Diagnosis of enteric pathogens in children with gastroenteritis.

The aim of this study was to determine the isolation trends of common and emerging pathogens in children over a 12-month period. The study group included 412 children under 6 years with diarrhoea who were either hospitalised, or seen in the outpatients department of The Sydney Children's Hospital. Pathogens were detected in 137 (33%) samples, with rotavirus most common (40%), followed by adenovirus (26%), astrovirus (12%), Campylobacter jejuni (12%), Salmonella spp. (10%) and Giardia lamblia (< 1 %). Giardia-specific antigen (GSA) was detected in 11 of 382 (3%) using an enzyme immunoassay (EIA), and this included four samples in which cysts of G. lamblia were detected by microscopy. Using electron microscopy (EM), viruses were detected in 29 of 120 (24%) samples from hospitalised children and 53 of 171 (31%) outpatients (P = 0.23). Amongst this subset, Norwalk-like viruses (NLVs) were detected by RT-PCR in 10 samples including six of 14 with small round viruses, one of seven with small viral-like particles (SVLPs), and three of 126 EM-negative samples. Lactoferrin, detected by EIA, was 59% more likely to be positive in samples infected with salmonella/campylobacter than in samples in which bacterial pathogens were not isolated. As an indicator for infection with these bacterial agents, the assay showed a sensitivity and specificity of 95 and 40.3%, respectively. A routine microbiological analysis of stools from children of this age group should include a screen for foodborne bacterial agents and rotavirus. Tests for adenovirus, astrovirus and NLVs should be secondary. The cost-effectiveness of including the EIAs for lactoferrin and G. lamblia in the routine testing protocol needs to be evaluated.

Detection of astrovirus gastroenteritis in children.

A commercial enzyme immunoassay (EIA) for the detection of astrovirus antigen was used to detect the virus during a 12-month survey of enteric pathogens in children in outpatient (n = 238) and hospital (n = 176) settings. It was found to have a 100% sensitivity and 98.6% specificity. Nineteen astrovirus isolates were detected and confirmed by northern hybridization, cell culture, and RT-PCR. The virus was detected mainly amongst outpatients although a comparison of the detection rate with that in hospitalised children did not demonstrate a statistically significant difference (p = 0.1347). In contrast, there was a strong association between hospitalization and rotavirus infection (p = 0.0371), and a strong association between infection detected in outpatients and adenovirus infection (p = 0.0193). Strains of astrovirus were sequenced, genotyped and shown to be: type 1 (n = 11), type 3 (n = 1), and type 4 (n = 7). Maximum genetic variation in type 1 isolates was 8.6% and type 4 was 7.8%. Changes did not result in amino acid substitutions.

Visualisation of phenotypically mixed HIV-1 and HTLV-I virus particles by electron microscopy.

Virions produced after HIV-1 infection of HTLV-I transformed cells have an expanded tropism that has been attributed to the presence of HTLV-I glycoproteins in the envelope. This report now directly identifies these phenotypically mixed virions by immunogold labelling electron microscopy. Furthermore we estimate there are 2% of these in cell-free supernatant, which represents up to 1 x 10(7) particles/ml from an in vitro infection. HTLV-1 envelope labelling was localised to a single region, suggesting a defined event in packaging of foreign envelope proteins into HIV-1 virus particles.

Inactivation of feline calicivirus, a Norwalk virus surrogate.

Norwalk and Norwalk virus-like particles (NVLPs) [also known as small round structured viruses (SRSVs)] are members of the family Caliciviridae and are important causes of gastroenteritis in humans. Little is known about their survival in the environment or the disinfection procedures necessary to remove them from contaminated settings. As NVLPs cannot be grown in tissue culture, survival studies require the use of a closely related cultivable virus. This study assesses the survival of the surrogate feline calicivirus (FCV) after exposure to commercially available disinfectants and a range of environmental conditions. Disinfectants tested included glutaraldehyde, iodine, hypochlorite, a quaternary ammonium-based product, an anionic detergent and ethanol. Complete inactivation of FCV required exposure to 1000 ppm freshly reconstituted granular hypochlorite, or 5000 ppm pre-reconstituted hypochlorite solution. Glutaraldehyde and the iodine-based product effectively inactivated FCV whereas the quaternary ammonium product, detergent and ethanol failed to completely inactivate the virus. The stability of FCV in suspension and in a dried state was assessed after exposure to 4 degrees C, room temperature (20 degrees C) and 37 degrees C. With increasing temperature, the stability of FCV was found to diminish both in suspension and in the dried state. FCV in the dried state did not survive for one day at 37 degrees C. This study provides a basis for establishing guidelines for disinfection protocols to decrease the spread of NVLPs in a community setting.

