1 to 98.2%) and sensitivity (from 76.7 to 85.1), increased throughput and the possibility of simultaneously testing different kinds of samples collected from the potential sites of infection and utilizing different syndromic panels.

Evaluation of the new Sysmex UF-5000 fluorescence flow cytometry analyser for ruling out bacterial urinary tract infection and for prediction of Gram negative bacteria in urine cultures.

BACKGROUND: We evaluated the new flow cytometer UF-5000 with a blue semiconductant laser as a screening tool for ruling out urine samples negative for UTI and its ability to predict Gram negatives in culture. METHODS: Flow cytometry and microbiological analysis were performed on 2719 urine samples, sent to our microbiology laboratory with a request for urine culture. RESULTS: UF-5000 showed a very good performance in the screening process. Carryover and cross-contamination was negligible. 797 samples were culture positive at a cut-off of ≥105CFU/mL. ROC curve analysis for BACT count demonstrated AUC between 0.973, on 2714 samples, 0.959, on 1516 female samples, and 0.988 on 1198 male samples, respectively. At the cut-off of BACT ≥58/µL AND/OR YLC ≥150/µL, SE was 99.4%, SP 78.2%, PPV 65.4% and NPV 99.7%; false negatives were 0.6%, avoiding unnecessary cultures in 55.5% of specimens. "Gram Neg?" flag predicted Gram negatives in culture with a SE of 81.6% and SP of 93.3%. CONCLUSION: The new Sysmex UF-5000 showed high diagnostic accuracy in UTI-screening with a very low rate of false negatives. The instrument is capable of predicting Gram negatives with a good SE and a high agreement with the culture, even if this performance needs further evaluation.

Technical note: Discard the specimen collection swab directly at point of care to improve extensive automated processing in clinical microbiology laboratories.

We compared, in terms of microorganisms recovery, the discard of specimen collection swab, after swirling into its medium, directly at point of care, with its placing into the medium and vortexing on arrival in the laboratory. Our results show that these two procedures are overlapped in terms of bacterial recovery.

[Experimental evaluation of the Sysmex UF-1000i for ruling out non-gonococcal urethritis]. / Utilizzo sperimentale di Sysmex UF-1000i nello screening di uretrite non gonococcica.

Acute nongonococcal urethritis (NGU) is one of the commonest sexually transmitted infections affecting men and women. The diagnosis of NGU has traditionally required microscopic evidence of urethritis. However, a significant proportion of patients with urethral symptoms do not have microscopic evidence of urethritis. The purpose of the present study was to evaluate the analytical performance of the UF1000i, a recently introduced fluorescence flow cytometer intended for urinalysis purposes which provides new analytical features that seem particularly suitable for microbiological diagnostics, for ruling out NGU or predicting the presence of infection. The Sysmex UF1000i is a flow cytometry analyzer capable of quantifying a lot of particles, including bacteria (BACT) and white blood cells (WBCs). To evaluate the analytical performance of the UF1000i as a method for ruling out NGU, we examined 200 urethral smear samples, collected in a new liquid transport medium (Copan), and compared the UF1000i results with standard culture/molecular and microscopic Gram stain results. With instrument cut-off values of 200 BACT x 10^6/L and 500 WBCs x 10^6/L, we obtained a sensitivity of 84%, a specificity of 82%, and a high negative predictive value (96%). Culture/molecular detection of pathogens remains the gold standard technique for the diagnosis of NGU. However, the Sysmex UF1000i is capable of improving the efficiency of NGU presumptive diagnosis, providing results in a few minutes, with a high negative predictive value and high values of sensitivity.

Evaluation of the Sysmex UF1000i flow cytometer for ruling out bacterial urinary tract infection.

