Use of modified nucleotides and uracil-DNA glycosylase (UNG) for the control of contamination in the PCR-based amplification of RNA.

The inadvertent carryover of amplified fragments of nucleic acids (amplicons) is a potential source of contamination in the polymerase chain reaction. Recently, a method has been developed to generate amplicons with deoxyuracil triphosphate (dUTP) and to specifically hydrolyze these amplicons with uracil-DNA glycosylase (UNG) following the completion of the assay. We evaluated this system for the specific amplification of RNA from coxsackievirus A3 and B3. We found that RNA from both viruses could be amplified with dUTP, although the use of this triphosphate in place of TTP resulted in some loss of assay sensitivity. We also found that the dUTP-containing amplicons could be efficiently hydrolyzed by UNG, resulting in a 10,000,000-fold reduction in amplicon concentration with little effect on the native nucleic acid. The dUTP-UNG method has a great deal of potential for reducing amplicon contamination during the routine performance of nucleic acid amplification reactions.     

Persistent diarrhea and fecal shedding of retroviral nucleic acids in children infected with human immunodeficiency virus

Gastrointestinal dysfunction is a serious problem in many children infected with human immunodeficiency virus (HIV), the etiology of which has not been clearly defined. Quantitative nucleic acid amplification was used to study the correlation between shedding of HIV nucleic acids and gastrointestinal symptoms in HIV-infected infants and children. Many with HIV infection and persistent diarrheal disease shed HIV nucleic acids in their feces, as did an HIV-infected patient without apparent diarrheal disease. HIV nucleic acids were not found in feces of non-HIV-infected individuals. Intestinal infection with HIV appears to be important in the pathophysiology of gastrointestinal dysfunction in infants and children with HIV infection. Furthermore, the fecal shedding of HIV may play a role in HIV transmission in environments prone to high levels of fecal-oral contamination.     

Allergenicity of orally administered immunoglobulin preparations in food-allergic children

Passive immunization by the oral administration of immunoglobulin preparations derived from bovine milk, chicken egg, and human sera has been proposed as a method for the prevention and treatment of enteric diseases. However, the allergenic potential of these proteins may be a factor limiting their widespread use for disease prevention. An in vitro study with sera from milk- and egg-allergic children was performed to determine whether these immunoglobulin preparations have allergenic potential. Protein extracts of milk, bovine immunoglobulin, egg white, human immune globulin, and five egg yolk antiviral immunoglobulin preparations were bound to nitrocellulose paper. These preparations were probed for specific IgE binding with sera from milk- and egg-allergic patients. Of 22 milk-hypersensitive patients, 16 had specific IgE binding against the bovine immunoglobulin preparation. Of 28 egg-allergic patients 15 had specific IgE binding against one or more of the egg yolk-derived antiviral chicken immunoglobulins. Control sera were negative against the milk and egg preparations. Western blot analysis confirmed that milk- and egg-allergic patients had IgE-specific antibodies for bovine and chicken immunoglobulin molecules. Therefore, the removal of contaminating proteins from milk and egg antibody preparations would be unlikely to eliminate their allergenic potential. In contrast, sera from milk- and egg-allergic patients displayed no detectable IgE binding to human immunoglobulin preparations. These data indicate that the administration of antibody preparations derived from bovine and chicken sources may lead to severe allergic reactions in milk- or egg-sensitized patients and to sensitization in some nonallergic individuals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzyme Immunoassays for Measurement of Cytomegalovirus Immunoglobulin M Antibody

The diagnosis of congenital cytomegalovirus (CMV) infection is often accomplished by the detection of circulating antibody directed against CMV. We devised a method for measuring CMV-specific immunoglobulin M (IgM) based on the isolation of IgM antibody by reaction with a solid phase coated with antihuman IgM. The determination of IgM antibody specific for CMV was accomplished by the subsequent addition of CMV or control antigen and enzyme-labeled CMV antibody (solid phase-IgM method). We compared the sensitivity and specificity of this method with those of a conventional form of solid-phase enzyme immunoassay in which CMV antigen is bound to the solid phase (solid phase-antigen method). Both assay systems were capable of detecting CMV-specific IgM antibody in the sera of 10 babies with documented CMV infection and in those of the mothers of 4 of these babies. The solid phase-IgM method yielded negative results in all 66 sera available from babies who did not have congenital CMV infection. On the other hand, the solid phase-antigen system yielded false-positive results in 12 (18%) of these sera. In addition, the solid phase-antigen system yielded false-positive results in 8 of 12 sera obtained from patients with demonstrable rheumatoid factor. However, the solid phase-IgM system yielded negative results for the rheumatoid sera, provided that appropriate control reactions were performed. The solid phase-IgM system is thus a specific and sensitive method for the determination of CMV IgM antibody.     

