Hand hygiene: virucidal efficacy of a liquid hand wash product against Ebola virus.

BACKGROUND: Hand washing is an important targeted hygiene intervention for limiting the spread of infectious agents, including the Ebola virus, which continues to re-emerge. We have assessed the virucidal efficacy of a commercially available liquid hand wash product (LHW) for inactivating Ebola virus. METHODS: The ASTM E1052-11 Standard was used to evaluate the efficacy of an LHW containing the microbicidal active salicylic acid for inactivating Ebola virus - Makona variant suspended in an organic load. Three concentrations (12.5%, 25%, 50%) of three lots of LHW prepared in 440 ppm hard water were evaluated at room temperature for 20, 30, and 60 s contact time. RESULTS: A 25% solution of the LHW caused 4.5 log10 and 4.8 log10 reduction in Ebola virus titer within 20 and 30 s, respectively. The efficacy of a 12.5% LHW solution was lower (1.9 and 2.0 log10 reduction in titer within 20 and 30 s, respectively). The efficacy of the 50% LHW solution could not be measured, due to inability to sufficiently neutralize the LHW at the end of exposure. CONCLUSION: These results suggest the potential utility of an appropriately formulated liquid hand wash agent during Ebola virus disease outbreaks for use within healthcare, community, and home settings. Such an LHW should also be effective against other enveloped viruses, such as the pandemic coronavirus SARS-CoV-2.

Use of a small-scale, portable test chamber for determining the bactericidal efficacy of aerosolized glycol formulations.

This study aimed to understand the efficacy and mechanisms of action of an aerosolized glycol-ethanol formulations against bacteria. We validated a small-scale in-house test chamber to determine the microbicidal efficacy of four aerosolized formulations combining dipropylene glycol and ethanol against Staphylococcus aureus and Escherichia coli embedded in alginate. The aerosolized glycol/ethanol formulation decreased bacterial viability by 3 log10 and was more efficacious than an ethanol only control formulation. Electron microscopic examination indicated extensive structural damage in both bacteria, and membrane damage was confirmed with potassium release in S. aureus and DNA release in E. coli. The development of a small test chamber facilitated the measurement of the microbicidal efficacy and experiments to understand the mechanism of action of an aerosolized microbicidal formulation. SIGNIFICANCE AND IMPACT OF THE STUDY: There is an increased interest in developing effective microbicidal-aerosolized formulations. The development of a small in-house test chamber allowed the measurement of the microbicidal efficacy of an aerosolized glycol/ethanol formulation at a low cost. We showed that a glycol/ethanol aerosolized formulation caused extensive structural damage in Gram-negative and -positive bacteria resulting in a 3 log10 reduction in viability.

A quantitative method to assess the role of indoor air decontamination to simultaneously reduce contamination of environmental surfaces: testing with vegetative and spore-forming bacteria.

Indoor air can spread pathogens, which can be removed/inactivated by a variety of means in healthcare and other settings. We quantitatively assessed if air decontamination could also simultaneously reduce environmental surface contamination in the same setting. Two types of vegetative bacteria (Staphylococcus aureus and Acinetobacter baumannii), and a bacterial spore-former (Geobacillus stearothermophilus) were tested as representative airborne bacteria. They were separately aerosolized with a Collison nebulizer into a 24-m3 aerobiology chamber and air samples collected with a programmable slit-to-agar sampler. Settling airborne particles were collected on culture plates placed at, and collected from, five different locations on the floor of the chamber with a custom-built remote plate-placement and -retriever system. Experimentally contaminated air in the chamber was decontaminated for 45 min with a device based on HEPA filtration and UV light. The plates were incubated and CFU counted. The device reduced the viability levels of all tested bacteria in the air by >3 log10 (>99·9%) in 45 min. Based on two separate tests, the average reductions in surface contamination for S. aureus, A. baumannii and G. stearothermophilus were respectively, 97, 87 and 97%. We thus showed that air decontamination could substantially and simultaneously reduce the levels of surface contamination in the same setting irrespective of the type of pathogen present. SIGNIFICANCE AND IMPACT OF THE STUDY: The innovative and generic test protocol described can quantitatively assess the reduction in environmental surface contamination from microbial decontamination of indoor air in the same setting. This added advantage from air decontamination has implications for infection prevention and control in healthcare and other settings without the need for additional expense or effort. Continuous operation of an air decontamination device, such as the one tested here, can lead to ongoing reductions in pathogens in air and on environmental surfaces.

