Evaluation of rapid diagnostic test for influenza

In high risk patients such as in eldery, newborns and immunosuppressed subjects, a timely diagnosis of influenza is required for the most appropriate antiviral strategy in order to avoid severe secondary respiratory complications and viral spreading. Influenza is preventable by vaccination and chemoprophylaxis and is treatable by specific antiviral indications. The need for a timely diagnosis has led to the introduction of numerous rapid diagnostic tests.These are mostly antigen detection test giving results within 30 minutes, a clinically relevant time-frame to complement with the use of antiviral medications or chemoprophylaxis strategy. When evaluating performances of rapid test for influenza viruses, it is important to consider the type and quality of specimen to be tested, as well as sensitivity and specificity of the assays. Nasal/nasopharyngeal swabs are the most frequently submitted specimens, but nasal/nasopharingeal aspirates and washs can improve the diagnostic sensitivity of the test. Only some rapid assays can be successful used with broncoalveolar washings. In this review,we evaluated the sensitivity, specificity, reproducibility and feasibility of the most currently licensed rapid tests for influenza virus A and B. A flow-chart for the laboratory diagnosis of influenza with rapid test in combination with confirmatory test is proposed

Ruolo del sequenziamento diretto nella tipizzazione di HCV-RNA a fini clinico-epidemiologici

Background/Aims: Genotyping and subtyping of hepatitis C virus (HCV) is epidemiologically and clinically relevant to prognosis and therapeutical management of HCV infection.Aim was to study the feasibility of an “in house” direct sequencing method for the genotyping and subtyping of HCV strains compared with a commercially available genotyping assay (Inno-LiPA,VERSANT® HCV Genotype 2.0 Assay, Bayer). Methods: 74 clinical plasma samples cross-representing HCV genotypes 1 to 5 typed with the line probe assay Inno-LiPA (LiPA) were subjected to a laboratory-developed 5’UTR direct sequencing protocol (5’UTR Seq).A NS5B direct sequencing protocol (NS5B Seq) was apply to 23/74 samples (17 negative by the 5’UTR).Two libraries of 5’UTR and NS5B HCV prototypes were constructed; BioEdit 7.0.0 and ClustalW were used for alignments and phylogenetic analysis. Results: 5’UTR Seq typed 47/74 LiPA positive samples (64%). Concordance between the two assays was 91% (43/47) and 72% (31/43) for HCV genotyping and subtyping, respectively. LiPA and 5’UTR Seq discordant samples were typed by NS5B Seq. NS5B Seq typed 13/17 samples negative with 5’UTR Seq.All genotypes and subtypes detected with NS5B Seq were concordant with LiPA. 5’UTR and NS5B direct sequencing pointed out a wider HCV subtype distribution than LiPA for genotype 1, 2 and 4. By the combination of 5’UTR and NS5B direct sequencing, 81% (60/74) of LiPA positive samples were typed. Conclusion: HCV typing and subtyping with the line probe assay Inno-LiPA is highly recommended for clinical purposes.A more detailed HCV typing for epidemiologic purposes can by achieved by the direct sequencing of a region with a moderate degree of genetic variability such as NS5B. HCV NS5B analysis is more efficient in resolving viral strain genetic variability than direct sequencing of a highly conserved region such as 5’UTR

Antimicrobial Activity of Euplotin C, the Sesquiterpene Taxonomic Marker from the Marine Ciliate Euplotes crassus

Strains of the marine ciliate protist Euplotes crassus produce exclusive terpenoids called euplotins that play an ecological role. Among these derivatives, euplotin C is the main of four secondary metabolites isolated from cultures of this protozoon and represents the sesquiterpene taxonomic marker from E. crassus. Because different terpenoid metabolites of plant origin showed a certain antimicrobial activity, we assessed the compound euplotin C, purified by high-pressure liquid chromatography and solubilized in two solubility enhancers, against the protozoa Leishmania major and Leishmani infantum, the fungus Candida albicans, and nine strains of gram-positive and gram-negative microorganisms. An activity of euplotin C against Leishmania promastigotes was demonstrated (50% lethal doses were 4.6 or 8.1 μg/ml depending on the agent used to solubilize the compound), while the effect was less evident on Candida and nearly absent on bacteria. A nonsignificant cytotoxicity (50% lethal dose, >200 μg/ml) against the J774 cell line was observed. A leishmanicidal activity was also shown by the living, euplotin-producing cells of E. crassus cultured together with promastigotes; this activity increased with time from 10 min to 6 h of incubation. This study provides an initial rationale for the evaluation of euplotin C and other similar natural products as alternative or possibly synergistic compounds for current antiprotozoon chemotherapeutics

Real-time ed end-point Polymerase Chain Reaction per la quantizzazione del DNA di Citomegalovirus: confronto tra metodi e con il test per l’antigene pp65

