Impaired innate immunity in the newborn: newborn neutrophils are deficient in bactericidal/permeability-increasing protein.

OBJECTIVE: The mechanisms by which newborns are at increased risk for invasive bacterial infections have been incompletely defined. A central element of innate immunity to bacterial infection is the neutrophil-a cell that contains cytoplasmic granules replete with antibiotic proteins and peptides. The activity of adult neutrophils against gram-negative bacteria is believed to depend to a significant degree on the presence in neutrophil primary (azurophilic) granules of the 55-kDa bactericidal/permeability-increasing protein (BPI), which binds with high affinity to bacterial lipopolysaccharides and kills gram-negative bacteria. In light of the importance of BPI to antibacterial host defense and to investigate possible factors underlying the risk of neonatal bacterial infections, we determined the relative content of BPI in the neutrophils of adults and newborns. DESIGN: The cellular content of BPI was determined by Western blotting of neutrophils derived from full-term newborn cord blood (n = 21; mean gestational age: 38.6 weeks) and from adult peripheral blood (n = 22; mean age: 29 years). Extracellular levels of BPI in adult and newborn plasma were assessed by enzyme-linked immunosorbent assay. Neutrophil content of other azurophil granule markers also was assessed: myeloperoxidase by Western blotting and defensin peptides by acid-urea polyacrylamide gel electrophoresis and Coomassie staining. Acid extracts of newborn and adult neutrophils were analyzed for antibacterial activity against serum-resistant encapsulated isolate Escherichia coli K1/r. RESULTS: The neutrophils of newborns contain at least threefold to fourfold less BPI per cell than adult neutrophils (67 +/- 13 ng per 10(6) cells vs 234 +/- 27 ng per 10(6) cells). The relative BPI-deficiency of newborn neutrophils apparently was not attributable to perinatal stress-related degranulation of intracellular BPI stores because: 1) newborn and adult neutrophils contained nearly identical amounts of 2 microbicidal constituents derived from the same primary (azurophil) granule compartment as BPI (the enzyme myeloperoxidase as well as defensin peptides), and 2) levels of extracellular BPI in newborn plasma, measured by enzyme-linked immunosorbent assay, represent only approximately 2% of cellular BPI content. As predicted by their lower BPI content, newborn neutrophil acid extracts demonstrated significantly lower antibacterial activity against E coli K1/r than did adult neutrophil acid extracts. CONCLUSION: These data suggest that the neutrophils of newborns are selectively deficient in BPI, a central effector of antibacterial activity against gram-negative bacteria. BPI deficiency correlates with decreased antibacterial activity of newborn neutrophil extracts against serum-resistant E coli and could contribute to the increased incidence of gram-negative sepsis among newborns relative to healthy adults.neonatal sepsis, gram-negative bacteria, endotoxin, neutrophil, polymorphonuclear leukocyte, innate immunity, bactericidal/permeability-increasing protein, defensin, myeloperoxidase.

PHA production, from bacteria to plants.

The genes encoding the polyhydroxyalkanoate (PHA) biosynthetic pathway in Ralstonia eutropha (3-ketothiolase, phaA or bktB; acetoacetyl-CoA reductase, phaB; and PHA synthase, phaC) were engineered for plant plastid targeting and expressed using leaf (e35S) or seed-specific (7s or lesquerella hydroxylase) promoters in Arabidopsis and Brassica. PHA yields in homozygous transformants were 12-13% of the dry mass in homozygous Arabidopsis plants and approximately 7% of the seed weight in seeds from heterozygous canola plants. When a threonine deaminase was expressed in addition to bktB, phaB and phaC, a copolyester of 3-hydroxybutyrate and 3-hydroxyvalerate was produced in both Arabidopsis and Brassica.

Immune response to Escherichia coli O157:H7 in hemolytic uremic syndrome following salmonellosis.

