Etiology and outcome of diarrhea after marrow transplantation: a prospective study.

BACKGROUND/AIMS: Acute diarrhea after marrow transplant is usually ascribed to acute graft-vs.-host disease (GVHD) or infection, with a reported 40%-50% incidence of infection. The aim of this study was to determine the incidence of acute diarrhea after transplantation, its causes, and its outcome. METHODS: Two hundred ninety-six patients were followed up; patients with diarrhea were studied using standard evaluation of stool plus immunoelectron microscopy; assays for astrovirus, picobirnavirus, and Norwalk virus; and gene-probe methods for toxin-producing Escherichia coli. In 38 patients with diarrhea, intestinal biopsy specimens and duodenal fluid were also analyzed. RESULTS: One hundred fifty acute diarrheal episodes developed in 126 patients (an incidence of 43%). Intestinal infection was found in 20 of 150 episodes: viruses (astrovirus, adenovirus, cytomegalovirus, and rotavirus) in 12 patients, nosocomially acquired bacteria (Clostridium difficile and Aeromonas) in 7 patients, and mixed infection in 1 patient. Acute GVHD was responsible for 72 of 150 episodes (48%). Clinical signs and symptoms of infection and GVHD were similar. In 58 of 150 episodes (39%), no clear etiology could be found for self-limited diarrhea. CONCLUSIONS: Intestinal infection accounted for 13% and acute GVHD for 48% of diarrheal episodes. The most common infecting organisms were astrovirus, C. difficile, and adenovirus. Most cases of diarrhea after marrow transplant are not caused by infection.

Direct detection of Molluscum contagiosum virus in clinical specimens by in situ hybridization using biotinylated probe.

Molluscum contagiosum virus (MCV) is an unclassified poxvirus which has recently become recognized as causing a major sexually transmitted disease. At present no assay is available for specific detection of MCV because the virus cannot be serially propagated in cell culture. Since MCV produces an abortive, limited growth with some cytopathic effect in certain cell lines, we were able to develop an in situ hybridization assay for detection of MCV genome in clinical specimens. Human fetal diploid lung cell monolayers were infected with clinical specimens, and after proper incubation and fixation in paraformaldehyde, hybridization was performed under full stringency conditions with a molecularly cloned biotinylated probe. Only MCV infected cells showed homology to the MCV probe with a purple-brown cytoplasmic staining. Additionally, we have described an in situ hybridization assay for direct detection of MCV genome in formalin-fixed, paraffin-embedded biopsies. Characteristic intracytoplasmic Molluscum bodies (Henderson-Paterson bodies) were detected in stratum spinosum cells of the epidermis. Striking staining similarities have been observed between in situ hybridization and haematoxylin-eosin cytostaining. These procedures are the first successful identification of MCV genome in clinical samples by molecular hybridization, with sensitivity and specificity equal to or greater than electron microscopy.

The isolation and characterization of a Norwalk virus-specific cDNA.

Norwalk virus, an important cause of epidemic, acute, nonbacterial gastroenteritis in adults and children, has eluded adaptation to tissue culture, the development of an animal model, and molecular cloning. In this study, a portion of the Norwalk viral genome encoding an immunoreactive region was cloned from very small quantities of infected stool using sequence-independent single primer amplification. Six overlapping complementary DNA (cDNA) clones were isolated by immunologic screening. The expressed recombinant protein from a representative clone reacted with six of seven high titer. Norwalk-specific, postinfection sera but not with corresponding preinfection sera. Nucleic acid sequence for all clones defined a single open reading frame contiguous with the lambda gt11-expressed beta-galactosidase protein. Only oligonucleotide probes specific for the positive strand (defined by the open reading frame) hybridized to an RNaseA-sensitive, DNaseI-resistant nucleic acid sequence extracted from Norwalk-infected stool. Furthermore, RNA extracted from serial postinfection, but not preinfection, stools from three of five volunteers hybridized to a Norwalk virus cDNA probe. Clone-specific oligonucleotide probes hybridized with cesium chloride gradient fractions containing purified Norwalk virion. In conclusion, an antigenic, protein-coding region of the Norwalk virus genome has been identified. This epitope has potential utility in future sero- and molecular epidemiologic studies of Norwalk viral gastroenteritis.