BACKGROUND: Urine culture is one of the most frequently requested tests in microbiology, and it represents the gold standard for the diagnosis of UTIs. Considering the high prevalence of negative results and the long TAT of the culture test, the use of a rapid and reliable screening method is becoming more and more important, as it reduces the workload, the TAT of negative results, and above all, unnecessary antibiotic prescription. METHODS: The Sysmex UF1000i is a new urine flow cytometry analyzer capable of quantifying urinary particles, including BACT, WBCs, and YLCs. To evaluate the analytical performance of the UF1000i as a method for ruling out UTIs, we examined 1349 urine samples and compared the UF1000i results with standard urine culture results. RESULTS: With instrument cut-off values of 170BACTx10(6)/L and 150WBCsx10(6)/L, we obtained a sensitivity of 98.8%, a specificity of 76.5%, a NPV of 99.5%, and four false negative results (1.2%), avoiding the culture of 57.1% of samples. CONCLUSION: The Sysmex UF1000i was capable of improving the efficiency of a routine microbiology laboratory by processing 100samples/h and providing negative results in a few minutes, thus reducing unnecessary testing with an acceptable number of false negative results. In addition, the preliminary evaluation of B_FSC and B_FLH parameters from bacteria histograms seems to be useful for the distinction of bacterial strains detected (Gram-negatives versus Gram-positives). In fact when B_FSC was less than 30 ch, it allowed the distinction of Gram-negative bacteria in 97% of the samples.

Molecular identification of bloodstream pathogens in patients presenting to the emergency department with suspected sepsis.

The rapid detection of pathogens in blood is critical for a favorable outcome of patients with suspected sepsis. Although blood culture (BC) is considered the criterion standard for diagnosis of bloodstream infection, it often takes several days to detect the causative organism. In this study, we compared BC with a commercially available multiplex real-time polymerase chain reaction (PCR) assay to detect bacteria and fungi in blood samples from 144 patients admitted to the emergency department with suspected sepsis. Of 144 blood samples examined, 91 (63%) were negative by both methods and 53 (37%) were positive by at least one of the two methods. In 30 among all positive cases (56.6%),both methods identified the same organisms, in 13 cases (24.5%), BC identified organisms not detected by real-time PCR,and in 10 cases (18.9%), SeptiFast PCR assay gave positive results, whereas the BC was negative. In this study, we wished to compare SeptiFast results obtained by standard procedures, but future clinical studies are necessary to define SeptiFast PCR as support for BC in the early diagnosis of severe bloodstream infections.

Direct antifungal susceptibility testing of positive Candida blood cultures by sensititre YeastOne.

Invasive candidiasis is associated with high morbidity and mortality. Differences in the virulence and susceptibility of the various Candida spp. to antifungal drugs make the identification and rapid MIC determination very important for clinical management. The aim of this study was to improve the turnaround time (TAT) for antimicrobial test generation by susceptibility testing directly from the bottle of blood culture positive for yeasts, circumventing the isolation process and thereby generating an accurate antifungal MIC determination as quickly as possible. Sensititre YeastOne was used by direct inoculation from positive blood culture bottles in 40 cases of candidaemia. All the results were compared with those obtained using standard laboratory procedures after subculturing from a positive bottle onto solid media. The results obtained from direct inoculation of Sensititre YeastOne compared with tests carried out using standard procedures show that out of a total of 40 strains tested no very major errors or major errors and only 4 minor errors occurred (98% agreement rate out of a total of 240 drug/bug combinations tested), thus generating an accurate antifungal MIC determination and saving an average time of 24 hours compared with the time required for the standard procedures traditionally used.

Improving the efficiency and efficacy of pre-analytical and analytical work-flow of urine cultures with urinary flow cytometry.

Quantitative urine culture is one of the most frequently requested tests in microbiology laboratories. An automated system for screening purposes is strongly needed to save technical staff time and obtain rapid results. Our study investigated 1.047 urine samples collected from inpatients and outpatients with a commercial vacutainer system (Becton Dickinson, Milan, Italy) and compared a second-generation flow cytometry (Sysmex UF100, Dasit, Cornaredo, Italy) with standard urine culture tested on agar plated by means of 10 microliter loop. The specimens were screened and cultured on receipt. The results obtained with Sysmex UF-100 are very interesting, especially if this analyzer is used as a screening method for negative urine samples, and comparable to data obtained from culture examination. In fact, considering together bacteria and leukocyte count (> 4500 bacteria and/or > 50 leukocytes/microL) we obtained a negative predictive value of 99.5% in comparison with the standard culture method. The classical culture method needs 24 hours for a result, whereas the Sysmex UF-100 analyzer gives results in a few minutes, thus reducing the microbiology turn around time (TAT) with obvious benefits for patients and physicians.