Escherichia coli O157:H7, a Shiga-like toxin (SLT)-producing enteric pathogen, has been implicated in most cases of post-diarrheal hemolytic uremic syndrome (D + HUS). Infection with other bacterial pathogens such as Salmonella has also preceded D + HUS episodes, leading to speculation that these organisms may also be etiological. We present two children with unrelated D + HUS following salmonellosis. Both children had negative stool cultures on sorbitol-MacConkey agar soon after the onset of diarrhea. After the diagnosis of HUS, both patients had repeat stool cultures positive for Salmonella alone. Polymerase chain reactions for SLT I and II gene sequences in Salmonella isolates were negative. Enzyme-linked immunosorbent assay for specific humoral response to E. coli O157:H7 lipopolysaccharide in acute and convalescent serum samples revealed evidence of heretofore undetected E. coli O157:H7 infection contemporaneous with each D + HUS episode. These cases demonstrate that isolation of only non-SLT-producing microbes from children with D + HUS should raise suspicion of concurrent undetected infection with SLT-producing organisms. Assaying specific immune response to E. coli O157:H7 can be an important epidemiological adjunct. Bacterial infection with non-SLT-producing Salmonella may represent concomitant enteric pathology rather than D + HUS-instigating infection.

Chronic parvovirus B19 infection resulting in chronic fatigue syndrome: case history and review.

The spectrum of disease caused by parvovirus B19 has been expanding in recent years because of improved and more sensitive methods of detection. There is evidence to suggest that chronic infection occurs in patients who are not detectably immunosuppressed. We report the case of a young woman with recurrent fever and a syndrome indistinguishable from chronic fatigue syndrome. After extensive investigation, we found persistent parvovirus B19 viremia, which was detectable by polymerase chain reaction (PCR) despite the presence of IgM and IgG antibodies to parvovirus B19. Testing of samples from this patient suggested that in some low viremic states parvovirus B19 DNA is detectable by nested PCR in plasma but not in serum. The patient's fever resolved with the administration of intravenous immunoglobulin.

Primary antral gastritis in young American children. Low prevalence of Helicobacter pylori infections.

Although Helicobacter pylori (H. pylori) is considered by many to be the major cause of primary antral gastritis (PAG), several important questions concerning its pathogenetic role remain unanswered. The most basic unresolved issue relates to the low prevalence of H. pylori in children in developed countries. If H. pylori is the cause of PAG, the prevalence of PAG should also be low, but previous studies have not provided data on this issue. To answer this question, we prospectively studied 408 children who underwent esophagogastroduodenoscopy and biopsy from January 1, 1988, to December 31, 1990, for symptoms consistent with peptic disease or immunocompromise. Although the prevalence of PAG was similar (about 20%) in the four age groups of patients studied (< 5, 5-9, 10-14, and 15-20 years), the prevalence of H. pylori infections was significantly greater in the oldest age group (67%, P < 0.0001). Only four of 39 children < 10 years old with PAG had evidence of H. pylori. H. pylori is an uncommon finding in our population of young American children with PAG, indicating that it does not play an important role in the pathogenesis of this disorder in this age group.

Humoral immune responses to Shiga-like toxins and Escherichia coli O157 lipopolysaccharide in hemolytic-uremic syndrome patients and healthy subjects.