Biochemical and genetic characterization of autoagglutinating phenotypes of Aeromonas species associated with invasive and noninvasive disease.

The genetic characteristics and biochemical and structural properties of a number of autoagglutinating (AA) strains of Aeromonas associated with invasive and noninvasive disease in humans and infections in animals and from environmental sources were investigated. Of 27 strains analyzed by multilocus enzyme typing and DNA hybridization studies, 25 (93%) were confirmed to belong to either hybridization group 1 (phenospecies and genospecies Aeromonas hydrophila) or 8 (phenospecies Aeromonas sobria; genospecies Aeromonas veronii). Further analysis of 19 of these strains indicated that four major groups could be identified on the basis of serologic and surface characteristics, protein and lipopolysaccharide composition, and virulence properties; these groupings held true regardless of the site of isolation or disease process involved. The major AA+ group identified was serogroup O:11, whose strains possessed an S layer, were resistant to the bactericidal activity of normal serum, and were pathogenic in mice. The results suggest a set of useful phenotypic and structural markers for identification of specific subsets of mesophilic Aeromonas involved in a wide range of infections in the animal kingdom.

Penetration and replication of Edwardsiella spp. in HEp-2 cells.

The ability of 22 Edwardsiella strains to penetrate and replicate in cultured epithelial cells was initially evaluated by light microscopy methods and by the recovery of gentamicin-resistant (Gmr) bacteria from the Triton X-100 cell lysates of HEp-2-infected monolayers. Giemsa-stained HEp-2 cells revealed the presence of numerous internalized bacteria 3 h postinfection, often appearing as parallel rows of replicated bacteria within the cytosol and sometimes obliterating the cytoplasm because of the large numbers of bacilli present. Invasive bacteria were also sometimes found within cytoplasmic vacuoles in infected cells; thin-section electron micrographs of HEp-2-infected cells supported these conclusions. Results of light microscopy studies and cell lysate assays indicated that most Edwardsiella tarda (92%) and some Edwardsiella hoshinae strains were invasion positive on one or more occasions, while Edwardsiella ictaluri isolates were uniformly negative. HEp-2 invasion by E. tarda was a microfilament-dependent (cytochalasin B- and D-sensitive) process, with maximum numbers of Gmr CFU recorded between 3 and 6 h postinfection. The small percentage (0.01 to 1.0%) of the challenge inoculum recoverable as Gmr progeny 3 to 6 h postinfection was attributed to a strong cell-associated (not filterable) hemolysin that was produced by a majority (85%) of the E. tarda strains but not by E. ictaluri and only minimally by E. hoshinae. This cytolysin/hemolysin was responsible for the toxic effects observed in HEp-2 cells during the infection-replication process of edwardsiellae and appears to play a role in the release of internalized and replicated bacteria from infected cells. The results suggest an invasion strategy with some similarities to and differences from those of other recognized enteroinvasive pathogens.

Foodborne Snow Mountain agent gastroenteritis with secondary person-to-person spread in a retirement community.

A variety of small round-structured viruses are being recognized with increasing frequency as a cause of gastroenteritis in the community, but have rarely been reported to cause outbreaks in hospitals or extended-care facilities. From March 20 through April 15, 1988, an outbreak of gastroenteritis occurred in a retirement facility in the San Francisco Bay area. Illness was characterized by diarrhea, nausea, and vomiting; two residents died. Attack rates were 46% (155 of 336) in residents and 37% (28 of 75) in employees. During the initial outbreak period, illness among residents was associated with two shrimp meals served in the facility dining hall (odds ratio = 6.7). Person-to-person transmission probably occurred: The risk of becoming ill one or two days after a roommate became ill was significantly greater than that of becoming ill at other times during the outbreak (risk ratio = 6.5). Microbiologic examinations for bacterial and parasitic enteric pathogens were negative; however, 27-nm viral particles were detected by immune electron microscopy and by blocking enzyme immunoassay to Snow Mountain agent in stools obtained at the onset of illness from one of six ill residents. Seroconversion (greater than fourfold antibody rise) to Snow Mountain agent was detected in acute- and convalescent-phase serum specimens from five of six ill residents as measured by enzyme immunoassay, but not for Norwalk agent as measured by radioimmunoassay. This report of an outbreak of Snow Mountain agent gastroenteritis in an extended-care facility documents that these difficult-to-identify 27-nm viruses can cause outbreaks in inpatient settings.

