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Biomedical subjects

R Yolken

Publications and source records attributed to R Yolken.

11 recordsLinked to original sources
PubMed1992

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.

Artifacts↗
PubMed1992

Detection of rotaviruses in the day care environment by reverse transcriptase polymerase chain reaction.

Group A rotavirus is an important cause of morbidity among infants and toddlers in day care centers. Transmission by the fecal-oral route is well established, but fomites and environmental surfaces may also play an important role in transmission. A highly sensitive polymerase chain reaction (PCR) assay was used to detect rotavirus RNA in day care environments. Areas sampled included floors, diaper change areas, toy balls, and other surfaces. In two centers undergoing outbreaks of rotavirus, 7 (39%) of 18 toy balls had detectable rotavirus as did 8 (21%) of 39 swabs from environmental surfaces. By comparison, only 1 (5%) of 21 toy balls and 1 (2%) of 44 environmental surface swabs had detectable rotavirus in centers without rotavirus outbreaks (P = .0001). Thus, rotaviruses are highly prevalent in day care centers during outbreaks of diarrhea. The monitoring of environments by sensitive nucleic acid amplification techniques may lead to strategies for the diminution of disease transmission within the day care environment.

Base Sequence↗
PubMed1991

Improved detection of rotavirus shedding by polymerase chain reaction.

To improve identification of children excreting rotavirus a method for the amplification of rotavirus RNA by the polymerase chain reaction (PCR) was developed. The assay was compared with a solid-phase enzyme immunoassay in the detection of rotavirus shedding by infants in hospital during the winter peak of rotavirus infections. Forty children were studied in an intermediate care unit after transfer from intensive care units. Only two were admitted primarily because of diarrhoea; the other thirty-eight were admitted for management of various other disorders. Rotavirus shedding was detected by enzyme immunoassay in twenty of the infants, and nine of these (aged 1 week to 8 months) remained in hospital for more than 5 days after the initial detection of rotavirus and could be studied long term. Of 103 faecal samples from the nine infants, 60 (58%) contained rotavirus RNA detected by reverse-transcriptase (RT)/PCR, whereas only 37 (36%) were positive for rotavirus antigen by the immunoassay (chi 2 = 10.3, p less than 0.002). The geometric mean time of rotavirus shedding was 9.5 (range 1-19) days as detected by RT/PCR and 5.7 (range 1-17) days by the immunoassay (p less than 0.018). In five of the nine children, RT/PCR detected rotavirus shedding for 2-7 days longer than the immunoassay and in four children RT/PCR was positive 1 or more days before rotavirus antigen was detected. Further studies should attempt to find out whether infected infants are capable of spreading wild-type virus during periods when they are not shedding antigen as detectable by enzyme immunoassay.

Antigens, Viral↗
PubMed1991

Efficacy and pharmacokinetics of intravenous immune globulin administration to high-risk neonates.

UNLABELLED: OBJECTIVE--To determine whether intravenous immune globulin (IVIG) administration modifies the incidence of infections in high-risk neonates. DESIGN--Randomized, double-blind study. SETTING--Neonatal intensive care unit at a tertiary care center. PARTICIPANTS: A total of 170 infants were enrolled, 82 of whom received IVIG and 88 of whom received the placebo preparation. Infants were stratified by birth weight into one of three groups (category 1, those weighing less than 1000 g; category 2, those weighing between 1000 and 1500 g; and category 3, those weighing more than 1500 g). INTERVENTIONS--Intravenous immune globulin (750 mg/kg of body weight), or albumin placebo was administered within 72 hours of admission to the tertiary care center and every 14 days thereafter until discharge from the neonatal intensive care unit or age 3 months. Serum IgG levels were measured and data collected relating to the incidence of systemic and localized infections and to the course of hospitalization. MEASUREMENTS AND MAIN RESULTS--The administration of IVIG had no major side effects and resulted in higher serum IgG levels in infants in all birth weight categories compared with infants receiving the placebo. Systemic infections developed in five IVIG-treated infants and five placebo-treated infants. Administration of immunoglobulin had no significant effect on the rate of localized infections or necrotizing enterocolitis. It also did not affect hospital course of the infants as measured by length of hospitalization or the number of days on assisted ventilation, supplemental oxygen, or antibiotics was required. CONCLUSIONS--The general administration of IVIG using this dosage regimen has limited effects on the clinical course of infants in a neonatal intensive care unit.

Bacteremia↗
PubMed1991

Detection of animal and human group B rotaviruses in fecal specimens by polymerase chain reaction.