Shiga-like-toxin (SLT)-producing Escherichia coli strains, especially serotype O157:H7, are important causes of bloody diarrhea and are associated with the development of hemolytic-uremic syndrome (HUS). Enzyme-linked immunosorbent assays (ELISAs) were developed for the serologic detection of immunoglobulin M (IgM), IgG, and IgA to Shiga toxin (ST) and SLT-I, IgG to SLT-II, and IgM and IgG reactive against E. coli O157 lipopolysaccharide (LPS). Serum samples were collected from 27 HUS patients (25 pediatric and 2 adult) and tested in the ELISAs. Of 27 patients, 10 (37%) were positive for at least one class of antibody to ST/SLT-I. None of the patients were positive for IgG antibody to SLT-II. Twenty-one of the 27 patients (78%) were positive for antibody to E. coli O157 LPS; 19 of 27 (70%) were positive for IgM, and 20 of 27 (74%) were positive for IgG. None of 48 control serum samples were positive in any of the toxin assays, and only 1 of 48 (2%) and 2 of 48 (4%) were positive for IgM and IgG, respectively, to E. coli O157 LPS. Twelve of the 24 patients (50%) from whom stool specimens were collected were positive by culture for E. coli O157. Overall, serology and culture produced confirmation of infection by SLT-producing organisms in 23 of 27 (85%) HUS patients. A combination of ELISA for antibodies to E. coli O157 LPS and culture provided evidence for 22 of 27 (82%) of these patients. The results indicate that while ELISAs for ST/SLT-I and SLT-II antibodies were of limited diagnostic value, the ELISAs for IgM and IgG to E. coli O157 LPS provided valuable and sensitive adjuncts to culture.

Evaluation of an automated immunodiagnostic assay, VIDAS Rotavirus, for detection of rotavirus in fecal specimens.

Four hundred sixty-four fecal specimens were tested for rotavirus by three immunoassays, VIDAS Rotavirus (bioMérieux Vitek, Hazelwood, Mo.), Rotaclone (Cambridge Biotech Corporation, Worcester, Mass.), and Pathfinder Rotavirus (Sansfi Diagnostics Pasteur, Chaska, Minn.). Twenty-seven discordant specimens were evaluated by electron microscopy. The sensitivities, specificities, positive predictive values, and negative predictive values were 98, 99.3, 98.7, and 99%, respectively, for VIDAS Rotavirus; 100, 99, 98.1, and 100%, respectively, for Rotaclone; and 100, 92.4, 87.3, and 100%, respectively for Pathfinder Rotavirus.

Multicenter evaluation of a new enzyme immunoassay for detection of Clostridium difficile enterotoxin A.

The Premier Clostridium difficile toxin A enzyme immunoassay (PTA EIA) (Meridian Diagnostics, Inc., Cincinnati, Ohio) for rapid diagnosis of antibiotic-associated colitis (AAC) was evaluated in a multicenter study. Stool samples from 421 patients suspected of having AAC were tested for toxin A by the PTA EIA and for toxin B by three tissue culture assays (TCA) employing WI-38 cells (New England Deaconess Hospital) in conventional tubes or foreskin fibroblasts (Children's Hospital) or Vero cells (Beth Israel Hospital) in microwells. The tubes and plates were examined at 24 and 48 h for cytotoxicity. Clinical criteria, repeat testing at another site, and culture of frozen stool samples for C. difficile were used to evaluate discrepant results. Of 504 samples, 66 were positive and 409 were negative by both tests. Eight samples had indeterminate PTA EIA results and were excluded from this analysis. Of 21 discrepancies, 9 were PTA EIA positive and TCA negative and 12 were PTA EIA negative TCA positive. Following resolution of the discrepancies, 11 of 12 PTA EIA-negative-TCA-positive and 5 of 9 PTA EIA-positive-TCA-negative samples were considered true positive for AAC. The sensitivity and specificity were, respectively, 86.6 and 99.0% for the PTA EIA and 93.9 and 99.8% for TCA. The predictive values of positive and negative tests were, respectively, 94.7 and 97.4% for the PTA EIA and 98.7 and 98.8% for TCA. We conclude that the PTA EIA is a rapid, simple EIA technique whose accuracy in detecting enterotoxin A approaches that of reference TCA methods for detection of cytotoxin B.

Rapid detection of group A streptococci: comparative performance by nurses and laboratory technologists in pediatric satellite laboratories using three test kits.