Duck hepatitis B photoinactivation bydimethylmethylene blue in RBC suspensions.

BACKGROUND: Dimethylmethylene blue (DMMB) has been used to photoinactivate a number of model viruses, including VSV, in RBC suspensions under conditions that preserve in vitro RBC properties during storage. The relative sensitivity of duck HBV (DHBV) and VSV to photoinactivation by DMMB was investigated by performing an indirect immunofluorescence assay (IFA) using primary duck hepatocyte (PDH) cultures or a standard plaque assay for the respective viruses. STUDY DESIGN AND METHODS: DMMB was added to 45-percent Hct, WBC-reduced, oxygenated AS-3 RBCs at 10-, 1-, and 0.1-microM concentrations. Samples (1-mm thick) were illuminated with 5.4-mW per cm(2) of red light for 2 or 9 seconds. Unilluminated samples without DMMB or with 10 microM DMMB served as control. RESULTS: DHBV and VSV were rapidly photoinactivated by DMMB in a concentration and light-dose-dependent fashion. Neither virus was substantially inactivated by incubation with DMMB in the dark. For a given light exposure, DHBV required a concentration of DMMB one-one hundredth that of VSV to achieve approximately the same level of inactivation. CONCLUSION: DHBV appears to be considerably more sensitive than VSV to DMMB photoinactivation. Photoinactivation in 45-percent Hct RBCs can be achieved in seconds by using micromolar quantities of dye.

Proto-oncogene ras GTPase-linked induction of glutathione-S-transferase by growth factors in PC12 cells.

This report provides evidence linking activation of Ras GTPase by growth factors and induction of glutathione-S-transferase isozymes in PC12 cells. Ras GTPase was activated by EGF, NGF, insulin and phorbolester in PC12 cells. Activation of Ras GTPase was found to be associated with induction of the expression of GST mu and pi isoenzymes while there was no detectable induction of GST alpha expression. GST pi was found to be induced by all the Ras GTPase activating agents tested while activation of Ras by phorbolester and insulin induced expression of GST mu only. These results suggest a role of Ras, at least in part, in controlling the expression of GST and that there might be independent signalling pathways for the expression of different GST isoenzymes. GST activity was found to be very high (4-fold) in the lysate obtained from retinoic acid treated PC12 cells when compared with untreated cells. Induction of GST expression was found to be initiated within 30 min of retinoic acid treatment in PC12 cells reaching a maximum level at 4 h. However, immunoblot analysis showed that retinoic acid (RA), unlike mitogens/growth factors, weakly induced the expression of GST pi but not the expression of alpha, mu and microsomal GSTs. Overxpression of inhibitory polypeptides that block signals generated from Ras and Cdc42 was found to reverse the retinoic acid activation-dependent induction of GST expression in PC12 cells. These results provide evidence for the first time suggesting a novel role of Ras GTPase in the regulation of GST expression which might have a significant implication in developing drug resistance and/or growth of cancer cells.

Inhibition of rotavirus infection in vitro and in vivo by a synthetic peptide from VP4.

A synthetic peptide corresponding to bovine rotavirus C486 (BRV) VP4 amino acid sequence 232-255 (VP4-peptide) was studied with the objective of defining the origin of the protective immune response reported previously by Ijaz et al. (J. Virol. 1991, 65, 3106-3113). Pretreatment of MA-104 cells with the VP4-peptide before infection with rotavirus prevented both the attachment of 35S-labelled virus and plaque formation in vitro. In vivo studies using a murine rotavirus model demonstrated that intragastric administration of VP4-peptide protected subjects from challenge with virulent rotavirus. These results clearly indicate the importance of this epitope in virus-cell interactions and their potential as a rotavirus vaccine candidate.