Combined infection of Norwalk-like virus and verotoxin-producing bacteria associated with a gastroenteritis outbreak.

Detection of multiple pathogens, particularly a combination of viruses and bacteria, is infrequently documented in outbreaks of gastroenteritis. This paper reports the presence of Norwalk-like virus (NLV) and enterohaemorrhagic verotoxin-producing Escherichia coli in one individual, and NLV and verotoxin-producing Aeromonas sobria in another individual, both part of a large gastroenteritis outbreak. The causes of gastroenteritis in such outbreaks may be more complex than previously thought.

Small round-structured (Norwalk-like) viruses and classical human caliciviruses in southeastern Australia, 1980-1996.

A total of 6,226 fecal samples collected from 1980 to 1996 in the Australian states of Victoria, New South Wales, and Tasmania from individuals with gastroenteritis were tested for small round-structured viruses (SRSVs) and classical human caliciviruses (ClHuCVs) by electron microscopy. There were 223 samples positive for SRSVs, and nine positive for ClHuCVs. SRSVs were detected in individuals of all ages and were commonly associated with gastroenteritis outbreaks in nursing homes and hospitals. SRSVs were detected throughout the year, but were more common in the period from late winter to early summer in Australia (August to December). There were peaks of virus activity in the early 1980s and more recently in 1995 and 1996. Analyses by RT-PCR and sequencing of a segment of ORF1 encoding the putative RNA polymerase for SRSVs and ClHuCVs showed the presence of viruses belonging to several genogroups. Viruses of genogroup 1 (Norwalk/Southampton-like) and genogroup 3 (ClHuCVs) were relatively rare. Viruses of genogroup 2 (Snow Mountain-like) were common, and could be divided into two subgroups, one containing Toronto/Mexico-like viruses, the other Lordsdale/Camberwell-like viruses. The majority of viruses detected belonged to this latter subgroup.

Hospital outbreak of Norwalk-like virus.

An outbreak of gastroenteritis caused by Norwalk-like virus occurred in two areas of the hospital: area 1, consisting of three adjacent and interconnected wards, with mostly elderly patients; and area 22, an acute ward in a separate building with elderly patients. In area 1, 40 patients and 20 staff were affected; in area 2, 18 patients and 14 staff were affected. Infection control measures were instituted in consultation with the government health authority. These measures did not appear to affect the course of the outbreak, but may have prevented spreads to the other wards.

Parvovirus B19 in HIV infection: a treatable cause of anemia.

We describe the case of an adult male patient with AIDS who presented with severe anemia and on investigation was found to have red cell aplasia due to parvovirus B19 infection. Bone marrow examination revealed absence of erythroid development and rare giant pronormoblasts. Repeated serological examinations revealed a low level of parvovirus IgM but no IgG. Viremia was demonstrated by electron microscopy and by the polymerase chain reaction (PCR). The patient's initial hemoglobin was 45 g/l and over a four month period he required twenty units of blood. He was treated with intravenous immunoglobulin (Intragam, CSL) at a dose of 400 mg/kg/day for five days. This led to an increase in his hemoglobin to 135 g/l. Parvovirus remained detectable by PCR but not by electron microscopy. Six months later the patient relapsed (Hb 65 g/l). Again he was transfused and treated with intravenous immunoglobulin for five days. His hemoglobin rose to 153 g/l and remained stable. He subsequently received maintenance treatment with 30 g of intagram once a month. We recommend that parvovirus be considered in any HIV infected patient with recurrent anemia.

Molecular characterization of Camberwell virus and sequence variation in ORF3 of small round-structured (Norwalk-like) viruses.

