Investigation by bioassay of the efficacy of sodium hydroxide treatment on the inactivation of mouse-adapted scrapie.

Sodium hydroxide (NaOH) has been shown to reduce the infectivity of transmissible spongiform encephalopathy (TSE) agents. This study investigated the efficacy of sodium hydroxide at 0.1M, 0.25M and 0.5M concentrations for the inactivation of mouse-adapted scrapie strain ME7. Times and temperatures modelled conditions used in an industrial plasma fractionation plant for sanitisation of ultrafilters, and the sodium hydroxide component of Clean In Place sanitisation. The concentration of scrapie ME7 brain homogenate in NaOH test solutions was 1% (w/v). At the end of incubation periods, the samples were adjusted to neutral pH prior to intracerebral inoculation into mice for bioassay. The conditions of 0.1M NaOH at 60 degrees C for 2min and 0.25M NaOH at 30 degrees C for 60min were found to inactivate 3.96 and 3.93logs of scrapie, respectively. Use of 0.5M NaOH at 30 degrees C for 60 or 75min was found to inactivate >or=4.23 and 4.15logs of scrapie. This indicates that the use of these conditions in an industrial process would substantially reduce prion infectivity.

Prion-removal capacity of chromatographic and ethanol precipitation steps used in the production of albumin and immunoglobulins.

BACKGROUND AND OBJECTIVES: Although there is no epidemiological evidence to suggest that classical Creutzfeldt-Jakob disease (CJD) is transmitted through blood or blood products, the variant form (vCJD) has been implicated in transmission via packed red blood cells. The potential threat of the infectious agent contaminating plasma pools has led to manufacturing processes being examined for capacity to remove prions. The objective of these studies was to examine the prion-removal potential of the chromatographic purification and ethanol precipitation steps used to fractionate immunoglobulins and albumin from human plasma. MATERIALS AND METHODS: Western blot assay was used to examine the partitioning of proteinase K-resistant scrapie prion protein (PrPsc) over DEAE Sepharose, CM Sepharose and Macro-Prep High Q chromatographic columns, utilizing microsomal scrapie 263K spiked into each scaled down feedstream and assayed after each chromatographic step. In further studies, bioassay in C57 black mice was used and spikes of 10 000 g clarified brain homogenate of scrapie ME7 were added to feedstreams before sequences of scaled down chromatographic or Cohn fractionation process steps. RESULTS: The microsomal spiking study with Western blot detection demonstrated substantial partitioning of PrPsc away from the target proteins in all ion exchange chromatographic steps examined. The log10 reduction factors (LRF) across DEAE Sepharose and CM Sepharose columns for albumin were > or = 4.0 and > or = 3.0 respectively. The reductions across DEAE Sepharose and Macro-Prep High Q for intravenous immunoglobulin were 3.3 and > or = 4.1 respectively. Bioassay demonstrated LRFs of >or = 5.6 across the combination of DEAE Sepharose and CM Sepharose columns in the albumin process and > or = 5.4 across the combination of DEAE Sepharose and Macro-Prep High Q columns in the intravenous immunoglobulin process. Bioassay studies also demonstrated a LRF of > or = 5.6 for immunoglobulin produced by Cohn fractionation. CONCLUSIONS: Using rodent-adapted scrapie as a model, the studies indicated that ion exchange chromatography, as well as Cohn immunoglobulin fractionation have the potential to effectively reduce the load of TSE agents should they be present in plasma pools.

Investigation of prion removal/inactivation from chromatographic gel.

BACKGROUND AND OBJECTIVES: Concerns about the potential for prions to be retained on chromatography gels during the manufacture of plasma products prompted development of an investigational strategy for detecting infectious prions bound to gels. The objective was to firstly examine methods of implanting gels intracerebrally (IC) in mice, then to examine prion cleaning from a scaled-down version of the DEAE Sepharose column used in a production process to fractionate immunoglobulins and albumin from human plasma. MATERIALS AND METHODS: The study consisted of two parts: (i) the pathophysiological impact by IC inoculation of ground gel beads was compared to whole gel beads; (ii) the feedstreams to two DEAE Sepharose columns were spiked with scrapie ME7. One column was subjected to the protein loading and elution portions of the chromatography cycle. The other column was subjected to the full cycle of protein loading and elution, followed by regeneration with 0.5 m NaCl, 1 m NaOH and solvent/detergent washes. The gels were unpacked and bioassayed by IC implantation in mice to quantify infectivity. RESULTS: IC inoculation of ground gel beads resulted in unacceptably high pathological impact in the mice whereas whole gel bead inoculation resulted in a reduced affect. Accordingly, the whole bead model system was used to assess prion removal/inactivation from chromatography gels at the pre- and postcleaning stage of the chromatography cycle. Infectious prions were detected on the DEAE Sepharose prior to the cleaning step; however, the gel cleaning cycle reduced infectivity by a log reduction factor (LRF) of > or = 2.75, thus reducing infectivity by bioassay to below detectable limits. CONCLUSIONS: A model system for assessment of prion inactivation/removal from chromatography gels has been established. Spiked prion infectivity does bind to DEAE Sepharose gel; however, the cleaning cycle removed infectivity to levels below that detectable by bioassay.

