Christine Rozand

Prevalence, characterization and clonal analysis of Escherichia coli O157: non-H7 serotypes that carry eae alleles

We examined O157:non-H7 strains isolated from various sources and geographical locations and found 15/57 strains to carry eae alleles, including alpha, beta, epsilon and kappa/delta, suggesting that these strains may be prevalent. All strains were serologically and genetically confirmed to be O157, but none were the H7 serotype or carried any trait virulence factors of the Escherichia coli O157:H7 serotype. Genetic H typing of the eae-positive strains showed that the alpha-eae-bearing strain was H45, while the beta- and epsilon-eae strains were H16 and the kappa/delta-eae strains were H39. The beta- and epsilon-eae-bearing O157:H16 strains shared approximately 90% pulsed-field gel electrophoresis (PFGE) similarity and were distinct from the other strains that had other eae alleles. Interestingly, an epsilon-eae O157:H16 strain isolated from meat in France shared PFGE similarity to the O157:H16 strains from water in the United States. Multilocus sequence typing showed that there is clonal diversity within the O157 serogroup, as some O157:non-H7 strains clustered with EPEC clonal groups, while others clustered within the ST-171 group of diverse strains and serotypes that had not previously included any strains from the O157 serogroup. Clonal analysis also showed that none of the eae-positive O157:non-H7 strains we examined were closely related to the pathogenic O157:H7 serotype.

Novel real-time PCR method to detect Escherichia coli O157:H7 in raw milk cheese and raw ground meat.

Raw milk, raw milk cheeses, and raw ground meat have been implicated in Escherichia coli O157:H7 outbreaks. Developing methods to detect these bacteria in raw milk and meat products is a major challenge for food safety. The aim of our study was to develop a real-time PCR assay to detect E. coli O157:H7 in raw milk cheeses and raw ground meat. Well-known primers targeting a mutation at position +93 of the uidA gene in E. coli O157:H7 were chosen, and a specific TaqMan-minor groove binder probe was designed. This probe targets another mutation, at position +191 of the uidA gene in E. coli O157:H7. The first step in the study was to evaluate the specificity of this probe with 156 different O157:H7/NM strains and 48 non-O157:H7/NM strains of E. coli. The sensitivity of the method was evaluated by pre- and postinoculation of cheeses and meat enrichments with different E. coli O157:H7 strains. All the E. coli O157:H7 isolates tested were positive, and none of the other bacteria were detected. Our results indicate that this method is sensitive enough to detect 10(2) E. coli O157:H7 isolates per ml of cheese or meat enrichment broth (24 h at 41.5° C) and is more sensitive than the International Organization for Standardization reference method. We can conclude that this new real-time PCR protocol is a useful tool for rapid, specific, and sensitive detection of E. coli O157:H7 in raw milk and raw ground meat products.

Simplified detection of food-borne pathogens: an in situ high affinity capture and staining concept.

Food industries need simple, rapid and cost-effective solutions for pathogen detection in food and environmental samples. In this paper, we describe a simple but novel detection concept combining an affinity capture surface and intracellular metabolic marker to visualize the bacterial presence on the affinity surface. The surface of a Solid Phase Support (SPS) is functionalized with specific phage tail proteins targeted to the bacterial pathogen of interest. The SPS is placed directly into the primary food enrichment bag after stomaching. Following incubation, the captured bacteria are visually detected in situ as a result of the bacterial reduction of the colorless soluble substrate triphenyltetrazolium chloride (TTC) (present in the primary culture medium) to an intracellular red insoluble formazan product. Detection on the SPS is observed as an intense red color after 22 to 40 hours of enrichment. This is not impaired by the presence of food particles and the natural background microflora. The in situ method significantly simplifies pathogen detection by eliminating any post-enrichment intervention that is necessary in the traditional methods of analysis. We have demonstrated the application of this new approach for the detection of Escherichia coli O157: H7, Listeria spp. and Salmonella spp. in artificially contaminated food samples.

Specificity analysis of a novel phage-derived ligand in an enzyme-linked fluorescent assay for the detection of Escherichia coli O157:H7.

An assay using a phage-derived ligand to capture Escherichia coli O157:H7 prior to antibody detection was evaluated for assay specificity. Analysis of 200 strains showed that the assay was highly specific for the O157 serogroup. It detected all the O157:H7 strains including Shiga toxin-producing O157 nonmotile strains as well as O157 non-H7 strains. In addition, the assay detected various O157:H7 phenotypic variants that are not easily detected by routine analytical methods, as well as a rough strain that did not express O157 antigen and therefore is undetectable serologically. The phage ligand assay showed no cross-reactivity to the other E. coli serotypes. Isolates of Salmonella group N and a few Citrobacter freundii strains that cross-reacted with anti-O157 sera also showed cross-reactivity with the phage ligand. However, other strains that cross-reacted serologically with anti-O157 sera were correctly identified as negative with the phage ligand assay, including several strains of E. coli that nonspecifically autoagglutinate latex reagents.