Rapid tests for detecting group A streptococci in throat swabs are often performed outside hospitals or commercial laboratories by individuals with little or no technical training. We compared the abilities of nurses and technologists to perform and interpret three commercial kits (Directigen 1-2-3, ICON Strep A, and Culturette Brand 10-Minute Strep A ID) in three hospital satellite locations (the emergency department, a walk-in emergency clinic, and a general pediatric clinic). When the three tests were compared with culture, the sensitivities of the tests as performed by nurses and technologists, respectively, were 39 versus 44% for Directigen, 55 versus 51% for Culturette, and 72 versus 39% for ICON. A significant difference in sensitivity was found only with ICON tests. This result was largely explained by the tendency of technologists to test moist swabs, while nurses generally processed dry swabs; ICON test sensitivity was significantly greater with dry swabs. The specificities of Directigen and ICON tests performed by nurses and technologists were high (97 to 100%). The difference in the specificities of the Culturette test as determined from results obtained by nurses and technologists (80 versus 98%) was due to the tendency of one nurse to overinterpret the latex agglutination reaction. Analysis of the accuracies of the tests during practice periods compared with the accuracies of the tests during the study periods revealed statistically significant improvement in test performance. We conclude that these tests are specific but not sensitive when performed by nurses and technologists in satellite laboratories. With one exception, nurses and technologists performed the tests with comparable accuracy after brief training periods.

Enzymatically labelled nucleic acid (NA) probe assays for detection of Campylobacter spp. in human faecal specimens and in culture.

Seventy-two stock clinical isolates of C. jejuni were tested with the SNAP Culture Identification Test Kit (Molecular Biosystems, Inc., San Diego, CA), an enzyme labelled nucleic acid probe colony blot assay, and with the Campyslide (BBL Microbiology Systems, Cockeysville, MD), a latex agglutination test. The sensitivity and overall agreement of both the SNAP and Campyslide tests were 100% in comparison with standard culture and identification tests. In addition, 14 C. jejuni, two C. laridis, and one 'C. upsaliensis', and one C. hyointestinalis, all fresh isolates from acutely ill patients, were detected with both tests. Ninety-eight faecal specimens from acutely ill patients were tested within 48 h using the probe based SNAP Diagnostic Test Kit (Molecular Biosystems, Inc.). Results were compared with standard culture. The probe test detected 4/6 specimens positive by culture; sensitivity was 66%. However, the two culture positive specimens, undetected by the probe test, had pathogens present at levels well below the probe test sensitivity (10(5) CFU g-1 stool). Specificity of the probe test was 98% (90/92 negative specimens) compared to culture. The results of this study demonstrate the accuracy of enzyme labelled oligonucleotide probes in the identification of C. jejuni isolated on culture and suggest a potential role for such probes in the direct detection of campylobacter in clinical faecal specimens.

Microbial challenge of a blood cell separator outside-seal bowl system.

The ability to store platelets beyond 24 hours requires a functionally closed system. This study tested the ability of a cell separator bowl seal system to resist penetration of microbial contamination under normal running conditions and under extreme environmental stress. Three test organisms, Micrococcus luteus, Serratia marcescens, and Staphylococcus epidermidis, were applied directly to the bowl at the edge of the seal or aerosolized and passed through the centrifuge chamber while the cell separator was run through a simulated platelet collection. A sterile, bacteriologic nutrient medium was perfused through the tubing set, thus simulating the flow of blood fractions. Following the procedure, the medium was examined for microbial growth. The concentration of aerosolized bacteria ranged from 5.2 x 10(1) to 3.9 x 10(3) colony-forming units (CFU) per mL, and the concentration of bacteria applied to the edge of the seal ranged from 1.9 x 10(5) to 2.8 x 10(9) CFU per mL. The positive control, direct inoculation of S. marcescens into the circulating medium (50 CFU/500 mL), resulted in recovery of the identical organism after 24 hours' incubation. No contamination of the system was detected in 40 experiments with aerosolized bacteria or in 32 experiments in which bacteria were applied directly to the seal. This study demonstrates that this sealed-bowl system resists microbial contamination.