Inactivation and clearance of viruses during the manufacture of high purity factor IX.

Haemophilia is a bleeding disorder characterised by a deficiency in Factor IX. Replacement therapy in the form of a Factor IX concentrate is a widely accepted practice. In this paper we describe a double virus inactivated chromatographic process for producing a high purity Factor IX product, MonoFIX((R))-VF. The process involves separation of the prothrombin complex by cryoprecipitation, fraction I precipitation and DEAE-cellulose adsorption, further ion-exchange chromatography of crude Factor IX, followed by solvent/detergent treatment. Heparin affinity chromatography is then used to further purify Factor IX. Final nanofiltration is sequential through 35 nm then 15 nm membrane filters. The principal virus inactivation/removal steps are solvent/detergent treatment and nanofiltration and the partitioning of relevant and model viruses provides further reduction in virus load through the production process.Solvent/detergent treatment was shown to achieve log reduction factors of 4.5 for HIV-1, 5.1 for Sindbis virus, 6.1 for vesicular stomatitis virus (VSV), 5.1 for bovine viral diarrhoea virus (BVDV) and 5.3 for pseudorabies virus (PRV). BVDV is a model for hepatitis C virus (HCV), and pseudorabies virus (PRV), like hepatitis B virus (HBV) is an enveloped DNA virus. Using scaled down models of the production process, we have also demonstrated the neutralization/partitioning of at least 6 logs of hepatitis A virus (HAV) during cryoprecipitation, Fraction I precipitation, and the DEAE adsorption and elution step, and a further 1.6 log reduction in HAV load as a result of heparin affinity chromatography. The log reduction factors for HAV as a result of the second ion-exchange chromatography step and as a result of enhanced neutralisation associated with solvent/detergent treatment were not significant. Nanofiltration was shown to contribute a further log reduction factor of 6.7 for HAV and 5.8 for BVDV indicating that log reduction factors of this order would be obtained with other viruses of a similar or larger size, such as HIV, HBV and HCV.Overall, these studies indicate that MonoFIX-VF is a product with an extremely high level of viral safety.

Etiology of acute gastroenteritis in hospitalized children in Melbourne, Australia, from April 1980 to March 1993.

Acute infectious diarrhea is common in children. Control requires knowledge of causes. Few comprehensive long-term studies of etiology have been undertaken in developed countries. This report is of a 13-year survey of 4,637 children from 0 to 14 years of age, admitted to a large children's hospital for treatment of gastroenteritis, in which viruses, bacteria, and parasites were sought. A recognized enteric pathogen was identified in 56.6% of children. Group A rotaviruses occurred in 39.6% of children overall and in 55% of children 12 to 23 months of age. They were a frequent cause (18.7%) of acute gastroenteritis in children under 6 months and in those aged 5 to 13 years (16%). Rotaviruses were almost entirely responsible for winter admission peaks. Enteric adenovirus types 40 and 41 (6% overall) were more frequent in children under 12 months (9.4%). Salmonella spp. (5.8%) and Campylobacter jejuni (3.4%) were more common in children over 5 years (13.1% and 6.7%, respectively). The 43.5% of cases (60% in children under 6 months) where no enteric pathogen was identified are cause for concern. The involvement of small viruses (including caliciviruses and astroviruses) may be clarified when molecular biology techniques are utilized to address this gap in our knowledge. This comprehensive 13-year study of the cause of acute infectious diarrhea in children in developed countries reinforces the importance of rotavirus and highlights a large group for whom the etiology remains unknown, an issue of particular concern with babies under 6 months of age. New techniques have the potential to identify old and new pathogens causing disease in these vulnerable infants.

Inactivation kinetics of model and relevant blood-borne viruses by treatment with sodium hydroxide and heat.

To determine the efficacy of a clean-in-place system for the inactivation of viruses present in human plasma, the effect of 0.1 M sodium hydroxide at 60 degrees C on viral infectivity was investigated. Inactivation of the following model and relevant viruses were followed as a function of time: human hepatitis A virus (HAV), canine parvovirus (CPV; a model for human parvovirus B-19) pseudorabies virus (PRV, a model for hepatitis B virus), and bovine viral diarrhoea virus (BVDV, a model for hepatitis C virus and human immunodeficiency virus). Infectivity of CPV was determined by a novel in situ EIA method which will prove useful for studies to validate parvovirus inactivation or removal. Infectivity of BVDV, PRV and CPV were shown to be reproducibly inactivated below the limit of detection by 0.1 M NaOH at 60 degrees C within 30 s. HAV was inactivated to below the limit of detection within 2 min. Treatment with heat alone also resulted in some log reduction for all viruses tested except for CPV which remained unaffected after heating at 60 degrees C for 16 min. Treatment of HAV with hydroxide alone (up to 1.0 m) at 15 degrees C did not lead to rapid inactivation. Collectively, these data suggest that 0.1 M NaOH at 60 degrees C for two min should be sufficient to inactivate viruses present in process residues.

