Short report: detection of Entamoeba histolytica and E. dispar directly in stool.

Current diagnosis of Entamoeba histolytica infection requires the direct microscopic identification of the parasite, a technique that is insensitive and cannot distinguish pathogenic E. histolytica from noninvasive E. dispar. Enzyme-linked immunosorbent assay (ELISA) antigen detection tests were developed to distinguish E. histolytica from E. dispar infection in stool specimens. The ELISA result for E. histolytica antigen was positive in 26 of 27 E. histolytica-positive stool specimens, three of 25 E. dispar-positive stools, and one of 30 stools with other or no intestinal parasites, giving a specificity and sensitivity for the detection of E. histolytica infection of 93% and 96%, respectively. The assay result used to detect both E. dispar and E. histolytica was positive in 26 of 27 E. histolytica-positive stools, 19 of 25 E. dispar-positive stools, and one of 30 stools negative by microscopy and culture for Entamoeba, giving a specificity and sensitivity of 97% and 87%, respectively. Because these ELISAs can be completed in several hours, they offer promise as rapid and sensitive means of detecting amebic infection.

A Salmonella Typhimurium 197 outbreak linked to the consumption of lambs' liver in Sydney, NSW.

We identified an increase in the number of cases of Salmonella Typhimurium phage type 197 in New South Wales in February 2005. Cases were predominantly of Lebanese descent. To identify risk factors for illness, we conducted an unmatched case-control study including 12 cases and 21 controls. Eight of 12 cases (67%) and no controls reported eating lambs' liver (OR incalculable, P<0.05), and seven of nine cases (78%) and one of 21 controls (5%) reported eating fresh fish (OR 70.0, P<0.05). Among participants who did not eat liver, there was a strong association between eating fish and illness (OR 60.0, P<0.05). The fish was from divergent sources. Five cases had bought the liver from two different butcher's shops, which obtained the lambs' liver from a single abattoir. Consumption of liver is a risk for salmonellosis. Traditional dishes may place some ethnic groups at increased risk of foodborne disease.

Genetic characterization of toxin A-negative, toxin B-positive Clostridium difficile isolates by PCR.

Toxin-specific enzyme immunoassays, cytotoxicity assays, and PCR were used to analyze 48 toxin A-negative, toxin B-positive Clostridium difficile isolates from various geographical sites around the world. All the isolates were negative by the TOX-A TEST and positive by the TOX A/B TEST. A deletion of approximately 1.7 kb was found at the 3' end of the toxA gene for all the isolates, similar to the deletion in toxinotype VIII strains (e.g., C. difficile serotype F 1470). Additional PCR analysis indicated that the toxin B encoded by these isolates contains sequence variations downstream of the active site compared to the sequence of reference strain VPI 10463. This variation may extend the glucosylation spectrum to Ras proteins, as observed previously for closely related lethal toxin from Clostridium sordellii and toxin B from toxin A-negative, toxin B-positive strain F 1470. Toxin A-negative, toxin B-positive isolates have recently been associated with disease in humans, and they may be more common than was previously supposed.

Rapid diagnosis of Entamoeba infection by using Entamoeba and Entamoeba histolytica stool antigen detection kits.

Humans are infected by two morphologically identical species of Entamoeba: Entamoeba histolytica causes amebic colitis and liver abscess, and Entamoeba dispar is noninvasive. Several weeks of culture and isoenzyme (zymodeme) analysis are required to differentiate E. histolytica from E. dispar. Here we report a field trial of commercial antigen detection kits designed to rapidly detect and differentiate E. histolytica from E. dispar in stool specimens. Stool specimens from 202 patients with diarrhea were examined for E. histolytica and E. dispar by microscopy, culture, and antigen detection. Compared with culture, microscopic identification of the E. histolytica-E. dispar complex was 60% sensitive and 79% specific, while the screening antigen detection test for the E. histolytica-E. dispar complex was 80% sensitive and 99% specific. Differentiation of E. dispar from E. histolytica by the E. histolytica-specific test was 95% sensitive and 93% specific compared with zymodeme analysis. We conclude that the antigen detection test for the E. histolytica-E. dispar complex is more sensitive and specific than microscopy and that the E. histolytica-specific antigen detection test is as reliable and much more rapid than zymodeme analysis for the differentiation of E. histolytica from E. dispar.

Characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile responsible for a nosocomial outbreak of Clostridium difficile-associated diarrhea.

