The production of heat-labile (LT) and heat-stable (ST) enterotoxins, colonisation factor antigens (CFAs) and haemagglutinins was investigated amongst 310 Escherichia coli (E. coli) isolates obtained from 62 children under the age of five, with diarrhoea. Twenty-one isolates were found to produce enterotoxins, of which fifteen (71%) isolates produced ST only, 2 (10%) produced LT only and 4 (19%) produced both LT and ST. However, none of the isolates demonstrated any of the common CFAs identified to date, but 8 out of the 21 isolates demonstrated haemagglutination with rabbit, sheep or human group A erythrocytes, suggesting the presence of putative CFAs, yet unidentified.
A gene associated with lipopolysaccharide (LPS) transport was cloned from a local clinical Vibrio cholerae O1 strain of the Ogawa serotype by using the Lactococcus lactis nisin-controlled expression (NICE) system. The V. cholerae wzm gene, which codes for an integral membrane transporter protein, was expressed and targeted to the cytoplasmic membrane, and was crudely isolated through simple centrifugation and SDS solubilization. To examine seroreactivity of this construct, rabbits were orally fed with 10(9) cfu/ml of live, recombinant L. lactis carrying the wzm gene, induced with nisin prior to administration. Recombinant plasmids were retrieved from L. lactis cultured directly from stool samples of inoculated rabbits. Reverse-transcriptase PCR of wzm using the retrieved plasmids confirmed transcription of this gene, indicating viability and stability of the recombinants in vivo. The L. lactis-Wzm construct elicited substantial levels of IgG and sIgA, and challenge with virulent V. cholerae O1 evoked severe diarrhoea in the naive, non-immunised control group, but not in those fed with either recombinant or non-recombinant L. lactis. Oral administration with recombinant L. lactis expressing the V. cholerae wzm gene increases both systemic and mucosal immunity, whereas L. lactis itself appears capable of protecting against the diarrhoeal symptoms caused by V. cholerae. Wzm is a conserved membrane protein associated with the LPS endotoxin, and together with the food-grade L. lactis, represent an attractive target for the development of a safer, live anti-infective therapy against V. cholerae.
A blocking test was incorporated into the commercial IDEIA Adenovirus test (DAKO Diagnostics Ltd., Cambridgeshire, UK) to detect false positive results when faecal specimens were tested for adenovirus antigen. Immune rabbit serum raised against pooled adenovirus particles from human faecal specimens, together with the pre-immune serum, was used. Assessment of positive showed that false positives were produced under two different conditions: when results were based on visual determination instead of a cut-off value determined from photometric reading, and when absorbance values were not immediately read at the end of the test. Under the optimum condition for reading and assessment of test results (immediate reading and photometric determination), 11% of 65 adenovirus-positive samples were checked by the blocking ELISA as false positives. The rest of the specimens showed blocking of positive absorbance values by 70 to 98%. ELISA was found to be more sensitive than immune electron microscopy on samples with lower antigen concentration.
Microsporidia are obligate intracellular parasitic fungi causing chronic diarrhea, particularly among immunocompromised patients. The current method used for diagnosis is based on conventional microscopy, which does not differentiate parasites at the species level. The present study was carried out to identify microsporidian species in immunocompromised patients. From March 2016 to March 2017, a total of 289 archived stool samples were examined microscopically for microsporidian spores using Gram-chromotrope Kinyoun (GCK) stain. Positive stool samples by microscopy were subjected to polymerase chain reaction and sequencing for species identification. Based on microscopy examination, the overall prevalence of microsporidian infections was 32.2% (93/289; 95% CI = 27.1-37.8). Of these stool samples, 45 samples were successfully amplified and confirmed as Enterocytozoon bieneusi. No Encephalitozoon intestinalis was detected. Accurate identification of species might help clinicians to decide appropriate management strategies as dissemination risks and treatment response vary for different species, hence improving the management of microsporidian infections.