Our law firm actively investigates most E. coli outbreaks in the United States, fighting for victim compensation. One of the issues we run into is testing for E. coli O157:H7 long after onset of symptoms. Patients often experience renal failure (E. coli kidney failure) before a stool sample has been tested for E. coli. By that time, any E. coli may have cleared from the body, making a positive test for E. coli impossible.

It is critical to an E. coli case to get a positive test for E. coli O157:H7 and then to further test the sample of E. coli to obtain a genetic fingerprint of the bacteria that can be matched to other victims and food sources.

Below is a description of a complex process taken to obtain a positive test for E. coli O157:H7 on a stool sample taken on day 30 of illness--an unusually long time for E. coli bacteria to remain in the body. The patient was a 40-year-old male who presented with several days of abdominal cramps, vomiting, and loose stool with interspersed blood. The patient’s medical condition continued to deteriorate, and he experienced renal failure. On day 9 of his hospitalization, he commenced hemodialysis and plasmapherisis. The working diagnosis was thrombotic thrombocytopenic purpura (TTP). Several days later, doctors suspected that the patient had developed hemolytic uremic syndrome (HUS), which is generally caused by an E. coli O157:H7 infection. By the time doctors reached this determination, finding E. coli in the stools was questionable, but persistence resulted in a positive test:

On the day the fecal sample was referred to our laboratory, it was screened for the presence of preformed Shiga toxin by the Vero cell cytotoxicity assay (4, 11). In addition, an investigational 10-min chromatographic immunoassay for Stx1 or Stx2 detection was performed. This handheld Shiga toxin detection (HHSTD) system, manufactured at the Naval Medical Research Center (Silver Spring, Md.), incorporates rabbit polyclonal anti-Stx1 or anti-Stx2 antibody immobilized on filter paper to capture Stx-Stx1 or Stx2 and Stx2 toxin variants, respectively, as they migrate across the membrane in sample buffer. Colloidal-gold-labeled toxin-specific monoclonal antibodies (described elsewhere [10, 12]) were also used to impregnate the filter paper but not immobilized. The monoclonal antibodies bind the antigen, if it is present, and form a visible band where the capture antibodies are fixed to the filter paper. We tested the sensitivity of the kits with purified toxin and stool specimens seeded with known numbers of toxin-producing organisms.

The level of detection was 3 to 6 ng of toxin (for Stx2 and Stx1, respectively) or approximately 107 CFU of Shiga toxin-producing E. coli (STEC). The Stx2 kit was less sensitive for the Stx2 variant Stx2c or Stx2d and detected 20 ng of toxin or 108 CFU. The kits were specific for toxin type and showed no cross-reactivity between Stx1 or Stx2 and its variants or with negative human stool components in our trials.

The results of the HHSTD immunoassays and the conventional cytotoxicity assay of the primary stool sample were negative. However, we reasoned that very low numbers of STEC bacteria would be shed at this late point after disease onset. We also suspected that if O157:H7 organisms were present, their appearance would be masked by the predominant normal flora seen when stool is plated directly onto sorbitol MacConkey (SMAC) agar (3). Similarly, toxin production would be below detectable levels. Two approaches were used to enhance STEC recovery and increase toxin detection.

First, in order to observe greater numbers of organisms in the stool, we suspended approximately 1 ml of sample in 10 ml of brain heart infusion broth (Remel, Lenexa, Kans.) and incubated the mixture overnight at room temperature to slow the growth of normal fecal flora, as is done when culturing Yersinia enterocolitica. The enrichment broth was diluted in serial 10-fold increments on the next day, and 100 _l of each dilution was spread plated onto Difco Luria-Bertani (LB) agar and SMAC agar (Becton Dickinson, Sparks, Md.) and incubated overnight. Clearly discernible individual sorbitol-negative and –positive colonies were seen in the 10_6 and 10_7 dilutions plated on SMAC agar.

Sweeps of growth from the corresponding dilutions of overnight broth plated on LB agar were collected with a sterile cotton swab and mixed in 1 ml of the HHSTD kit buffer, and 300 _l was tested for toxin (isolates from SMAC agar were not used owing to the concern that false-positive reactions in the HHSTD kit might occur because of carryover of neutral red from the MacConkey base).

The HHSTD kit gave positive reactions for Stx1 and Stx2 on the mixed bacterial sweeps, which prompted us then to screen the sorbitol-positive and -negative colonies on SMAC agar for toxin production. A Shiga toxin gene probe was used to identify individual toxin-producing organisms by colony blot hybridization. One hundred fifty colonies of both the sorbitol-positive and –negative phenotypes were transferred with sterile toothpicks onto three LB agar plates (50 colonies each) in a grid pattern. The colonies were blotted onto nitrocellulose membranes after overnight incubation.

The membrane-bound colonies were lysed, and the DNA was denatured by saturation with 0.5 N NaOH. The nitrocellulose membranes were then dried at 80°C in a vacuum oven overnight and probed with PCR-amplified DNA of the Stx2-encoding gene (14). The colony blots were screened for hybridization with the Enhanced Chemiluminescence nucleic acid detection kit (Amersham Life Science, Buckinghamshire, England) in accordance with the manufacturer’s instructions.

The sorbitol-negative phenotype correlated 100% with hybridization to the Stx2 gene probe. The sorbitol-negative colonies were identified as E. coli with an API 20E strip (BioMe´rieux, Durham, N.C.), and the serotype was determined by agglutination with O157 and H7 antisera (Centers for Disease Control and Prevention, Atlanta, Ga.). Clarified overnight culture lysates of the E. coli O157:H7 isolate (designated WR30) produced 106 50% cytotoxic doses/ml by Vero cell assay and were positive for both Stx1 and Stx2 when tested with the HHSTD kit.

At the same time that the broth enrichment stool culture was inoculated, a second broth culture was prepared to enhance toxin expression by induction of lysogenic toxin-converting bacteriophages. One milliliter of the fecal sample was diluted in 10 ml of brain heart infusion broth to which 5 _l of mitomycin C (Sigma, St. Louis, Mo.), an alkylating agent known to induce the lytic cycle of lambdoid phages, was added (1, 6). After overnight incubation at 37°C with shaking, the broth culture supernatant was tested for Shiga toxin with the investigational test kit. A weak positive reaction for Stx2 was obtained, whereas a similarly prepared mitomycin-free broth culture was negative.

Source: Teel, Louise D., et. al, Shiga Toxin-Producting Escherichia coli-Associated Kidney Failure in a 40-Year_old Patient and Late Diagnosis by Novel Bacteriologic and Toxin Detection Methods, Journal of Clinican Microbiology, July 2003, Vol. 41, No. 7, p. 3438-3440.

Labels: E coli lawyer,Kidney Failure, Renal Failure