Immunological characterization of the Marin County strain of astrovirus.

Marin County virus (MCV) was isolated from a stool suspension and serially propagated in human embryonic kidney cell cultures. MCV particles in stool and cell-propagated virus stocks showed reactivity by immune electron microscopy (IEM) with rabbit antiserum to astrovirus type 5. MCV antigen was also detected in two MCV stool samples by enzyme immunoassay (EIA) with an astrovirus group-specific monoclonal antibody. Acute and convalescent sera from 3 of 3 MCV-infected patients showed seroconversion to cell-propagated MCV by EIA. Immunofluorescence of MCV propagated in cell culture showed positive reactivity with an astrovirus group specific monoclonal antibody and astrovirus type 5 antiserum, with some cross-reactivity with astrovirus type 1. Similar results were obtained with the prototype strain of astrovirus type 5. However, in plaque-reduction assays, both the prototype astrovirus type 5 and MCV were neutralized by type 5 antiserum only. We conclude that MCV can be serially propagated by techniques used for previously described astroviruses and is serotypically an astrovirus type 5.

Prevalence of acute enteric viral pathogens in acquired immunodeficiency syndrome patients with diarrhea.

Diarrhea due to enteric pathogens is an important complication of advanced human immunodeficiency virus infection. Whereas numerous bacterial and parasitic agents have been implicated, the role of pathogenic enteric viruses is less clear. Stools from 153 human immunodeficiency virus seropositive men were tested by electrophoresis, enzyme-linked immunosorbent assay, and immune electron microscopy for the presence of rotaviruses (group A and non-group A), adenoviruses, and Norwalk agent. Virus was detected in 9% of the patients with acquired immunodeficiency syndrome, 3% of the patients with acquired immunodeficiency syndrome-related complex, and none of the seropositive men without these diagnoses. Virus detection was not more likely in stool from patients with diarrhea.

Surface properties of autoagglutinating mesophilic aeromonads.

The surface characteristics of 24 autoagglutinating (AA+) mesophilic aeromonads were investigated. One group of 16 was found to be highly related serologically by their reactive pattern against O antisera generated against three reference strains. Subsequent characterization of 11 of these isolates (group 1) indicated that they had the following properties in common: precipitation after boiling (PAB+), membership of serogroup O:11 (typing scheme of Sakazaki and Shimada), resistance to lysis by bacteriophage Aeh1, and possession of a surface layer (S layer) as determined by transmission electron microscopy. Strains not exhibiting the same serologic reactivity pattern belonged to diverse serogroups (other than O:11), were generally susceptible to lysis by Aeh1, and were S layer negative by transmission electron microscopy (group 2). Analysis of selected isolates representing both groups indicated that group 2 strains were usually more hydrophobic than group 1 isolates in several different assays; both groups, however, possessed high surface charge as determined by binding to DEAE-cellulose. Group 1 isolates were more virulent than group 2 strains tested as determined by lower 50% lethal doses for mice. On the basis of the results of the kinetics of autoagglutination in broth, relative surface hydrophobicity, uptake of Congo red, agglutination of yeast cells, and electrophoretic protein profiles of whole-cell extracts, the surface layer associated with O:11 mesophilic aeromonads appears to be distinct from that of Aeromonas salmonicida. The results suggest that a new pathogenic group of mesophilic aeromonads linked through a common AA phenotype, serogroup, and S layer cause serious infections in both humans and animals (fish).

Virulence markers of mesophilic aeromonads: association of the autoagglutination phenomenon with mouse pathogenicity and the presence of a peripheral cell-associated layer.

