<em>Background</em> <em>and</em> <em>Aims</em>. Bacterial meningitis and sepsis are medical emergencies where tests with a high sensitivity and short turn around time (TAT) are crucial for an early targeted therapy. Aim of this study was the evaluation of an optimal diagnostic strategy for infectious meningitis/sepsis management, assessing seven methods: cerebrospinal fluid (CSF) physical-chemical examination, CSF cultural tests (CCT), Gram stained smears (GSS), CSF latex agglutination test (CLAT), blood culture (BC), Real-Time (RT)-PCR and FilmArray Technology (FAT) performed directly on CSF/blood. <br /><em>Materials</em> <em>and</em> <em>Methods</em>. Samples of CSF (240), blood (180) and cavitary fluids (9) were tested by commercial RT-PCR (Eurospital and Liferiver kits) and traditional methods. Positive samples (BC and RT-PCR) were tested by FAT (Blood Culture Identification Panel, Biofire, Salt Lake City, UT, USA) performed directly on CSF, blood and cavitary fluids. <br /><em>Results</em>. In CSF, GSS, CLAT, CCT, RT-PCR and FAT sensitivity was 41%, 35%, 41%, 100% and 62,5%, respectively. In blood, BC, RT-PCR and FAT sensitivity was 96%, 70% and 44%, respectively. TAT was 48-96 hrs, 3 hrs and 1 hr and NPV was 98%, 89% and 57%, respectively. <br /><em>Conclusions</em>. For sepsis management, RT-PCR is faster than BC (3 hrs vs 24-72 hrs), but limited by a low overall sensitivity (70%), due to the low number of detectable pathogens; FAT, performed directly on positive BC should replace biochemical identification (Vitek 2, Biomérieux Marcy-l’Étoile, France) reducing TAT (1 hr <em>vs</em> 12 hrs). For meningitis management, RT-PCR is the most sensitive and rapid method used in routine and emergency regimen. It is cost effective and it represents the gold standard for diagnosis and follow-up of patients under treatment. For meningitis management, FAT, with a higher sensitivity and rapidity and an easier and objective interpretation, should replace CLAT and GSS in emergency regimen.
SummaryPre-analytical and post-analytical evaluation in the era of molecular diagnosis of sexually transmitted diseases: cellularity control and internal control.Background. Increase of molecular tests performed on DNA extracted from various biological materials should not be carried out without an adequate standardization of the pre-analytical and postanalytical phase.Materials and Methods. Aim of this study was to evaluate the role of internal control (IC) to standardize pre-analytical phase and the role of cellularity control (CC) in the suitability evaluation of biological matrices, and their influence on false negative results. 120 cervical swabs (CS) were pre-treated and extracted following 3 different protocols. Extraction performance was evaluated by amplification of: IC, added in each mix extraction; human gene HPRT1 (CC) with RT-PCR to quantify sample cellularity; L1 region of HPV with SPF10 primers. 135 urine, 135 urethral swabs, 553 CS and 332 ThinPrep swabs (TP) were tested for C. trachomatis (CT) and U. parvum (UP) with RT-PCR and for HPV by endpoint-PCR. Samples were also tested for cellularity.Results. Extraction protocol with highest average cellularity (Ac)/sample showed lowest number of samples with inhibitors; highest HPV positivity was achieved by protocol with greatest Ac/PCR. CS and TP under 300.000 cells/sample showed a significant decrease of UP (P<0.01) and HPV (P<0.005) positivity. Female urine under 40.000 cells/mL were inadequate to detect UP (P<0.05).Conclusions. Our data show that IC and CC allow optimization of pre-analytical phase, with an increase of analytical quality. Cellularity/sample allows better sample adequacy evaluation, crucial to avoid false negative results, while cellularity/PCR allows better optimization of PCR amplification. Further data are required to define the optimal cut-off for result normalization.
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