OBJECTIVE: Hemolytic uremic syndrome (HUS) is a life-threatening disease caused by Shiga toxin-producing Escherichia coli (STEC) infection. The treatment approaches for STEC-mediated typical HUS and atypical HUS differ, underscoring the importance of rapid and accurate diagnosis. However, specific detection methods for STECs other than major serogroups, such as O157, O26, and O111, are limited. This study focuses on the utility of PCR-based O-serotyping, serum agglutination tests utilizing antibodies against the identified Og type, and isolation techniques employing antibody-conjugated immunomagnetic beads for STEC isolation. By employing these methods, we successfully isolated a STEC strain of a minor serotype, O76:H7, from a HUS patient.

The aim of this study was to evaluate the occurrence of Cryptosporidium oocysts in a drinking water treatment plant (DWTP) located in a rural area of northern Italy. Influent and effluent samples were collected at the DWTP over three years (2013-2016). In parallel, tap water samples from a public drinking fountain were collected as well. All samples were analyzed for the presence of Cryptosporidium spp. oocysts by a common method based on an immunomagnetic separation (IMS)/immunofluorescence assay (IFA), complemented by 4,6-diamidino-2-phenylindole (DAPI) staining. A reverse transcriptase-PCR (RT-PCR) protocol was added to evaluate oocyst viability. The results highlighted a high variability of oocyst concentrations across all samples (mean 4.3 ± 5.8/100 L) and a high variability in the percentage of DAPI-positive specimens (mean 48.2% ± 40.3%). Conversely, RT-PCR did not reveal the presence of viable C. parvum and C. hominis oocysts. A nested PCR targeting Cryptosporidium 18S ribosomal DNA, carried out in two water samples, confirmed the presence of a Cryptosporidium genotype associated with wild animals in the river and in tap water. The results obtained underline the vulnerability of the investigated surface water to Cryptosporidium spp. contamination. Although the recovered Cryptosporidium genotype is not a human pathogen, its presence demonstrates the existence of a potential pathogen Cryptosporidium spp. contamination risk. Moreover, these results underline the importance of also considering unconventional (not bacterial) biological contaminations (protozoa) in water resources in rural areas, including those of developed countries.

This study investigated whether changes in circulating tumor cell (CTC) numbers reflect tumor progression and treatment efficacy in esophageal squamous cell carcinoma (ESCC). A 47-year-old male patient with ESCC is presented in this case study. The patient was evaluated for a series of serum tumor markers and subjected to radiological examinations before and after surgery and during follow-up over the course of five years. In addition, the CTCs in 7.5 mL of peripheral blood were enriched by magnetic-activated cell sorting negative selection and identified by immunofluorescence staining. Serum tumor markers remained within normal ranges and were discordant with imaging scans during the follow-up. Initially, one CTC was detected in the peripheral blood sample, and 14 were observed seven days after the operation. After 12 wk, subcutaneous metastases and bone metastases occurred, and the number of CTCs increased to 84. After 48 wk, lung metastases were noted, and the CTC level was 21. At 104 wk, the number of CTCs was 14, and disease recurrence was detected by positron emission tomography-computed tomography. The CTC counts were in accord with the imaging studies at several time points. The additional information provided by CTC enumeration could thus facilitate monitoring of disease status and treatment efficacy and provide support for treatment decisions.

Concentration of mycobacteria from sputum by centrifugation prior to acid-fast microscopy increases case finding compared to direct microscopy of the sputum (direct smear). However, centrifugation has to be performed outside the safety cabinet and many laboratories do not have access to a centrifuge. Magnetic bead extraction of the mycobacteria is an alternative method that can be performed in a cabinet with just a magnet. Magnetic TB-Bead (Microsens Medtech Ltd) extraction of mycobacteria from sputum prior to microscopy was compared to direct smear on 78 sputum samples. Microscopy of the TB-Bead extracts identified all of 26 of the direct smear positive samples either with the same microscopy score or, in 19/27 of samples, with an increased microscopy score which aided microscopy detection. In addition, microscopy of the TB-Bead extracts identified 10 additional positive samples compared to direct smear; which represents a statistically significant increase in case finding of 38% (p = 0.002) compared to direct smear. In a separate study, TB-Beads enabled further 4 positive samples to be detected from 30 centrifuged pellets that were originally smear negative; two of these were subsequently found to be positive when the original deposits were reinvestigated by smear microscopy. By concentrating mycobacteria from sputum and sputum deposits, TB-Beads have been demonstrated to increase the number of positive sputum samples which could increase case-finding. The TB-Bead method is simple and rapid and compatible with use within a safety cabinet.

We describe a novel method for detecting micrometastasis in the blood stream of cancer patients based on RT-PCR amplification of tumor-associated carcinoembryonic antigen (CEA) mRNA. To increase sensitivity and specificity of RT-PCR, CEA transcript was selectively up-regulated in cancer cells by exposure of peripheral blood to non-toxic concentrations of staurosporine (ST). Thereafter, polyA(+) RNA was extracted from tumor cells captured by means of magnetic beads coated with a monoclonal antibody against a common human epithelial antigen. Finally, RNA was subjected to RT-PCR analysis of CEA transcript. Using this approach, we demonstrated an ST-mediated increase in CEA transcript in blood specimens collected from a patient with metastatic colon cancer before receiving treatment with 5-fluorouracil/leucovorin. After a few cycles of chemotherapy, CEA-positive tumor cells were no longer detected. Clinical follow-up of this patient indicated that treatment with chemotherapy induced a dramatic reduction in liver metastasis. Therefore, it can be hypothesized that lack of CEA transcript detection might be consistent with disappearance or at least marked reduction of circulating tumor cells.

OBJECTIVE: To investigate whether a sheep flock was the original reservoir of a Shiga toxin-producing Escherichia coli (STEC) O103 strain causing a clinical human case and to compare the two diagnostic methods automated immunomagnetic separation (AIMS) and AIMS-ELISA.
RESULTS: AIMS detected Escherichia coli O103 in 36.5% of the samples and AIMS-ELISA detected E. coli O103 in 52.1% of the samples. Polymerase chain reaction detected stx1 and eae in three of 109 E. coli O103 isolates. Pulsed field gel electrophoresis showed that the sheep and human STEC O103 were characterized by distinctly different profiles.
CONCLUSIONS: The sheep flock was shown to carry STEC O103, although an association between the sheep flock and the clinical human case could neither be proven nor eliminated. Substantial agreement was found between AIMS and AIMS-ELISA, but AIMS-ELISA was less time consuming and resulted in a higher recovery of E. coli O103.
CONCLUSIONS: The study shows that sheep may be carriers of STEC that are associated with human disease and that the methods described can be used to increase the sensitivity of STEC detection.