Solid-phase radioimmunoassay for the detection of rotavirus.

A solid-phase radioimmunoassay method has been developed for the detection of rotavirus in the form of a purified antigen and in stool. The parameters of the radioimmunoassay were examined and optimized to give high sensitivity and same-day results. Compared with electron microscopy, the assay is up to 10 times as sensitive for detection of the virus in stool and up to 128 times as sensitive for detection of a purified virus antigen. In a field study on stool specimens it was at least as efficient as electron microscopy.     

Surface properties of the Vero cytotoxin-producing Escherichia coli O157:H7.

Strains of Escherichia coli serotype O157:H7 are Vero cytotoxin-producing enteric pathogens which have been associated with sporadic cases and outbreaks of hemorrhagic colitis and with the hemolytic uremic syndrome in humans. In addition to toxin production, adherence of many pathogenic bacteria to intestinal mucosal surfaces is a critical primary step in the pathogenesis of diarrheal diseases. Although E. coli serotype O157:H7 organisms adhere to intestinal epithelia of orally infected animals in a pattern morphologically identical to that previously described in adherent, effacing E. coli infections, the mechanisms of bacterial adherence are not known. To determine the cell surface adhesins which mediate attachment of E. coli O157:H7 to epithelial surfaces, we evaluated the surface properties of these organisms. Five strains isolated from children with the hemolytic uremic syndrome were grown both in broth cultures and on agar media. Adherence and invasion of E. coli O157:H7 in Intestine 407 and HEp-2 epithelial cell lines was quantitated using an enteroinvasive E. coli strain (serotype O164:NM) as a control. Cell surface properties of E. coli O157:H7 were evaluated by agglutination of a series of erythrocytes, transmission electron microscopy, DEAE-ion-exchange chromatography, and hydrophobic interaction chromatography. E. coli O157:H7 strains adhered to but did not invade either Intestine 407 or HEp-2 cells. Homologous O157:H7 rabbit antiserum blocked attachment of bacteria to tissue culture cells, in contrast to heterologous antiserum and preimmune rabbit serum, which did not inhibit attachment of E. coli O157:H7. None of the five O15:H7 isolates mediated mannose-resistant hemagglutination under any of the in vitro culture conditions. One isolate mediated mannose-sensitive hemagglutination after serial passage in broth cultures. Pili and fibrillae were not visualized by electron microscopy on nonhemagglutinating organisms, but pili were demonstrated on the one isolate which mediated mannose-sensitive hemagglutination. All O157:H7 strains demonstrated high anionic surface charge (DEAE) but low surface hydrophobicity properties (hydrophobic interaction chromatography). The findings suggest that surface structures other than pili can mediate attachment of serotype O157:H7 bacteria to epithelial cells in vitro.     

Fulminant disseminated Varicella Zoster virus infection without skin involvement.

BACKGROUND: Varicella Zoster virus (VZV) infection is potentially very serious in bone marrow transplant recipients, and may manifest as a disseminated visceral infection. This condition is generally accompanied by a vesicular rash. OBJECTIVES: We review here a case of fulminant fatal disseminated VZV infection, not accompanied by skin involvement, and the laboratory approaches currently available to diagnose this disease. STUDY DESIGN: Post mortem tissue samples were subjected to histopathological examination, and tested for herpesviruses by electron microscopy and PCR. RESULTS: Intranuclear inclusions were noted by histological examination in the lungs, liver, kidneys and bone marrow. Particles with a herpesvirus morphology were visualized in liver tissue. VZV DNA was detected in liver and bone marrow by PCR followed by sequencing of the amplicons. Viremia was documented by retrospective testing of the serum by PCR. CONCLUSIONS: A disseminated VZV infection which proved rapidly fatal was demonstrated in a case without skin manifestations. This rare presentation of VZV infection is potentially underdiagnosed. Testing for VZV viremia by PCR can at the very least suggest the diagnosis although whether plasma-associated viremia is truly pathognomonic of visceral disseminated infection remains to be established.     

Immunochemical analysis of immune response to Chlamydia trachomatis in Reiter''s syndrome and nonspecific urethritis.

Chlamydia trachomatis (Ct) has been proposed as a causative agent in Reiter's syndrome (RS) when an infection occurs in a susceptible host. To assess whether this susceptibility is reflected in a characteristic humoral immune response we compared patients with complicated (RS) and uncomplicated courses of nonspecific urethritis (NSU). Geometric mean titres of antibodies to C. trachomatis by immunofluorescence were 89.6 for RS, 80.0 for NSU and 16.0 for normals. 125I-Protein A probing of immunoblotted antigens of C. trachomatis revealed no band unique to RS. 125I-anti-IgA probing of immunoblots demonstrated reactivity with the 59,000 dalton antigen in 11/11 RS and 2/6 NSU. The major outer membrane protein of C. trachomatis (40,000 daltons) bound immunoglobulin nonspecifically. There was no clearly differentiating feature between HLA-B27-positive and B27-negative RS. One sequentially studied patient revealed an augmentation in synovial fluid IgA reactivity during the course of disease. No pattern of humoral immune response to C. trachomatis could be regarded as specific for RS nor for HLA B27-positivity. The study did not identify a Reiter's-specific antigen in C. trachomatis but demonstrates the usefulness of applying blotting techniques to population studies of HLA modulation of immune response to infectious agents.     