General method for site-directed mutagenesis in Escherichia coli O18ac:K1:H7: deletion of the inducible superoxide dismutase gene, sodA, does not diminish bacteremia in neonatal rats.

A defined deletion in the Escherichia coli K-12 sodA gene (encoding manganese-superoxide dismutase) linked to a nontransposable selectable marker was generated by transposon Tn5 insertion in combination with in vitro mutagenesis. This mutant allele was used to replace the wild-type sodA gene in an E. coli clinical isolate of serotype O18ac:K1:H7 by bacteriophage P1 transduction. The O18ac:K1:H7 sodA mutant contained no manganese-superoxide dismutase and no hybrid manganese-iron-superoxide dismutase. The sodA mutant was more sensitive to paraquat toxicity than were the parental strain and an isogenic mutant bearing an analogously constructed sodA+ Tn5 insertion allele. In a suckling rat model for bacteremia following oral inoculation of E. coli K1, the sodA mutant was undiminished in its capabilities both to colonize the gastrointestinal tract and, surprisingly, to cause bacteremia. In conjunction with the rat model for E. coli K1 pathogenesis, the method for site-directed mutagenesis described in this paper permits determination of the role played in colonization and bacteremia by any K1 gene which either has a homolog in E. coli K-12 or can be cloned and manipulated therein.

Diagnosis of infectious diarrheal diseases.

For many years, microbiologic examination of feces was focused on the isolation of two members of the family Enterobacteriaceae--Salmonellae and Shigellae. Over the past two decades, other enteric pathogens such as the various classes of diarrheagenic E. coli, Campylobacter, Vibrio spp., Yersinia enterocolitica, and Clostridium difficile have gained prominence. A newly recognized protozoan parasite, Cryptosporidium is now known to infect both immunocompetent and immunodeficient individuals. Added to these is the growing list of viruses incriminated in diarrheal diseases and gastritis. This article provides some of the basic information needed to allow for the isolation and identification of many of the currently recognized enteric pathogens. It also provides information on some of the currently available rapid test procedures that will speed up stool specimen testing and allow for more timely reporting to the physician.

Performance of a solid phase enzyme immunoassay for detection of group A streptococci in a pediatric office laboratory as refereed by a hospital laboratory.

We evaluated the performance of a new rapid solid phase enzyme immunoassay, SUDS Group A Strep (MUREX Corp., Norcross, GA) for the detection of Group A beta-hemolytic streptococci in a pediatric office practice. Duplicate throat swabs were obtained from 341 children with pharyngitis. One swab was used in the SUDS test and the other was cultured in the office laboratory. Office SUDS and culture (sheep blood agar plate, aerobic 24-hour incubation) were compared with culture using reference techniques (sheep blood agar plate, anaerobic 48-hour incubation) in a hospital laboratory. Compared with hospital laboratory culture, the sensitivity of office SUDS (73.8%) was superior to that of office culture (66.6%) at P = 0.05. Specificities were 93.1 and 98.6%, respectively; positive predictive values were 86.1 and 96.6%; and negative predictive values were 85.9 and 83.5%. The sensitivity and specificity of SUDS compared with office culture were 88.5 and 87.8%, respectively, but would have been 93 and 94% had hemolyzed media not been used on several occasions in the office culture procedure. We conclude that SUDS Group A Strep was significantly more sensitive than throat cultures as performed in a typical pediatric practice although the performance of office cultures could have been improved by standard quality control techniques.

Evaluation of a latex agglutination test for diagnosis of Clostridium difficile-associated colitis.