Quantitave Polymerase Chain Reaction (PCR) for Cytomegalovirus (CMV) DNA provides highly sensitive and specific data for detecting CMV as well as monitoring the infection and determining the appropriate antiviral strategy.To determine the clinical application of a recently introduced real-time (RT) PCR assay for CMV DNA quantitation in peripheral blood leukocytes (PBLs) and defining its correlation with the commercial quantitative end-point (EP) PCR method COBAS AMPLICOR CMV Monitor and pp65 antigen test. Sequential PBL samples (n=158) from 32 liver transplanted patients with CMV asymptomatic infection and positive for CMV DNA by EP-PCR were retrospectively analysed with RT-PCR and studied according to pp65 antigen levels. A good correlation was found between RT-PCR and pp65 antigen test (r=0.691) and between the two PCR assays (r=0.761). RT-PCR data were significantly higher in pre-emptive treated patients (those with >20 pp65+positive cells, median value: 3.8 log10 copies/500,000 PBLs) than in not-treated ones (2.9 logs).According to pp65 levels of 0, 1-10, 11-20, 21-50, 51-100 and >100 positive cells/200,000 PBLs, median CMV DNA load by RT-PCR was 2.6, 3.0, 3.6, 4.0. 4.2 and 4.8, log10 copies/ 500,000 PBLs, respectively (EP-PCR CMV DNA levels: 2. 8, 2.9, 3.8, 3.7, 3.9 and 4.0 logs). For samples with >20 pp65+cells, that is above the level at which pre-emptive therapy was started, RT-PCR values were significantly higher than in groups with less than 20 pp65+cells, whereas EP-PCR values did not significantly differ and showed a slower progression rate. Dilutions of DNA from CMV AD169 strain were used to probe RT-PCR reproducibility (between and intra-assay variability < 2%) and sensitivity (100% detection rate at 10 copies/reaction, 28.5% with EP-PCR). A significant improvement is coming from the introduction of RT-PCR to the study of CMV DNA dynamics in differently CMV infected patients due to a more reliable quantitation of CMV DNA for moderate and high DNA level compared to EP-PCR with better sensitivity and specificity. RTPCR gives more precise informations on viral load kinetics for evaluating the infection progress and assessing antiviral response, significantly simplifying and accelerating the process of producing a reliable quantification of CMV DNA for clinical purposes

Real-time PCR per HBV DNA: valutazione del nuovo sistema automatizzato COBAS AMPLIPREP™/COBAS TAQMAN™ HBV

Success of antiviral therapy for chronic hepatitis B is supported by highly sensitive PCR-based assays for Hepatitis B virus (HBV) DNA. Nucleic acid extraction from biologic specimens is technically demanding and reliable PCR results depend it. Performances of the fully automatic system COBAS AmpliPrep™/COBAS TaqMan™ 48 (CAP/CTM) (Roche, Branchburg, NJ) for HBV DNA extraction and real -time PCR quantification were assessed and compared with the end-point PCR COBAS AMPLICOR HBV Monitor (CAHBM, Roche). Analytical evaluation with a proficiency panel showed that CAP/CTM quantitated HBV DNA levels in one single run over a wide dynamic range (7 logs) with a close correlation between expected and observed values (r=0.976, interassay variability below 5%). Clinical evaluation as tested with samples from 92 HBsAg-positive patients, demonstrated excellent correlation with CAHBM (r=0.966, mean difference in quantitation: 0.36 log10 IU/ml). CAP/CTM detected 10% more viremic patients and longer period of residual viremia in those on therapy. In lamivudine (LAM)-resistant patients, reduction of HBV DNA after 12 months of Adefovir (ADF) was higher in the combination (LAM+ADF) schedule than in ADF monotherapy (5.1 vs. 3.5 logs) suggesting a benefit in continuing LAM. In conclusion,CAP/CTM can improve the management of HBV infection, the assessment of antiviral therapy and drug resistance, supporting further insights in the emerging area of drug resistance

COBAS AmpliPrep-COBAS TaqMan Hepatitis B Virus (HBV) Test: a Novel Automated Real-Time PCR Assay for Quantification of HBV DNA in Plasma

Success in antiviral therapy for chronic hepatitis B is supported by highly sensitive PCR-based assays for hepatitis B virus (HBV) DNA. Nucleic acid extraction from biologic specimens is technically demanding, and reliable PCR results depend on it. The performances of the fully automatic system COBAS AmpliPrep-COBAS TaqMan 48 (CAP-CTM; Roche, Branchburg, NJ) for HBV DNA extraction and real-time PCR quantification were assessed and compared to the endpoint PCR COBAS AMPLICOR HBV monitor (CAHBM; Roche). Analytical evaluation with a proficiency panel showed that CAP-CTM quantitated HBV DNA levels in one single run over a wide dynamic range (7 logs) with a close correlation between expected and observed values (r = 0.976, interassay variability below 5%). Clinical evaluation, as tested with samples from 92 HBsAg-positive patients, demonstrated excellent correlation with CAHBM (r = 0.966, mean difference in quantitation = 0.36 log(10) IU/ml). CAP-CTM detected 10% more viremic patients and longer periods of residual viremia in those on therapy. In lamivudine (LAM)-resistant patients, the reduction of HBV DNA after 12 months of Adefovir (ADF) was higher in the combination (LAM+ADF) schedule than in ADF monotherapy (5.1 logs versus 3.5 logs), suggesting a benefit in continuing LAM. CAP-CTM detected HBV DNA in liver biopsy samples from 15% of HBsAg-negative, anti-HBcAg-positive graft donors with no HBV DNA in plasma. The amount of intrahepatic HBV DNA was significantly lower in occult HBV infection than in overt disease. CAP-CTM can improve the management of HBV infection and the assessment of antiviral therapy and drug resistance, supporting further insights in the emerging area of occult HBV infection