Five small round-structured viruses (SRSVs) associated with gastroenteritis in Victoria, Australia, from January to November 1994 were examined by sequencing cDNA prepared from faecal samples using RT-PCR. The sequence of the 3' half (3.8 kb) of the genome of one of these viruses, Camberwell, was determined. Camberwell virus was related most closely to Bristol and Lordsdale viruses, and belonged to the genetic group of SRSVs containing Bristol, Lordsdale, Toronto, OTH-25, Mexico, and Hawaii viruses. The amino acid identities between Camberwell and Bristol viruses for proteins encoded by ORF1 (partial), ORF2, and ORF3 were 99%, 98%, and 90%, respectively. A highly variable region in ORF3 corresponding to amino acid residues 123 to 169 (Bristol and Camberwell numbering) were identified. Short segments of ORF1 (polymerase region) and the highly variable ORF3 region was analysed for the other four viruses. The results obtained indicated the potential usefulness of the variable region in distinguishing between closely related viruses.

In vitro growth of the microsporidian Septata intestinalis from an AIDS patient with disseminated illness.

A new species of microsporidian, Septata intestinalis, was recently recognized as an opportunistic pathogen of AIDS patients. In this study, it was cultured from the nasopharyngeal aspirate of a human immunodeficiency virus type 1-infected patient with disseminated microsporidiosis. In human embryonic lung cells exposed to S. intestinalis, a cytopathic effect appeared within 28 days as foci of rounded up cells. Thin-section electron microscopy showed a variety of developmental stages of the microsporidium within parasitophorous vacuoles. In monocyte-derived macrophages, evidence of infection and development of the parasite was demonstrated by light and electron microscopy. In both infected human embryonic lung cells and monocyte-derived macrophages, a network of septa separated individual spores. Partial sequencing of the RNA small-subunit gene (16S rDNA gene) confirmed the identity of this parasite as S. intestinalis. This is the first report of the isolation of S. intestinalis in vitro and provides evidence that the parasite can be disseminated by macrophages.

A new trichrome-blue stain for detection of microsporidial species in urine, stool, and nasopharyngeal specimens.

Detection of microsporidia in clinical specimens has relied on electron microscopy, histology, or staining. This article describes further alterations to the modified trichrome staining method which make it easier to identify microsporidial spores. The changes are a decrease in the phosphotungstic acid level and the substitution of a colorfast counterstain, aniline blue, for the fast green of the original stain. The modified stain provides good contrast between microsporidial spores and background material including human and fungal cells. Stool specimens from 139 human immunodeficiency virus-seropositive patients revealed that 5 patients were infected with Enterocytozoon bieneusi and 6 patients had larger spores. Thin-section electron microscopy of the larger spores showed a structure consistent with that of either Encephalitozoon or Septata species. Three of the patients with Encephalitozoon- or Septata-like species had disseminated infection, with spores detected in nasopharyngeal aspirates and urine samples.

A case of acute hepatitis E in Victoria.

OBJECTIVE: To report a case of acute hepatitis E in Victoria, confirmed by laboratory investigations. CLINICAL FEATURES: A 10-year-old boy presented for medical attention with a seven-day history of anorexia and jaundice, 17 days after arriving from Pakistan. The diagnosis of acute hepatitis E was suspected after exclusion of the known causes of viral hepatitis, and was further established by specific antibody testing and identification of hepatitis E virus-like particles in a faecal sample collected three weeks after the onset of illness. INTERVENTION AND OUTCOME: The patient was managed at home, treated symptomatically and made a complete recovery. CONCLUSION: In patients who arrive from countries where hepatitis E is endemic, and who develop non-A, non-B, non-C viral hepatitis, hepatitis E should be considered as a possible diagnosis.

A new electron microscope positive staining method for viruses in suspension.

A new procedure for the positive staining of viruses in suspension, the Tokuyasu staining procedure (TSP), was evaluated using a non-enveloped virus, rotavirus; an enveloped virus, rubella virus and two glutaraldehyde-treated enveloped viruses, Human T Cell Lymphotropic Virus Type I (HTLV-I) and Human Immunodeficiency Virus Type 1 (HIV-1) as models. The TSP involves an initial staining of the virus with uranyl acetate (UA) followed by thin embedding in a mixture of UA and polyvinyl alcohol (PVA). Using aqueous UA for the TSP, a combination of positively and negatively stained particles was seen for both rotavirus and rubella virus. With glutaraldehyde-fixed HTLV-I and HIV-1, stain penetration did not occur and only negative staining was observed. The substitution of methanolic UA for aqueous UA in the TSP resulted in only positive staining of rotavirus and rubella virus. The change in procedure also resulted in stain penetration of the glutaraldehyde-fixed HTLV-I and HIV-1 to give positively stained particles. Some novel morphological features of rotavirus and rubella virus structure were observed by the TSP.