PCR-Enzyme Immunoassay for Detection of Streptococcus pneumoniae DNA in Cerebrospinal Fluid Samples from Patients With Culture-Negative Meningitis

A PCR-based assay was developed to amplify a conserved region of the pneumococcal autolysin gene. The amplified product was labelled with digoxigenin-labelled dUTP and was detected with a biotin-labelled probe in an enzyme immunoassay (EIA). The assay was initially tested with suspensions of various serotypes of Streptococcus pneumoniae and other gram-positive and gram-negative bacteria and was then applied to cerebrospinal fluid (CSF) specimens from patients with meningitis and those with other neurological disorders. The assay detected all the serotypes of S. pneumoniae tested, whereas all the other bacterial strains tested were negative. Seven of the 8 CSF specimens positive for pneumococcus by culture or latex agglutination (LA) were positive by PCR-EIA, whereas all 10 specimens positive for other organisms were negative. Among 11 patients with clinically diagnosed meningitis but with negative culture and LA results, 5 were positive by PCR-EIA. The assay was negative for all but one patient without meningitis; it was positive with the CSF from a child with immunodeficiency and pneumococcal abscesses on the scalp. PCR-EIA is a useful tool for the diagnosis of meningitis, especially when culture and LA are negative because of prior antibiotic treatment.     

Comparison of fluorescent and colorigenic substrates for enzyme immunoassays.

A variety of substrates can be employed in enzyme immunoassays (EIAs) for the measurement of enzyme-labeled immunoreactants. We compared the sensitivities of a fluorescent and a colorigenic substrate in an EIA system for the measurement of Haemophilus influenza purified polyribose phosphate. After a 10-min substrate incubation, the EIA in which the fluorescent substrate was used could detect 10 pg of polyribose phosphate per ml, whereas the EIA in which the colorigenic substrate was used required the addition of 640 pg of polyribose phosphate per ml to generate a positive reading. However, the use of longer substrate incubation periods led to an increase in sensitivity of the colorigenic EIA. After an incubation period of 240 min, the sensitivity was equal to that of the EIA in which the fluorescent substrate was used. These results suggest that the ultimate limit of sensitivity of EIA systems is determined by the nature of the antigen-antibody reactions. However, the use of high-energy substrates in EIA systems can allow for the attainment of maximal sensitivity after short enzyme-substrate incubation periods.     

Whole body protein turnover in children with human immunodeficiency virus (HIV) infection

The purpose of this study was to determine the rate of whole body protein turnover (WBPT) in human immunodeficiency virus (HIV)-infected children, and to determine the relationship between WBPT and growth. The rate of WBPT was calculated from the cumulative excretion of labeled urinary ammonia after a single intravenous dose of 15N-glycine in three groups of children: 1) HIV+ with growth retardation (HIV+ Gr); 2) HIV+ with normal growth (HIV+); and 3) HIV-uninfected with normal growth (HIV-). Twenty-six children between 2 and 11 y of age were studied (10 HIV+ Gr, 12 HIV+, 4 HIV-). All children were afebrile and free of acute infection during the study. Rates of WBPT (mean +/- SD) for the study groups were: HIV+ Gr, 12.2 +/- 4.8; HIV+, 10.7 +/- 5.1; and HIV-, 8.6 +/- 2.1 g.protein.kg-1.d-1 (NS, P > 0.05). Although not statistically significant, mean WBPT was 42% greater in HIV+ Gr, and 24% greater in HIV+ compared to HIV-. Statistically significant correlations were found between WBPT and Z scores for height (r = -0.39, P = 0.05) and weight-for-age (r = -0.51, P = 0.01) and dietary intake of protein (r = 0.39, P = 0.05), and between protein balance (synthesis-catabolism) and intakes of energy (r = 0.47, P = 0.02) and protein (r = 0.40, P = 0.04). There was no statistically significant correlation between WBPT and resting energy expenditure (r = 0.27, P = 0.19), or CD4 cell number (r = 0.05, P = 0.82). These data suggest an association between increased rates of protein turnover and low weight and height-for-age Z scores, and that it may be possible to achieve positive protein balance given an adequate intake of nutrients.     

