In vitro evaluation of antiviral and virucidal activity of a high molecular weight hyaluronic acid.

BACKGROUND: hyaluronic acid (HA), a non-sulphated glycosaminoglycan, is present in synovial fluid, vitreous humour serum and many connective tissues. Pharmaceutical preparations of HA are used in clinical practice for wound healing, joint pain, kerato-conjunctivitis, asthma, mouth care, oesophageal-reflux, and gastritis. Moreover, it is used as a filler to counteract ageing and facial lipoatrophy. Our study aims at investigating the in vitro antiviral activity of a high molecular weight HA.METHODS: the MTT test was used to rule out the potential toxic effects of HA on the different cell lines used in the antiviral assays. The antiviral activity of HA against Coxsackievirus B5, Herpes simplex virus-1, Mumps virus, Adenovirus-5, Influenza Virus A/H1N1, Human Herpesvirus-6, Porcine Parvovirus, Porcine Reproductive and Respiratory Syndrome Virus was assessed by virus yield assays.RESULTS: the most effective inhibition was observed against Coxsackievirus B5, with 3Log reduction of the virus yield at 4mg/ml, and a reduction of 3.5Log and 2Log, at 2mg/ml and 1mg/ml, respectively: the selectivity index was 16. Mumps virus was highly inhibited too showing a reduction of 1.7Log at 1mg/ml and 1Log at 4mg/ml and 2mg/ml (selectivity index = 12). The selectivity index for Influenza Virus was 12 with the highest inhibition (1Log) observed at 4mg/ml. Herpes simplex virus-1 and Porcine Parvovirus were mildly inhibited, whereas no antiviral activity was observed with respect to Adenovirus-5, Human Herpesvirus-6, Porcine Reproductive and Respiratory Syndrome Virus. No HA virucidal activity was ever observed against any of the viruses tested. Kinetic experiments showed that both Coxsackievirus B5 and Herpes simplex virus-1 replication were consistently inhibited, not influenced by the time of HA addition, during the virus replication cycle.CONCLUSIONS: the spectrum of the antiviral activity exhibited by HA against both RNA and DNA viruses, known to have different structures (with or without envelope) and replication strategies, suggests a non specific mechanism of action, probably involving cell membrane-virus interaction steps. The results of the kinetic experiments support this hypothesis

Wide spectrum activity of a high molecular weight Hyaluronic Acid.

Background Hyaluronic acid (HA), a non-sulphated glycosaminoglycan, is present in synovial fluid, vitreous humour and many connective tissues. Pharmaceutical preparations of HA are used in clinical practice for wound healing, joint pain, kerato-conjunctivitis, asthma, mouth care, oesophageal-reflux, and gastritis. Moreover, it is used as a filler to counteract aging and facial lypoatrophy. Here we investigated the antiviral activity in vitro of a high molecular weight (1,8KD) HA used in aesthetic medicine.Methods The MTT test was used to investigate the cytotoxicity of HA on the different cell lines used for virus growth: VERO, MDCK, PK15, JJHAN, MARC145. With the aim of investigating whether HA may affect cell membrane stabilization, experiments were carried out in which VERO cells were pre-treated with HA and then exposed to two lysis solutions, HCl and Triton X-100. The antiviral activity of HA was assessed by viral yield assays against Coxsackievirus B5 (COXB5), Herpes simplex virus-1 (HSV-1), Mumps virus (MV), Adenovirus (ADV), Influenza Virus (WSN33-AH1N1), Human Herpesvirus-6 (HHV-6), Porcine Parvovirus (PPV), Porcine Respiratory Reproductive Syndrome Virus (PRRSV). The virucide activity was assessed by exposing the different viral inocula to HA for 30’ at room T° and then their residual infectivity was titrated on cells. Time course experiments were carried out with COXB5 and HSV-1 within a replication single cycle by adding. HA at different time points.Results The MTT test showed that HI displayed no significant toxicity up to 4mg/ml. In lysis experiments, HA was able to protect VERO cells from lysis by both TritonX-100 and HCl. HSV-1, COXB5, MV, WSN33, PPV were inhibited by HA, the most effective inhibition being observed against COXB5 (3,5 Log reduction) and MV (1.7Log reduction). No virucidal activity by HA was ever observed against any of the viruses tested. Kinetic experiments showed that both COXB5 and HSV-1 were consistently inhibited independently upon the time of HA addition during the virus replication cycle. Conclusions The wide spectrum of antiviral activity exhibited by HA against both RNA and DNA viruses, known to have different structures (with or without envelope) and replication strategies, suggests a non specific mechanism of action, likely involving cell membrane-virus interaction steps either in virus entry or exit. The results of the kinetic experiments and of the cell protection from lysis support this hypothesis

Clinical performance of a commercial real-time PCR assay for Aspergillus DNA detection in serum samples from high-risk patients : comparison with a galactomannan enzyme immunoassay

We investigated the clinical performance of a polymerase chain reaction (PCR)-based commercial platform, the Myconostica MycAssay\u2122 Aspergillus (MAP), for fungal DNA detection in the serum of patients at risk of invasive aspergillosis (IA). Sixty-four hospitalized patients were prospectively enrolled and a total of 71 different episodes were investigated (30 episodes were clinically/microbiologically classified as IA and 41 as control episodes). When MAP was compared to the galactomannan (GM) assay, no significant differences were found in terms of sensitivity (46.7 % vs. 50.0 %), specificity (97.6 % vs. 95.1 %), positive predictive value (PPV) (93.3 % vs. 88.2 %), and negative predictive value (NPV) (71.4 % vs. 72.2 %). The corresponding areas under the curve (AUC) of the receiver operating characteristic (ROC) curves were also superimposable. Overall, because of the good agreement between the two assays and considering the high specificity and PPV of the MAP, we suggest the use of this PCR-based platform as a second-level examination for the evaluation of clinically undefined cases where culture or GM have provided positive results

Evaluation of serum (1 → 3)-β-D-glucan clinical performance: kinetic assessment, comparison with galactomannan and evaluation of confounding factors

Purpose We investigated the clinical performance of (1 → 3)-β-d-glucan (BG), as an early marker of invasive fungal infections (IFI), in different clinical settings. Methods BG serum levels were assessed by Fungitell (Associates of Cape Cod, Inc), in parallel with galactomannan (GM) when requested by clinicians. By a prospective monocentric study, 270 episodes at risk or with suspect of IFI were enrolled, namely 58 proven-probable invasive aspergillosis (IA), 27 proven invasive candidiasis (IC), 11 possible IC, 16 P.jirovecii pneumonia (PJP), 4 episodes of other IFI and 154 non-IFI controls. Results We found that (a) the BG overall sensitivity, specificity, positive predictive value and negative predictive value (NPV) were 87.9, 80.5, 76.7 and 89.9 %, respectively; (b) the highest sensitivity was found in the IC groups, followed by PJP, IA and other IFI groups; (c) an association was observed between BG kinetics and patients outcome; (d) in the IA episodes, the combination of BG or GM vs GM alone increased sensitivity from 60.0 to 83.3 % in the haematological patients; (e) false-positive BG results were related to Gram-negative infections or infusion of polyclonal IgM-enriched immunoglobulins, where high levels of BG were indeed detected. Conclusion Besides strengthening its overall good clinical performance, we provide evidence that serum BG correlates with clinical outcome and that, once used in combination with GM, BG allows to enhance IFI diagnosis rate. The high sensitivity and NPV, observed in the Intensive Care Unit setting, open to BG validation as a marker for assessment of antifungal treatment