phosphorus-radioisotopes and Diarrhea

This report describes a DNA amplification procedure for routine identification of heat-labile-toxin-producing Escherichia coli. Two oligonucleotide primers were used in a polymerase chain reaction procedure to amplify a highly conserved region of the A subunit of the heat-labile enterotoxin gene. Amplifications were done directly on E. coli colonies from plates when Salmonella, Shigella, or parasite infections were excluded as agents of the severe diarrhea in the patients. The conditions for the polymerase chain reaction method were empirically determined, and the procedure is inexpensive, sensitive, and specific. Positive results can be obtained over a wide variation in bacterial numbers, with no inhibition of Thermus aquaticus DNA polymerase. Detection of the amplified product can be done by agarose gel electrophoresis, which is specific and sensitive enough for routine diagnosis of this pathogen in clinical isolates. If greater sensitivity and specificity are required, hybridization with 32P- or alkaline phosphatase-labeled oligonucleotide probes can be used. Our results suggest that heat-labile-toxin-producing E. coli is responsible for about 9% of nondiagnosed diarrhea cases in Tygerberg Hospital, Tygerberg, Republic of South Africa.

Topics: Alkaline Phosphatase; Bacterial Toxins; Base Sequence; Diarrhea; DNA, Bacterial; Electrophoresis, Agar Gel; Enterotoxins; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Genes, Bacterial; Humans; Infant; Molecular Sequence Data; Phosphorus Radioisotopes; Polymerase Chain Reaction; Reproducibility of Results; Sensitivity and Specificity

Alkaline phosphatase-conjugated (AP) 26-base oligonucleotide DNA probes were compared with the same probes labeled with gamma-32P for the identification of heat-labile (LT) and heat-stable (ST) enterotoxigenic Escherichia coli (ETEC). The AP oligonucleotide probes were as sensitive as the radiolabeled (RL) probes in detecting LT and STA-2 target cell DNA, but the AP ST probe, which differed from STA-1 by two bases, was less sensitive than the RL probe in detecting STA-1 DNA (6.25 versus 0.78 ng). Of 94 ETEC that were identified with the RL probe, the AP probes detected 93% (28 of 30) of ST, 73% (25 of 34) of LT, and 67% (20 of 30) of LTST ETEC. When colony lysates of these ETEC were examined, the AP probes identified all 94 ETEC. In examinations of stool blots, the RL and AP probes were shown to have sensitivities of 71 and 59%, specificities of 91 and 86%, positive predictive values of 87 and 73%, and negative predictive values of 86 and 74%, respectively. AP oligonucleotide probes to detect ETEC were less sensitive in detecting ETEC by colony or stool blot hybridization than the RL probes but could be used by laboratories without access to radioisotopes to examine colony lysates.

Topics: Alkaline Phosphatase; Bacterial Toxins; Child; Diarrhea; DNA, Bacterial; Enterotoxins; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Feces; Humans; Nucleic Acid Hybridization; Oligodeoxyribonucleotides; Phosphorus Radioisotopes; Predictive Value of Tests

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Adult; Animals; Cholera; Cyclic AMP; Cyclic GMP; Diarrhea; Diarrhea, Infantile; Enterotoxins; Epithelial Cells; Epithelium; Escherichia coli Infections; Fluorides; Guanosine Triphosphate; Guanylate Cyclase; Humans; Infant; Intestinal Mucosa; Phosphoric Diester Hydrolases; Phosphorus Radioisotopes; Prostaglandins; Temperature; Tritium

Topics: Adenylyl Cyclases; Animals; Cattle; Chemical Precipitation; Cholera; Chromatography, Ion Exchange; Diarrhea; Enterotoxins; Enzyme Activation; Escherichia coli; Filtration; Hydrogen-Ion Concentration; Injections, Intravenous; Intestines; Liver; Liver Glycogen; Mice; Phosphorus Radioisotopes; Rats; Serum Albumin, Bovine; Tritium