Diagnosis of fastidious enteric adenoviruses 40 and 41 in stool specimens

Thirty-five stool specimens, collected over a 14-week period from pediatric gastroenteritis patients and shown to contain adenovirus by electron microscopy, were inoculated onto 293 and HeLa cells. Virus isolates were characterized by serum neutralization and restriction endonuclease cleavage analysis of viral DNA from infected cells. Adenovirus was isolated upon primary inoculation of 293 cells from all 35 specimens shown to contain adenovirus by electron microscopy. Fastidious adenoviruses 40 and 41 (Ad40 and Ad41) were found in 17 (49%) of the stool specimens, and 4 of these specimens contained a conventional species (Ad1, Ad1, Ad18, Ad31) as well as Ad40. This was first manifest by the observation that four of the isolates which initially grew only in 293 cells acquired the capacity to grow in HeLa cells upon subsequent passage. In each case, the conventional species was undetectable by DNA analysis in the original inoculum but was selected in 293 cells and became the only one detectable by the second passage. Four other specimens, containing Ad1 or Ad31 alone, failed to grow initially in HeLa cells but did grow in 293 cells. The results of this study demonstrate therefore that (i) 293 cells are more sensitive than HeLa cells for the isolation of conventional as well as fastidious enteric adenovirus species and (ii) identification of viruses from patient specimens should involve minimal passage of the virus in cell culture, as a single passage can result in misdiagnosis of the virus associated with the infection.     

Counter-immunoelectro-osmophoresis for the detection of infantile gastroenteritis virus (orbi-group) antigen and antibody.

A moderatley sensitive, rapid, and economical test scheme for the detection of infantile gastroenteritis virus (IGV) in stool or antibody in serum has been developed and evaluated. The test scheme with minor modifications was an adaptation of a counter-immunoelectro-osmophoresis system we once used for the detection of hepatitis B antigen. Large numbers of stool samples may be screened during half a working day for the presence of IGV using reference antiserum to IGV prepared in guinea-pigs. Serological studies of a diagnostic but not epidemiological nature may also be performed with equal facility by this same test scheme using highly purified IGV antigen derived from stool.     

Evaluation of cell line 293 for virus isolation in routine viral diagnosis.

Cell line 293, a continuous line of transformed human embryonic kidney cells, has been recognized for its sensitivity in the isolation of adenoviruses, particularly the fastidious species 40 and 41, from stool specimens. To explore the possibility of using this cell line for the isolation of other viruses from clinical specimens, 293 cells were tested for their susceptibility to a variety of viruses including herpes simplex virus, parainfluenza viruses, respiratory syncytial virus, and the enteroviruses ECHO 11, coxsackie B5, and coxsackie B6. All of the viruses induced a cytopathic effect in 293 cells. Consequently, 293 cells were introduced into the diagnostic laboratory and used along with primary African green monkey kidney (AGMK) cell cultures for the inoculation of all respiratory and stool specimens. The study represents a retrospective analysis of the performance of 293 cells over a 22-month period. It was confirmed that 293 cells were more sensitive than AGMK cells for the isolation of adenoviruses from both respiratory and stool specimens. The 293 cells were also sensitive for the isolation of enteroviruses (untyped) but more so from stool specimens than from respiratory specimens. Parainfluenza virus and respiratory syncytial virus were only rarely isolated in 293 cells. Herpesvirus isolates were obtained with equal frequency in both 293 and AGMK cells. This retrospective analysis confirms the value of 293 cells for the isolation of adenoviruses and demonstrates that 293 cells are also useful for the isolation of certain enteroviruses from both respiratory and stool specimens.     

The homogeneous photopolymerization of methyl methacrylate by colloidal cadmium sulfide

A completely new method of initiating the homogeneous radical polymerization of methyl methacrylate in bulk or in solution via the photogeneration of an electron hole pair in colloidal cadmium sulfide is presented. A polymerization mechanism involving an excited cadmium sulfide particle in both the initiation and termination steps is proposed. In the initiation a methyl methacrylate molecule is oxidized by a positive hole photogenerated in a CdS particle, which results in a novel chain-end structure of the poly(methyl methacrylate) (PMMA). Degradative chain transfer to reduced excited cadmium sulfide particles is responsible for chain termination. Thus, for the first time, a detailed polymerization mechanism in which all states of the polymerization, i.e., initiation, propagation, chain transfer and termination, is presented for the polymerization of vinyl monomers initiated by semiconductors. Thermogravimetry (TG) showed that the newly synthesized PMMA has greatly enhanced thermal stability when compared to normal radically prepared PMMA. In fact, the thermal stability approaches that of anionically prepared PMMA but is experimentally much easier to prepare. This technique enables the homogeneous embedding of CdS particles in a polymer matrix.