Comparative studies on the sensitivity of polymerase chain reaction and microscopic examination for the detection of Trypanosoma evansi in experimentally infected mice.

Trypanosoma evansi, a protozoan parasite in the blood of camels is routinely diagnosed by finding the flagellates in the wet films or stained smear of peripheral blood, examined under a microscope. Although specific, this method is not sensitive at early stages of infection. We have tested the use of polymerase chain reaction (PCR) in the identification of T. evansi in different stages of infection in mice and compared its sensitivity with that of the standard microscopic examination method. Using a specific pair of primers, it was possible to identify T. evansi in the blood of infected mice. Experimentally, groups of mice were infected with T. evansi, isolated from a naturally infected local camel and blood samples were collected every day for 30 days post-infection. Direct microscopy or PCR was applied to detect parasitaemia. Results showed that during the acute phase of infection, parasites were detected by PCR three days earlier than by microscopy. Furthermore, the infected mice were consistently positive by PCR during the chronic phase while the parasites could not be demonstrated during this period using microscopic examination. These findings suggest that PCR may be applied to camel trypanosomosis during both acute and chronic phase of infection. Furthermore, it would provide an excellent tool in the evaluation of treatment of anti-trypanocidal drugs.

Respiratory syncytial virus infection among hospitalized young children with acute lower respiratory illnesses in Al Ain, UAE.

To establish the frequency and clinical pattern of Respiratory Syncytial virus (RSV) infection in the region, children under 3 years of age admitted for acute lower respiratory illness during two winter seasons of the years 1993-94 and 1994-95 were studied prospectively. Seventy two cases were diagnosed to have RSV infection among the 252 studied, representing 28.57% of these patients. The overall infection rate was 32.1% and 36.5% respectively for the two studied winter seasons. Among these children, 90% were under 12 months of age. A clinical diagnosis of sepsis and respiratory distress was entertained in five RSV positive cases and they were < 1 month of age. The clinical pattern of RSV infection included bronchiolitis in 58.3% of cases, bronchopneumonia (19.4%) and pneumonia (11.1%). RSV activity was detected throughout the year with predominance during cooler months with an associated relative humidity (RH) between 50-60%. These results indicate that RSV plays a significant etiologic role among ALRI in hospitalized infants and young children in the Oasis region of the UAE. Factors such as RH, environmental temperature and lifestyle probably play an additional role in our region for the maintenance and dissemination of infection around the year.

Characterization of a synthetic peptide mimicking trypsin-cleavage site of rotavirus VP4.

A synthetic peptide corresponding to the trypsin cleavage site on the 84 k protein of bovine rotavirus was synthesized (VP4-peptide). This synthetic peptide could be cleaved by trypsin and therefore possessed the enzyme binding site present on the authentic protein. Further proof that this peptide mimicks the authentic trypsin cleavage site was the specific reaction of anti-peptide serum with the 84 k protein. The reaction of anti-peptide serum with infectious virus neutralized infectivity thereby supporting the biological importance of this site. Another interesting characteristic of this peptide was its ability to bind to the nucleocapsid protein resulting in a laddering effect on the nucleocapsid monomer (45 k), dimer (90 k) and trimer (135 k) [Gorzilia et al., J. Gen. Virol. 66, 1889-1900 (1985); Sabara et al., J. Virol. 53, 58-66 (1985); Sabara et al., J. Gen. Virol. 67, 201-212 (1986)]. Definitive proof of binding was provided by the fact that the increments in the ladder corresponded to the molecular weight of the synthetic peptide and that anti-peptide serum specifically reacted with the ladder formations. The laddering of the nucleocapsid could be eliminated by incubation with trypsin thus further supporting the formation of a synthetic peptide-nucleocapsid complex. Due to the ability of the peptide to bind to trypsin and to the nucleocapsid protein its biological activity was investigated. It appeared that increasing concentrations of the peptide reduced the rate of virus plaque formation, thereby suggesting that virus replication was inhibited. These results illustrate two features of this synthetic peptide which warrant further investigation; (1) its capacity to mimic an enzyme cleavage site and, (2) its ability to complex tightly to another protein. In protection-challenge experiments performed using a murine model, animals immunized with VP4-peptide provided protection passively, to neonates suckling on the immune dams, against a virulent rotavirus. The potential applications of this peptide in rotavirus diagnosis, therapy and synthetic peptides based vaccine is discussed.