Autoagglutination (AA phenotype) of mesophilic aeromonads in broth was found to be a virulence-associated marker. There were two kinds of AA+ strains: those that spontaneously pelleted (SP+), and those that pelleted only after boiling (PAB+). Of 79 strains tested, 24 (30%) were AA+, and 18 of these were recovered from clinical specimens. Most of the AA+ strains (n = 21) were identified as either Aeromonas sobria or Aeromonas hydrophila. Of the well-documented clinical isolates of A. sobria and A. hydrophila available, 5 (46%) of 11 from invasive disease and 4 (14%) of 29 from noninvasive disease were SP- PAB+. The SP- PAB+ phenotype was significantly associated with invasive infections (e.g., bacteremia and peritonitis [chi 2, P less than 0.05]). All seven of the SP- PAB+ A. sobria and A. hydrophila strains tested killed mice within 48 h after intraperitoneal infection with 1 x 10(7) to 3 x 10(7) CFU, whereas only two of four SP+ PAB+ strains tested were lethal. All of the SP- PAB+ A. sobria and A. hydrophila isolates examined shared common O somatic antigens and possessed an external layer peripheral to the cell wall as determined by thin-section electron micrography. The LL1 strain of A. hydrophila used by Dooley et al. (J. S. G. Dooley, R. Lallier, and T. J. Trust, Vet. Immunol. Immunopathol. 12:339-344, 1986) to demonstrate an S membrane protein component in aeromonads virulent for fish also was SP- PAB+ and possessed the peripheral membrane, suggesting an association between these two components. Seven AA- and three SP+ strains tested lacked this layer; furthermore, 22 (71%) of 31 such isolates did not kill mice. The AA phenotype was a stable characteristic upon long-term passage of isolates in vitro. Study of SP+ and PAB+ aeromonads by surface charge and hydrophobicity analyses indicated that neither property correlated with either virulence or the presence of an external layer.

Isolation of lymphocytopathic retroviruses from San Francisco patients with AIDS.

Infectious retroviruses have been detected in 22 of 45 randomly selected patients with acquired immune deficiency syndrome (AIDS) and in other individuals from San Francisco. The AIDS-associated retroviruses (ARV) studied in detail had a type D morphology, Mg2+-dependent reverse transcriptase, and cytopathic effects on lymphocytes. The viruses can be propagated in an established adult human T cell line, HUT-78. They cross-react with antiserum to the lymphadenopathy-associated retrovirus isolated from AIDS patients in France. Antibodies to ARV were found in all 86 AIDS patients and in a high percentage of 88 other homosexual men in San Francisco. This observation indicates the widespread presence of these lymphocytopathic retroviruses and their close association with AIDS.

Major polypeptide of duck hepatitis B surface antigen particles.

The 40- to 50-nm pleomorphic particles found in the sera of domestic Pekin ducks infected with duck hepatitis B virus were purified by rate zonal and isopycnic centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic polypeptide analysis of these particles, called duck hepatitis B surface antigen particles, revealed the major component to be a single 17,500-dalton polypeptide. This result is in contrast to polypeptide analyses of the surface antigens of related mammalian viruses, including hepatitis B, in which a major doublet of polypeptides is seen with molecular weights ranging from 23,000 to 29,000. Tryptic maps of 17,500-dalton polypeptide resembled that of the major non-glycosylated polypeptide of the adw subtype of hepatitis B surface antigen. A serological assay for antibody to the purified duck virus particles is also described.

Main Drain virus as a cause of equine encephalomyelitis.

Main Drain virus, which is thought to be transmitted normally among rabbits and various rodents by its natural vector, Culicoides variipennis, was isolated repeatedly from brain tissue of a sick horse from Sacramento County, California, and was implicated as the causative agent. Signs of illness were incoordination and ataxia, stiff neck, head pressing, inability to swallow, fever, and tachycardia.

A 27-nm virus isolated during an outbreak of acute infectious nonbacterial gastroenteritis in a convalescent hospital: a possible new serotype.