Single-antibody in situ enzyme immunoassay for infectivity titration of hepatitis A virus.

Hepatitis A virus (HAV) establishes a persistent infection in cultured cells, with minimal effect on host cell metabolism. As a result, the virus produces very little, if any, cytopathic effect (CPE), even with cell culture-adapted strains. This feature precludes the use of a plaque or standard endpoint assay (using CPE as an indicator of infection) for the titration of infectious virus. The radioimmunofocus assay (RIFA) is the standard method for HAV titration, though this method is labour intensive and requires the use of radioisotopes. To this end, a single-antibody in situ enzyme immunoassay (EIA) has been developed, using binding of a perioxidase-labelled monoclonal antibody to fixed cell monolayers as an indicator of infection. This novel assay is highly reproducible, can be read by eye, and is suitable for high throughput situations. Furthermore, the assay has been validated against the RIFA making it suitable for use in studies validating the safety of therapeutic biologicals for human use.

Long-term ganciclovir chemotherapy for congenital duck hepatitis B virus infection in vivo: effect on intrahepatic-viral DNA, RNA, and protein expression.

Long-term antiviral chemotherapy using the nucleoside analogue ganciclovir was undertaken with the aim of eliminating hepadnaviral covalently closed circular (CCC) DNA from the livers of ducks that were congenitally infected with the duck hepatitis B virus (DHBV). Twenty-four weeks of ganciclovir therapy caused a substantial reduction in viremia, intrahepatic viral DNA replicative intermediates, and viral core proteins. Unfortunately, ganciclovir therapy did not substantially affect CCC DNA or viral RNA levels, and the treatment resulted in an increase in the intrahepatic expression of the viral envelope proteins, pre-S and S. By the completion of therapy, the viral envelope proteins had assembled into large aggregates within the cytoplasm of most hepatocytes. Viral replication in the bile duct epithelial cells and in the extrahepatic sites was likewise not affected by long-term ganciclovir therapy. In conclusion, 24 weeks of ganciclovir therapy decreased most viral replication markers within the liver, except for those of viral CCC DNA, RNA, and envelope proteins. Long-term therapeutic strategies using nucleoside analogs such as ganciclovir should be used with caution in chronic hepatitis B virus (HBV) infection. The careful monitoring of serum and hepatic markers of viral replication may therefore be important to avoid possible toxic consequences, such as the selective accumulation of viral proteins.

Herpes simplex virus encephalitis in pediatrics: diagnosis by detection of antibodies and DNA in cerebrospinal fluid.

Between 1983 and 1991, 16 cases of herpes simplex encephalitis were diagnosed at the Royal Children's Hospital, Melbourne by virus isolation from the brain or cerebrospinal fluid (CSF) (2 cases), by detection of herpes simplex virus-specific IgM, IgA or IgG by enzyme immunoassay (12 cases) or by polymerase chain reaction (PCR) and herpes simplex virus-specific antibodies (2 cases). Specific antibody was detected in 4 of 13 CSF samples taken on Days 1 to 4 after onset of neurologic symptoms compared with 15 of 17 samples taken after the fourth day of illness. PCR was retrospectively applied to 20 stored CSF samples from 11 patients; 5 of 8 samples taken less than 4 days after onset of symptoms were positive compared with 2 of 12 taken after Day 4. In contrast all 5 fresh unfrozen CSF samples taken from Days 2 to 21 were positive by PCR. These results indicate that PCR is more sensitive for early diagnosis of herpes simplex encephalitis than detection of specific antibody in CSF which is most useful after the fourth day of illness.

Culture-positive tuberculosis at St Vincent's Hospital, Melbourne, 1962-1989.

The disease type and demography of patients with culture confirmed tuberculosis (TB) diagnosed at St Vincent's Hospital, Melbourne between the years 1962 to 1989 were reviewed. Four hundred and eighty-two patients with culture-positive TB were identified whose origins were as follows: Australia 194; Northern Europe 38; The Mediterranean 98; Asia 60 and other or unknown 92. Patients whose country of birth was in Asia or the Mediterranean area accounted for 57% of patients in the 1980s; they presented at a younger age, with a higher proportion of extrapulmonary disease and a more equal sex distribution than did Australian born patients. The main types of extrapulmonary disease also differed for the various ethnic groups. The overall proportion of patients with an isolate resistant to at least one of the anti-TB drugs was 10.0% but in the Asian born was 21.7%. This survey, the longest series of bacteriologically confirmed cases of TB reported from a single institution in Australasia, has identified several changes in how TB is presenting for diagnosis.