Suppression of transplant immunity in experimental trichinellosis.

Skin allograft rejection in Balb/c and C57BL/6J mice following experimental infection with 300 larvae of Trichinella spiralis or Trichinella pseudospiralis was studied. Skin grafts from normal C57BL/6J mice were transplanted to infected Balb/c mice and vice versa at days 3, 10, 20 and 30 post-infection. The clinical criteria for graft rejection, scarring and graft falling, were followed. The results indicated that T. spiralis and T. pseudospiralis infections induced a significant delay in graft rejection when compared to the control groups. A maximum rejection time of 24 days was observed in T. spiralis infected C57BL/6J mice which received skin grafts from Balb/c mice on day 3 post-infection. The rejection in the uninfected control group was on day 7 post transplant. The mean rejection times for transplants on various days post-infection, with both species were very similar. Also, the rejection profiles in Balb/c mice were comparable to that observed in C57BL/6J mice, with a maximum delay of 26 days to rejection again obtained in mice transplanted on day 3 post-infection, for both species. When the skin grafts were performed 5 or 10 days prior to infection, the rejection occurred on day 7, as in the control group. The effect of T. spiralis and T. pseudospiralis soluble larval extracts (TSE or TPE) on graft rejection was also examined. Four intraperitoneal injections of 50 micrograms each of TSE or TPE every 48 h for 7 days did not induce any significant delay in graft rejection. In contrast, secretory antigens prepared from cultured larvae in vitro induced significant delays in graft rejection.(ABSTRACT TRUNCATED AT 250 WORDS)

Priming and induction of anti-rotavirus antibody response by synthetic peptides derived from VP7 and VP4.

Synthetic peptides derived from bovine rotavirus C-486 (BRV) outer capsid (VP7 and VP4) and inner capsid (VP6) proteins were tested to evaluate their ability to prime and induce an anti-rotavirus antibody response. Peptides corresponding to the amino acid residues 232-255 of VP4 (VP4-peptide), 275-295 of VP7 (VP7-peptide) and 40-60 of VP6 (VP6-peptide) of BRV were chemically synthesized. These peptides were coupled to carrier proteins (either keyhole limpet haemocyanin (KLH) or recombinant rotavirus inner capsid protein-VP6 assembled into virus-like particles (VP6-carrier) were used as carrier to link the synthetic peptides under study), and the resulting conjugates were used to immunize rotavirus seronegative mice. An enzyme-linked immunosorbent assay (ELISA) was used to determine anti-peptide and anti-rotavirus antibody titres in serum samples collected after immunization. All peptides were immunogenic in mice and induced the production of anti-peptide antibodies, but with the exception of VP6-peptide they were not able to induce anti-rotavirus antibodies as measured by ELISA. Western blot analysis indicated that antibodies against each peptide were able to react with the respective authentic viral proteins of various rotavirus serotypes. To determine if a peptide-primed animal would respond to native viral proteins, animals were subsequently injected with purified BRV. A rapid and high anti-rotavirus antibody titre, in addition to a rise in anti-peptide antibody titre, was observed in peptide-primed mice. Furthermore, the sera obtained from these mice neutralized the virus under in vitro conditions. The significance of these results in relation to a potential rotavirus synthetic peptide-based vaccine is discussed.

Studies on the rate of selective uptake of amino acids by Trichinella larvae in vivo.