An outbreak of acute infectious nonbacterial gastroenteritis began among elderly patients in a convalescent hospital in Marin County in northern California in March 1978 and persisted through May 1978. The overall clinical attack rate was 51% of 187 residents and 12% of 180 employees. A 27-nm viruslike particle was observed by immune electron microscopy in stools obtained at or near the onset of illness from four of 32 patients. Seroresponses to the 27-nm particles were found by immune electron microscopy in 16 of 18 patients. In addition, serologic evidence of infection with this or a related agent was demonstrated in persons who developed illness in another large outbreak of acute infectious nonbacterial gastroenteritis which occurred in a nearby county. This agent is morphologically similar to but serologically unrelated to the Norwalk and Hawaii gastroenteritis agents and has been designated the Marin agent pending further classification.

A virus in Beechey ground squirrels that is related to hepatitis B virus of humans.

A virus given the name ground squirrel hepatitis virus (or GSHV), with many of the unique characteristics of human hepatitis B virus (HBV), has been found in Beechey ground squirrels in northern California. Common features include virus morphology, viral DNA size and structure, a virion DNA polymerase that repairs a single-stranded region in the viral DNA, crossreacting viral antigens, and persistent infection with viral antigen continuously in the blood. Although similar, GSHV and HBV Are not identical. The ground squirrel virion has a slightly greater diameter, the viral surface antigens crossreact only partially and, thus, are not identical, and GSHV DNA has two restriction endonuclease EcoRI cleavage sites in contrast to the single site in HBV DNA. Thus, GSHV is a member of the virus group that includes HBV and the virus recently found in woodchucks in the eastern United States and named woodchuck hepatitis virus. It is not yet known how closely the ground squirrel and woodchuck viruses are related.

Antigenic comparisons of two new rotaviruses from rhesus monkeys.

Two rotavirus strains isolated in cell culture from infant rhesus monkeys with diarrhea were closely related to SA 11 virus and to each other by plaque reduction neutralization tests. However, results of immune electron microscopy suggested possible antigenic differences between the two rhesus rotavirus strains.

Virus detection in monkeys with diarrhea: the association of adenoviruses with diarrhea and the possible role of rotaviruses.

To explore the role of viruses in the etiology of diarrhea in colony-reared monkeys, direct electron microscopy, the fluorescent virus precipitin test and cell culture inoculation were used to examine the stools of monkeys with and without diarrhea. The animals were predominantly rhesus with a few macaques of other species, and included infants, juveniles and adults. Adenoviruses were isolated from a higher proportion of specimens from rhesus monkeys with diarrhea (73% of specimens from infants and 78% of specimens from juveniles and adults) than from control monkeys without diarrhea (22% of specimens from infants and 26% of specimens from juveniles and adults). SV 20 was the most frequently isolated simian adenovirus type; SV 17 and SV 32 also were recovered. Noncultivable adenoviruses detectable only by electron microscopy were not seen. Although adenovirus excretion was associated with diarrhea, the causal role of adenoviruses was difficult to assess. When serial specimens from animals with chronic or intermittent episodes of diarrhea were examined, sequential infections with different viruses were found to be common. Rotaviruses were detected by electron microscopy and isolated in cell cultures from two infant rhesus monkeys with diarrhea. However, the low detection rate, together with negative serologic data on 40% of infant monkeys with diarrhea, suggested that rotaviruses were not the major cause of gastroenteritis in the monkeys under study.

Cyclic expression of antigen and infectious virus in a BHK cell line (0-853) persistently infected with an SSPE strain of measles virus.

Establishment and characteristics of a baby hamster kidney cell line (BHK 0-853) persistently infected with a subacute sclerosing panencephalitis (SSPE) strain of measles virus (Lec strain) is described. The persistent infection was easily and repeatedly established and no special conditions were required. There was a predictable fluctuation in expression of virus intracellular and membrane antigens which varied from greater than 90% to less than 1% of the cells demonstrating these antigens during the first 6 or 7 passages. Thereafter, fluctuation of antigen and infectious virus expression continued in an unpredictable fashion.