Pneumonia associated with infection with pneumocystis, respiratory syncytial virus, chlamydia, mycoplasma, and cytomegalovirus in children in Papua New Guinea.

Paired serum samples were collected from 94 children with pneumonia admitted to Goroka Hospital, Papua New Guinea. All but three of the children were aged 1-24 months. Only nine children were malnourished, with weight for age less than 70% of the Harvard median (three had weight for age less than 60% of the Harvard median). Pneumocystis carinii antigen was detected in the serum of 23 children. Twenty two children had serological evidence of recent infection with respiratory syncytial virus. Five children were probably infected with Chlamydia trachomatis at the time of the study, and there was less convincing serological evidence of current infection in a further 11 children. Five children showed a fourfold rise in antibody to Mycoplasma pneumoniae. Although only one child showed a fourfold rise in antibody to cytomegalovirus, 86 children had this antibody. No child showed a fourfold rise in antibody to Ureaplasma urealyticum or Legionella pneumophila. P carinii, respiratory syncytial virus, C trachomatis, M pneumoniae, and cytomegalovirus may be important causes of pneumonia in children in developing countries.

Bronchodilator response during acute viral bronchiolitis in infancy.

Bronchodilator responsiveness was assessed by measuring specific respiratory conductance before and after inhalation of aerosolized bronchodilator in 50 infants who had acute bronchiolitis due to respiratory syncytial virus infection. Thirty per cent of the infants showed an improvement in specific conductance. Responders could not be differentiated from nonresponders by family histories of atopy, eosinophil counts, or immunoglobulin levels in blood and nasal secretions. Eighty-three per cent of the families and 54% of the mothers of the infants were smokers. Babies of smoking mothers had lower specific conductances than did those of nonsmoking mothers but showed no differences in bronchodilator response. The clinical significance of this bronchodilator-responsive sub-group has yet to be defined.

Respiratory syncytial virus and heart block--cause and effect?

A child aged 3 years who developed a respiratory syncytial virus infection is described. His admission was complicated by the development of an irregular pulse. He initially had a variable first or second degree heart block with transient electrocardiographic evidence suggestive of pericarditis and/or myocarditis and later developed complete heart block. He has remained asymptomatic for the past four years. A search for other causes of the heart block proved negative. It is tempting to suggest an association between the acute RSV infection and the development of the heart block but such an association at this stage remains speculative.

Respiratory viruses and sudden infant death.

Viruses were shown to be present in the respiratory tract in 200 of 763 cases of the sudden infant death syndrome studied in the nine years 1974-82. Epidemiological and pathological evidence suggested that the distribution of viruses in the sudden infant death syndrome differs between infants aged 3 months or less and those aged over 3 months: the incidence of detection of virus was 14% in the younger group compared with 39% in the older group. The distribution of the viruses in these two groups was compared with that in 1341 live infants with respiratory virus infections. Adenovirus, influenza virus, parainfluenza virus, and rhinovirus had similar distribution among the victims of the sudden infant death syndrome and live controls. The incidence of detection of respiratory syncytial virus was increased in the older infants dying of the sudden infant death syndrome (90% of the cases detected) compared with the older group of live infants (53%). Antibody studies, detection of virus, and epidemiological data suggest that respiratory syncytial virus may be a precipitating factor of sudden death in older infants.

Association of respiratory virus infections with sudden infant death syndrome.

From September, 1974, to September, 1979, 488 cases of sudden infant death syndrome (SIDS) in Melbourne were studied for evidence of viral infection. One hundred and eighty-eight infants (39%) yielded one or more viruses, with respiratory viruses being detected in 102 cases (21%). Further evidence of a respiratory virus association with SIDS was obtained by comparing the monthy respiratory virus isolation rates at the Royal Children's Hospital from 1973 to 1979 with the incidence of SIDS in the same period. A highly significant correlation was obtained between these isolation rates and the incidence of SIDS, which suggests that respiratory viruses play a role in SIDS in Melbourne.

Seasonal pattern in childhood viral lower respiratory tract infections in Melbourne.

Respiratory syncytial virus and parainfluenza viruses are the major pathogens in acute lower respiratory infection in infants and younger children. They show distinct seasonal patterns. An annual epidemic of respiratory syncytial virus infection is seen in Melbourne and this coincides with the coldest months of the year. Parainfluenza virus Type 1, the most frequent cause of laryngotracheobronchitis, occurs as an autumn epidemic every second year. Parainfluenza virus Types 2 and 3 are present most years and do not show a clear seasonal pattern.