Groups of C57BL/6J mice, orally infected with 300 larvae each of Trichinella spiralis or T. pseudospiralis were injected with [3H]-alanine, tyrosine, tryptophan or glycine. The incorporation of isotope labelled amino acids into larval proteins was measured at 2, 6, and 12 months post-infection. It was shown that there is a significant increase in the in vivo uptake of isotope labelled amino acids with time by the larvae of T. spiralis and T. pseudospiralis. The level of uptake was highest for tyrosine followed by tryptophan, alanine and then glycine, for both species. The in vivo uptake of amino acids by T. pseudospiralis larvae was always higher than T. spiralis or the host at 6 and 12 months post-infection. At 2 months post-infection, T. spiralis uptake of these amino acids was higher, except for tyrosine. This may be related to the special needs of these larvae during the process of encystation. The higher metabolic requirements of T. pseudospiralis may be related to the higher energy needs of these non encapsulated, highly motile and mobile muscle larvae.

Seasonality and prevalence of rotavirus in Al-Ain, United Arab Emirates.

BACKGROUND: Rotaviruses are the single most important causative agent of acute neonatal enteritis in most avian and mammalian species including humans. Rotaviruses infections have also been shown to be associated with the elderly, immunocompromised individuals and more recently with epidemic diarrheal illness in adults. OBJECTIVES: To study the incidence and the effect of seasonality on the prevalence of rotaviruses in Al-Ain, United Arab Emirates. STUDY DESIGN: A total of 650 stool samples submitted to the laboratories of two University Teaching Hospitals (Al-Ain and Tawam) and a private hospital (Oasis) were examined for the presence of rotaviruses from January 1990-December, 1992, using a commercially available latex agglutination assay. The meteorological data (temperature, relative humidity and rainfall) recorded during the sampling period was analyzed statistically to examine the effect of seasonality on the prevalence of rotavirus cases in Al-Ain, United Arab Emirates. RESULTS: Rotavirus was detected in 21.4% of the samples examined. The predominant number of positive cases (35%) were in the 7-12 months age group. It was interesting to find rotavirus-positive cases in as low an age group as < 3 months (3.6%) and as high as 10 years (8.04%). There was no significant difference on infection rates between male and female groups in the study. However, there was a significant difference between the national (38.18%) and non-national children (61.28%). The higher rate of the latter may be due to import of infections. There appeared to be a seasonal pattern of rotavirus occurrence in the cases studied, with a marked increase in the number of positive cases during the months when the relative humidity was low (25-45%) and there was no rainfall. CONCLUSIONS: Rotavirus was detected in all age groups with a predominance in 7-12 month age groups, and a higher incidence in non-nationals. There was a marked increase in the number of positive cases during the months when the relative humidity was low (25-45%) and there was no rainfall. These findings are discussed in relation to the epidemiology and prophylaxis of rotavirus infections.

Contractile characteristics of the flexor muscle of mice infected with Trichinella spiralis, T. nativa or T. pseudospiralis.

A comparative analysis of skeletal muscle isometric contractile characteristics was performed in vivo on the flexor muscle of mice infected 6 mo earlier with 400 larvae of Trichinella pseudospiralis, Trichinella spiralis, or Trichinella nativa. The control group consisted of age- and sex-matched uninfected mice. The mice were injected with 0.1 ml of 50% urethane in saline, and the skin of the left hind limb was cut open longitudinally. The exposed flexor muscle was freed from the adjacent tissue and left attached freely to the knee joint while the tendon was hooked to a transducer. The signals were amplified with an amplifier connected to a chart recorder. The sciatic nerve was exposed and attached to an electrode. Impulses were generated and muscle contraction recorded. The exposed muscle and nerve were bathed in normal Krebs solution at all times and the animals were kept alive during the experiment. The normal muscle twitch tension of uninfected mice reached an average of 2.26 +/- 0.24 (SD) g. Tetany was achieved at 15 Hz. Low-Ca2+ Krebs depressed the twitch tension to 2.0 +/- 0.08 g while tetany remained at 15 Hz. Muscle twitch tension in mice infected with T. pseudospiralis reached 2.47 +/- 0.17 g and tetany at 15 Hz. Low Ca2+ depressed twitch tension to 1.14 +/- 0.12 g. Tetany was achieved at 20 Hz. In contrast, the muscle twitch of mice infected with T. nativa was significantly reduced to 1.4 +/- 0.09 g and tetany at 15 Hz. Low Ca2+ depressed twitch tension to 0.9 +/- 0.16 g and tetany at 15 Hz.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the survival of aerosolized bovine rotavirus (UK) and a murine rotavirus.