Paper-based point-of-care testing for cost-effective diagnosis of acute flavivirus infections†

Flavivirus infections are a serious healthcare concern in tropical and subtropical countries. Although well‐established laboratory tests can provide early diagnosis of acute dengue or Zika infections, access to these tests is limited in developing countries, presenting an urgent need to develop simple, rapid, and robust diagnostic tools. Microfluidic Paper‐based Analytical Devices (μPAD), are typically rapid, cost‐effective, user‐friendly, and they can be used as diagnostic tools for the diagnosis of these infections at Point of Care settings. Early and prompt diagnosis is crucial to improve patient management and reduce the risk of complications. In the present study, we developed and evaluated a wax‐printed paper‐based device for the detection of the dengue and Zika non‐structural NS1 viral protein in blood and plasma. Experiments have been carried out to increase specificity, while maintaining the required sensitivity. As a consequence, the quality of the raw materials and the washing steps were proved to be crucial. The μPAD was able to detect specifically in 6–8 min 10 ng/mL of protein in various sample types. A prototype for the differential detection of dengue and/or Zika NS1 protein was developed. The reading of the results was simplified by using a dedicated application on a smartphone.

Application of a paper based device containing a new culture medium to detect Vibrio cholerae in water samples collected in Haiti

Cholera is now considered to be endemic in Haiti, often with increased incidence during rainy seasons. The challenge of cholera surveillance is exacerbated by the cost of sample collection and laboratory analysis. A diagnostic tool is needed that is low cost, easy-to-use, and able to detect and quantify Vibrio cholerae accurately in water samples within 18-24h, and perform reliably in remote settings lacking laboratory infrastructure and skilled staff. The two main objectives of this study were to develop and evaluate a new culture medium embedded in a new diagnostic tool (PAD for paper based analytical device) for detecting Vibrio cholerae from water samples collected in Haiti. The intent is to provide guidance for corrective action, such as chlorination, for water positive for V. cholerae epidemic strains. For detecting Vibrio cholerae, a new chromogenic medium was designed and evaluated as an alternative to thiosulfate citrate bile salts sucrose (TCBS) agar for testing raw water samples. Sensitivity and specificity of the medium were assessed using both raw and spiked water samples. The Vibrio cholerae chromogenic medium was proved to be highly selective against most of the cultivable bacteria in the water samples, without loss of sensitivity in detection of V. cholerae. Thus, reliability of this new culture medium for detection of V. cholerae in the presence of other Vibrio species in water samples offers a significant advantage. A new paper based device containing the new chromogenic medium previously evaluated was compared with reference methods for detecting V. cholerae from spiked water sample. The microbiological PAD specifications were evaluated in Haiti. More precisely, a total of 185 water samples were collected at five sites in Haiti, June 2014 and again in June 2015. With this new tool, three V. cholerae O1 and 17 V. cholerae non-O1/O139 strains were isolated. The presence of virulence-associated and regulatory genes, including ctxA, zot, ace, and toxR, was confirmed using multiplex PCR. The three V. cholerae O1 isolates were positive for three of the four virulence-associated and regulatory genes. Twelve of the V. cholerae non-O1/O139 isolates were found to carry toxR, but none were ctxA+, zot+, or ace+. However, six of the V. cholerae non-O1/O139 isolates were resistant to penicillin, ampicillin, trimethoprim/sulfamethoxazole, nalidixic acid, and ciprofloxacin. The paper based analytical device (PAD) provides advantages in that standard culture methods employing agar plates are not required. Also, intermediary isolation steps were not required, including transfer to selective growth media, hence these steps being omitted reduced time to results. Furthermore, experienced technical skills also were not required. Thus, PAD is well suited for resource-limited settings.

Laser-cut paper-based device for the detection of dengue non-structural NS1 protein and specific IgM in human samples

The incidence of flavivirus infections has increased dramatically in recent decades in tropical and sub-tropical areas worldwide, affecting hundreds of millions of people each year. Dengue viruses are typically transmitted by mosquitoes and can cause a wide range of symptoms from flu-like fever to organ impairment and death. Although conventional diagnostic tests can provide early diagnosis of acute dengue infections, access to these tests is often limited in developing countries. Consequently, there is an urgent need to develop affordable, simple, rapid, and robust diagnostic tools that can be used at ‘Point of Care’ settings. Early diagnosis is crucial to improve patient management and reduce the risk of complications. In the present study, a novel laser-cut device made of glass-fiber paper was designed and tested for the detection of the dengue Non Structural 1 (NS1) viral protein and specific IgM in blood and plasma. The device, called PAD, was able to detect around 25 ng/mL of NS1 protein in various sample types in 8 minutes, following a few simple steps. The PAD was also able to detect specific IgM in human plasmas in less than 10 minutes. The PAD appears to have all the potential to assist health workers in early diagnosis of dengue fever or other tropical fevers caused by flaviviruses.