Current methods for diagnosis of Clostridium difficile-associated colitis (CAC) based on detection of cytotoxin B by a tissue culture assay (TCA) require technical expertise and up to 48 hours incubation. Recently, a latex agglutination (LA) test (Marion Laboratories) for rapid diagnosis of CAC has become available. Although early evaluations have been favorable, new evidence suggests that the LA reagent binds a soluble bacterial antigen that is not unique to toxigenic strains of C. difficile. The authors examined 201 stools received for CAC testing by LA and a reference TCA and investigated discrepant results. They obtained 29 LA(+)/TCA(+) and 155 LA(-)/TCA(-) results. Eleven patients had LA(+)/TCA(+) and 155 LA(-)/TCA(-) results. Eleven patients had LA(+)/TCA(-) results and 6 had LA(-)/TCA(+) results. The sensitivity and specificity of the LA were 83% and 93%, respectively, compared with TCA. The predictive values of positive and negative results obtained with the LA were 72% and 96%, respectively. Concentrated broth supernatants and live suspensions of three C. difficile isolates with LA(+)/TCA(-) results were tested in a rabbit ileal loop assay. All failed to demonstrate ability to produce an enterotoxin. The authors conclude that the LA method is suitable for rapid screening, but LA(+) results require confirmation by testing with other methods.

Mutagenicity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in the granuloma pouch assay.

The rat granuloma pouch assay was used to assess the in vivo mutagenic potential of 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ), a heterocyclic aromatic amine which is formed during the frying of meat and broiling of fish. The assay was performed with and without pre-induction by Aroclor. In the initial experiment IQ was injected directly into the pouch of non-induced rats. A 10-fold increase in mutation frequencies was obtained with the 2.0 mg/pouch dose of IQ with uninduced cell populations. In a second study IQ was injected intraperitoneally and into the pouch of rats that had been pre-induced with Aroclor. The dose of IQ administered varied from 0.1 to 2.0 mg/pouch. A 10-fold increase in mutation frequencies was obtained with the 2.0 mg/pouch dose of IQ with uninduced cell populations. Aroclor treatment produced no significant increase in mutation frequencies over uninduced animals. Its mutagenic effect is about 10-fold weaker than that of benzo[a]pyrene or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).

Gastrointestinal infections--dietary interactions.

Considerable progress has been made in understanding the complexities involved in the production of bacterial diarrheal diseases. The general mechanisms of disease that have been recognized include enterotoxigenicity, enteroadherence, and invasiveness. The interplay of epithelial cell surface receptors with the surface components of the various bacterial pathogens or their toxins will be reviewed. Knowledge of the stereospecific interactions of bacterial ligands with the eukaryotic receptors has led to the development of new strategies for prevention and therapy. The presence of foodstuffs in the intestinal lumen can contribute by a number of mechanisms to interference with the invading organism's attack on the intestinal cell surfaces. The effects of milk fat and plant lectins on the colonization of the bowel by enteric organisms is discussed.

Effect of tetracycline on the attachment of K88+ enterotoxigenic Escherichia coli to porcine small-intestinal cells.

The attachment of six strains of K88+, porcine pathogenic, enterotoxigenic Escherichia coli to isolated porcine intestinal mucosal cells was decreased following growth in the presence of concentrations of oxytetracycline below the minimal inhibitory concentration (MIC). The decrease in binding by the wild-type strains was detected at concentrations of drug as low as 0.001 microgram/ml, which was greater than four orders of magnitude below the MIC. When drug resistance was induced in these six strains, there was still a decrease in binding when the bacteria were grown in the presence of tetracycline. This decrease was comparable to the decrease in binding capacity of the wild-type strains caused by growth in the presence of tetracycline. In contrast, when one strain (G1108E) was made tetracycline resistant by the introduction of the R16 plasmid, the antibiotic had less effect on the binding of this strain than on the wild-type strain; however, growth in the presence of antibiotic still decreased adhesion. Overall, oxytetracycline decreased the adhesion of wild-type, induced-resistant, and genetically resistant K88+ enterotoxigenic E. coli to porcine small-intestinal cells, and this effect occurred at antibiotic concentrations several orders of magnitude below the MIC.