A combined reverse transcriptase reaction-polymerase chain reaction (RT-PCR) was developed to achieve the sensitive detection of group B rotaviruses (GBR). Sequences derived from genomic segment 3 of the IDIR (intestinal disease of infant rats) strain of GBR permitted the detection of greater than or equal to 0.08 pg of purified IDIR genomic RNA (4,000 genome copies). Primers complementary to the terminal sequences of gene 11 of GBR strain ADRV (adult diarrhea rotavirus) allowed for the detection of as little as 0.008 pg of purified ADRV genomic RNA. Detection of heterologous strains of GBR was also observed with these primer pairs. IDIR gene 3 primers recognized greater than or equal to 8 pg of RNA from bovine GBR obtained from a variety of geographic locations. RNA from IDIR, but not bovine GBR, strains was detected by means of RT-PCR with ADRV gene 11 primers. Neither set of GBR primers was reactive in RT-PCR with fecal specimens containing group A rotaviruses or fecal specimens from uninfected controls. This RT-PCR assay permits the sensitive and specific detection of a variety of GBR in fecal specimens.

Animals↗
PubMed1990

Immunoglobulins and other modalities for the prevention and treatment of enteric viral infections.

Viral infections of the gastrointestinal tract constitute a major health problem during the first years of life. In addition to causing acute diarrhea, rotaviruses and other enteric viruses may be involved in the pathogenesis of necrotizing enterocolitis and other neonatal enteric diseases. There are several potential methods for the prevention and treatment of gastrointestinal viral infections. Antiviral immune globulins, administered by the parenteral or oral route, might prove useful for the inhibition of intestinal viral replication. Alternatively, specific glycoprotein inhibitors of viral-cell binding might be used to prevent the productive infection of intestinal epithelial cells. In addition, since many enteric viruses require proteolytic enzymes for protein cleavage, protease inhibitors may prove to be effective agents for the inhibition of intestinal viral replication. At this point in time, these methods have proven useful for the inhibition of rotavirus infection in experimental animals. The successful application of these and other methods for the prevention of enteric infections in humans might substantially reduce the morbidity and mortality associated with enteric diseases.

Animals↗
PubMed1990

Removal of inhibitory substances from human fecal specimens for detection of group A rotaviruses by reverse transcriptase and polymerase chain reactions.

A method was developed for the purification of rotavirus RNA from fecal extracts in order to permit the sensitive identification of group A rotavirus in fecal specimens by the polymerase chain reaction. Sequential reactions with reverse transcriptase and Taq polymerase with directed primers from rotavirus gene 6 yielded characteristic 259-base-pair fragments that were then visualized by silver stain on a polyacrylamide gel. As few as 500 genomic copies of purified rotavirus RNA could be detected in this manner. However, when the method was applied to fecal samples with added rotavirus virions, inhibition was noted in many of the fecal extracts which were tested. The inhibition could be reversed by dilution of the fecal extract, but sensitivity was also reduced by a corresponding dilutional factor. The inhibition was quantitatively removed by an added step in the extraction process that utilized chromatographic cellulose fiber powder (CF11 powder) to purify the rotavirus RNA during a series of rapid washing and elution steps. After CF11 purification, rotavirus RNA could be detected in experimental fecal samples at dilutions 1,000- to 10,000-fold beyond the detection limits of standard techniques such as enzyme immunoassay and the direct visualization of RNA following polyacrylamide gel electrophoresis. Furthermore, following purification by CF11, rotavirus RNA could be detected in all of seven enzyme-linked immunosorbent assay-positive fecal samples obtained from a child with rotavirus gastroenteritis; when CF11 purification was not performed, rotavirus RNA could be detected in only four of these samples, even after the removal of inhibitors by dilution of the extracts. Large-scale identification of rotavirus in fecal specimens may be possible by use of CF11 purification of viral RNA prior to sequential reactions with reverse transcriptase and Taq polymerase in a modified polymerase chain reaction.

Feces↗
PubMed1978

Congenital hypoplastic anemia (Diamond-Blackfan syndrome) terminating in acute myelogenous leukemia.

A 31-yr-old female with congenital hypoplastic anemia (Diamond-Blackfan syndrome) whose long course terminated in acute myelogenous leukemia is described. In contrast to Fanconi anemia, malignant transformation rarely occurs in congenital hypoplastic anemia. This patient's diagnosis of congenital hypoplastic anemia is supported by her clinical course, absence of renal abnormalities, a negative family history for hematologic disorders, normal chromosome studies, failure of her skin fibroblasts to transform in culture with SV-40 virus, macrocytic erythrocyte indices, erythrocyte enzyme studies, and bone marrow findings. Only two other cases of malignancy have been reported in patients with congenital hypoplastic anemia. The development of malignancy in these patients suggests that malignant transformation may be a concern in the long-term progression of congenital hypoplastic anemia.

Adult↗
PubMed1976

Acute fluoride poisoning.

Fluoride poisoning is a potentially severe environmental hazard for children. A case of fluoride poisong is presented which was manifested by severe hypocalcemia, ventricular arrhythmias, and respiratory failure. Treatment of this poisoning, including peritoneal dialysis, is discussed. The kinetics of fluoride distribution as measured in this patient suggest a rapid binding of ingested fluoride to bone, followed by gradual release and excretion. Peritoneal dialysis resulted in no significant fluoride removal.

Calcium Hydroxide↗