The effect of relative humidity (RH) and temperature on the survival of airborne bovine rotavirus UK isolate (BRV-UK) and a murine rotavirus (MRV) was studied. In any one experiment, the virus under test was suspended in tryptose phosphate broth (TPB) supplemented with uranine (physical tracer) and an antifoam, was aerosolized using a Collison nebulizer into the rotating drum with the RH at either low (30 +/- 5%), medium (50 + 5%) or high (80 +/- 5%) level at 20 +/- 1 degrees C. Following a 15-min period of viral aerosol stabilization, sequential samples of drum air were collected using an All-Glass Impinger (AGI) for 24 h post-aerosolization. Both of the rotavirus isolates were found to survive best at medium RH level and high RH was found least favorable for the survival of these aerosolized rotaviruses. The survival pattern of aerosolized MRV was found to be the best when compared with survival pattern of all animal and human rotavirus isolates studies performed under aerosolized conditions in our laboratory. The findings of these experiments confirm and extend our previous reports on the survival of other animal and human aerosolized rotaviruses and emphasize the fact that air may be one of the vehicles for their dissemination and could explain why it is difficult to control nosocomial outbreaks of rotavirus gastroenteritis and to keep animal colonies rotavirus-free.

Molecular determinants of rotavirus virulence: localization of a potential virulence site in a murine rotavirus VP4.

The molecular basis of pathogenesis in vivo for a virulent mouse rotavirus (MRV) and a less virulent bovine rotavirus (BRV) were compared under in vitro and in vivo conditions. Obvious differences in the mobility of several genomic RNA segments were observed in one-dimensional gels. Under in vitro conditions, partial proteolytic peptide mapping identified differences between the two outer capsid proteins of these virus and no difference in inner capsid protein was observed. Since it has been observed by us and others that the gene coding for VP4 protein plays a significant role in determining virulence, the variability observed in the present study between the 84 k proteins (VP4) provided a basis for further investigations in order to locate a potential virulence determinant. A comparison of the carboxypeptidase digests of the MRV- and BRV-VP4 revealed an area of variability between amino acids 307 and 407, which may represent a site of virulence determinant. Under in vivo conditions the virulence of both parenteral BRV and MRV isolates and their corresponding reassortants (with replaced gene 4) were studied in murine and bovine hosts. Like their parents, BRV and MRV isolates, reassortants obtained by replacement of gene 4 in BRV with MRV gene 4 indicated that the dose of the virus isolate used and the clinical outcome in vivo was determined by gene segment 4. The implications of these findings to elucidate the molecular basis of pathogenesis of rotaviruses are discussed.

Characterization of two rotaviruses differing in their in vitro and in vivo virulence.

The proteins, genomic RNA and disassembly conditions and pathogenesis in vivo for a virulent mouse rotavirus (MRV) and a less virulent bovine rotavirus (BRV) were compared. An obvious difference in the mobility of several genomic RNA segments were observed in one-dimensional gels. Reassortants obtained by replacement of gene 4 in BRV with MRV gene 4 indicated that the dose of the virus used and the clinical outcome in vivo was determined by gene segment 4. Under in vitro conditions, a comparison of the inner capsid proteins by partial proteolytic peptide mapping did not reveal any difference between corresponding proteins. However, this technique did identify differences between the two corresponding outer capsid proteins of these viruses. These differences, in turn, may account for the increased stability of MRV, as compared to BRV, when subjected to calcium-chelating and chaotropic agents and may be one of the mechanisms involved in conferring virulence on the virus. The observed variability between the 84K proteins (VP4) provided a basis for further investigations in order to locate a potential virulence determinant, since it has been observed by us and others that the gene coding for this protein plays a role in determining virulence. A comparison of the carboxypeptidase digests of the MRV and BRV VP4 revealed an area of variability between amino acids 307 and 407, which may represent the site of a virulence determinant.