Clostridium difficile-associated diarrhea (CAD) is a very common nosocomial infection that contributes significantly to patient morbidity and mortality as well as to the cost of hospitalization. Previously, strains of toxin A-negative, toxin B-positive C. difficile were not thought to be associated with clinically significant disease. This study reports the characterization of a toxin A-negative, toxin B-positive strain of C. difficile that was responsible for a recently described nosocomial outbreak of CAD. Analysis of the seven patient isolates from the outbreak by pulsed-field gel electrophoresis indicated that this outbreak was due to transmission of a single strain of C. difficile. Our characterization of this strain (HSC98) has demonstrated that the toxin A gene lacks 1.8 kb from the carboxy repetitive oligopeptide (CROP) region but apparently has no other major deletions from other regions of the toxin A or toxin B gene. The remaining 1.3-kb fragment of the toxin A CROP region from strain HSC98 showed 98% sequence homology with strain 1470, previously reported by M. Weidmann in 1997 (GenBank accession number Y12616), suggesting that HSC98 is toxinotype VIII. The HSC98 strain infecting patients involved in this outbreak produced the full spectrum of clinical illness usually associated with C. difficile-associated disease. This pathogenic spectrum was manifest despite the inability of this strain to alter tight junctions as determined by using in vitro tissue culture testing, which suggested that no functional toxin A was produced by this strain.

Multicenter evaluation of the Clostridium difficile TOX A/B TEST.

Clostridium difficile, the primary cause of nosocomial diarrhea in the United States and many other industrialized countries, is recognized as a major health concern because of its ability to cause severe intestinal disease leading to complications such as relapses and infections due to vancomycin-resistant enterococci. The disease results from two toxins, toxins A and B, produced by this pathogen. In this study, we evaluated the TOX A/B TEST, a new 1-h enzyme immunoassay (EIA) that detects toxins A and B. We compared the test with the tissue culture assay, which is recognized as the "gold standard" for C. difficile testing. Evaluations were performed in-house at TechLab, Inc. (Blacksburg, Va.) and off-site at four clinical laboratories. Of 1,152 specimens tested, 165 were positive by the TOX A/B TEST and tissue culture and 973 were negative by both tests. The sensitivity and specificity were 92.2 and 100%, respectively. The positive and negative predictive values were 100 and 98.6%, respectively, and the correlation of the TOX A/B TEST with tissue culture was 98.8%. When discrepant samples were resolved by culture, the sensitivity and specificity were 93.2 and 98.9%, respectively. The positive and negative predictive values were 100 and 98.8%, respectively, with a correlation of 99.0%. There were no specimens that were positive by the TOX A/B TEST and negative by tissue culture. Fourteen specimens were negative by the TOX A/B TEST but positive by tissue culture. Of these, two were negative by toxigenic culture, five were positive by toxigenic culture, and seven were not available for further testing. There were no indeterminate results, since the test does not have an indeterminant zone. In a separate study, 102 specimens that were positive by tissue culture and the TOX A/B TEST were examined in toxin A-specific EIAs. Two specimens that presumptively contained toxin A-negative, toxin B-positive (toxA-/toxB+) isolates were identified. One specimen was from a patient with a clinical history consistent with C. difficile infection. Isolates obtained from these specimens by selective culture on solid media and in broth tested toxA-/toxB+ when grown in brain heart infusion dialysis flasks, which stimulate in vitro production of both toxins. Our findings show that the TOX A/B TEST is suitable as a diagnostic aid for C. difficile disease because it correlates well with tissue culture and detects isolates that may be missed with toxin A-specific EIAs.

Substance P activation of enteric neurons in response to intraluminal Clostridium difficile toxin A in the rat ileum.

BACKGROUND & AIMS: Nerves have been suggested to mediate the effects of bacterial toxins in intestinal diseases. However, the mechanisms involved are unknown. This study examined endogenous substance P (SP) activation of the substance P receptor (SPR) on enteric neurons in the rat ileum after exposure to intraluminal Clostridium difficile toxin A. METHODS: After intraluminal injection of toxin A in ileal loops, tissue was examined for pathological changes by histology and for SPR activation by immunocytochemical analysis of SP-induced SPR endocytosis. RESULTS: After toxin A administration, > 70% of enteric neurons showed SPR endocytosis and became swollen with thickened dendrites. In contrast, SPRs in control rats were largely confined to the plasma membrane. Rats denervated of primary afferent fibers with neonatal capsaicin injection and animals pretreated with a nonpeptide SPR antagonist showed few endosomal SPRs, and the pathological inflammatory effects of toxin A were ablated. CONCLUSIONS: Intraluminal toxin A causes the release of SP from primary afferent neurons: this endogenous SP then acts on enteric neurons in the submucosal and myenteric plexuses. SP is the primary mediator of an axon reflex mediating neurogenic inflammation in the intestine. SPR blockade may prove to be a novel therapy used to prevent intestinal inflammation.