Disease-specific alterations in frontal cortex brain proteins in schizophrenia, bipolar disorder, and major depressive disorder. The Stanley Neuropathology Consortium

Severe psychiatric disorders such as schizophrenia, bipolar disorder and major depressive disorder are brain diseases of unknown origin. No biological marker has been documented at the pathological, cellular, or molecular level, suggesting that a number of complex but subtle changes underlie these illnesses. We have used proteomic technology to survey postmortem tissue to identify changes linked to the various diseases. Proteomics uses two-dimensional gel electrophoresis and mass spectrometric sequencing of proteins to allow the comparison of subsets of expressed proteins among a large number of samples. This form of analysis was combined with a multivariate statistical model to study changes in protein levels in 89 frontal cortices obtained postmortem from individuals with schizophrenia, bipolar disorder, major depressive disorder, and non-psychiatric controls. We identified eight protein species that display disease-specific alterations in level in the frontal cortex. Six show decreases compared with the non-psychiatric controls for one or more diseases. Four of these are forms of glial fibrillary acidic protein (GFAP), one is dihydropyrimidinase-related protein 2, and the sixth is ubiquinone cytochrome c reductase core protein 1. Two spots, carbonic anhydrase 1 and fructose biphosphate aldolase C, show increase in one or more diseases compared to controls. Proteomic analysis may identify novel pathogenic mechanisms of human neuropsychiatric diseases.     

Diarrhea associated with rotavirus in rural Guatemala: a longitudinal study of 24 infants and young children.

A population of 24 infants and young children followed prospectively during the first 3 years of life was studied for the occurrence of rotavirus infection by using enzyme-linked immunosorbent assay to detect virus in stools. Infection with rotavirus was associated with 26 (14.2%) of 183 selected diarrheal episodes. Twenty of the 24 infants and young children had diarrhea associated with rotavirus on at least one occasion and six had two such episodes. Rotavirus infection was documented in over 50% of the dehydrating episodes studied, thus further indicating the importance of rotavirus in this population.     

Influence of maternal infections on neonatal acute phase proteins and their interaction in the development of non-affective psychosis

Although primary infections with Toxoplasma gondii or herpes viruses during pregnancy are established teratogens, chronic maternal infections with these pathogens are considered far less serious. However, such chronic infections have been associated with neuropsychiatric disorders in the offspring. The risks of non-affective psychoses, including schizophrenia, in offspring associated with these exposures during pregnancy have not been completely defined. We used data from neonatal dried blood samples from 199 cases of non-affective psychosis and 525 matched controls (born 1975–1985). We measure immunoglobulin G antibodies directed at T. gondii, cytomegalovirus and herpes simplex virus type-1 and -2, as well as levels of nine acute phase proteins (APPs). We assessed the interaction between maternal antibodies and neonatal APP in terms of risk of non-affective psychosis. Among controls, maternal exposure to T. gondii or cytomegalovirus, but not to the other herpes viruses, was associated with significantly higher levels of neonatal APPs. Among cases, none of the maternal exposures were associated with any significant change in APPs. We observed increased RR for non-affective psychosis associated with maternal infection with T. gondii (odds ratio 2.1, 95% confidence interval 1.1–4.0) or cytomegalovirus (1.7, 0.9–3.3) only among neonates with low APP levels. These findings suggest that chronic maternal infection with T. gondii or cytomegalovirus affect neonatal markers of innate immunity. Deficient fetal immune responses in combination with maternal chronic infections may contribute to subsequent risk for psychosis. A greater understanding of the maternal–fetal immunological interplay may ultimately lead to preventive strategies toward neuropsychiatric disorders.