Development and performance characteristics evaluation of a new Bioelectric Recognition Assay (BERA) method for rapid Sars-CoV-2 detection in clinical samples.

INTRODUCTION: As the second wave of COVID-19 pandemic is in progress the development of fast and cost-effective approaches for diagnosis is essential. The aim of the present study was to develop and evaluate the performance characteristics of a new Bioelectric Recognition Assay (BERA) regarding Sars-CoV-2 detection in clinical samples and its potential to be used as a point of care test. MATERIALS AND METHODS: All tests were performed using a custom portable hardware device developed by EMBIO DIAGNOSTICS (EMBIO DIAGNOSTICS Ltd, Cyprus). 110 positive and 136 negative samples tested by RT-PCR were used in order to define the lower limit of detection (L.O.D.) of the system, as well as the sensitivity and the specificity of the method. RESULTS: The system was able to detect a viral concentration of 4 genome copies/µL. The method displayed total sensitivity of 92.7 % (95 %CI: 86.2-96.8) and 97.8 % specificity (95 %CI: 93.7-99.5). When samples were grouped according to the recorded Ct values the BERA biosensor displayed 100.00 % sensitivity (95 %CI: 84.6-100.0) for Ct values <20-30. For the aforementioned Ct values the Positive Predictive Value (PPV) of the method was estimated at 31.4 % for COVID-19 prevalence of 1% and at 70.5 % for 5% prevalence. At the same time the Negative Predictive Value (NPV) of the BERA biosensor was at 100.0 % for both prevalence rates. CONCLUSIONS: EMBIO DIAGNOSTICS BERA for the detection of SARS-CoV-2 infection has the potential to allow rapid and cost-effective detection and subsequent isolation of confirmed cases, and therefore reduce household and community transmissions.

Newly Developed System for the Robust Detection of Listeria monocytogenes Based on a Bioelectric Cell Biosensor.

Human food-borne diseases caused by pathogenic bacteria have been significantly increased in the last few decades causing numerous deaths worldwide. The standard analyses used for their detection have significant limitations regarding cost, special facilities and equipment, highly trained staff, and a long procedural time that can be crucial for foodborne pathogens with high hospitalization and mortality rates, such as Listeria monocytogenes. This study aimed to develop a biosensor that could detect L. monocytogenes rapidly and robustly. For this purpose, a cell-based biosensor technology based on the Bioelectric Recognition Assay (BERA) and a portable device developed by EMBIO Diagnostics, called B.EL.D (Bio Electric Diagnostics), were used. Membrane engineering was performed by electroinsertion of Listeria monocytogenes homologous antibodies into the membrane of African green monkey kidney (Vero) cells. The newly developed biosensor was able to detect the pathogen's presence rapidly (3 min) at concentrations as low as 102 CFU mL-1, demonstrating a higher sensitivity than most existing biosensor-based methods. In addition, lack of cross-reactivity with other Listeria species, as well as with Escherichia coli, was shown, thus, indicating biosensor's significant specificity against L. monocytogenes.

Cyclospora Cayetanensis-Major Outbreaks from Ready to Eat Fresh Fruits and Vegetables.

Cyclospora cayetanensis is a coccidian protozoan that causes cyclosporiasis, a severe gastroenteric disease, especially for immunocompromised patients, children, and the elderly. The parasite is considered as an emerging organism and a major contributor of gastroenteritis worldwide. Although the global prevalence of cyclosporiasis morbidity and mortality has not been assessed, global concern has arisen since diarrheal illness and gastroenteritis significantly affect both developing countries and industrialized nations. In the last two decades, an increasing number of foodborne outbreaks has been associated with the consumption of fresh produce that is difficult to clean thoroughly and is consumed without processing. Investigations of these outbreaks have revealed the necessity to increase the awareness in clinicians of this infection, since this protozoan is often ignored by surveillance systems, and to establish control measures to reduce contamination of fresh produce. In this review, the major cyclosporiasis outbreaks linked to the consumption of ready to eat fresh fruits and vegetables are presented.

Newly Developed System for Acetamiprid Residue Screening in the Lettuce Samples Based on a Bioelectric Cell Biosensor.

Population growth and increased production demands on fruit and vegetables have driven agricultural production to new heights. Nevertheless, agriculture remains one of the least optimized industries, with laboratory tests that take days to provide a clear result on the chemical level of produce. To address this problem, we developed a tailor-made solution for the industry that can allow multiple field tests on key pesticides, based on a bioelectric cell biosensor and the measurement of the cell membrane potential changes, according to the principle of the Bioelectric Recognition Assay (BERA). We developed a fully functional system that operates using a newly developed hardware for multiple data sources and an Android application to provide results within 3 min. The presence of acetamiprid residues caused a cell membrane hyperpolarization, which was distinguishable from the control samples. A database that classified samples Below or Above Maximum Residue Levels (MRL) was then created, based on a newly developed algorithm. Additionally, lettuce samples were analyzed with the conventional and the newly developed method, in parallel, revealing a high correlation on sample classification. Thus, it was demonstrated that the novel biosensor system could be used in the food supply chain to increase the number of tested products before they reach the market.

In Vitro Gene Transcription of Listeria monocytogenes after Exposure to Human Gastric and Duodenal Aspirates.

The aim of the present study was to assess, for the first time to our knowledge, Listeria monocytogenes CFU changes, as well as to determine the transcription of key virulence genes, namely, sigB, prfA, hly, plcA, plcB, inlA, inlB, inlC, inlJ, inlP, and lmo2672 after in vitro exposure to human gastric and duodenal aspirates. Furthermore, investigations of the potential correlation between CFU changes and gene regulation with factors influencing gastric (proton pump inhibitor intake and presence of gastric atrophy) and duodenal pH were the secondary study aims. Gastric and duodenal fluids that were collected from 25 individuals undergoing upper gastrointestinal endoscopy were inoculated with L. monocytogenes serotype 4b strain LQC 15257 at 9 log CFU·mL-1 and incubated at 37°C for 100 min and 2 h, respectively, with the time corresponding to the actual exposure time to gastric and duodenal fluids in the human gastrointestinal tract. Sampling was performed upon gastric fluid inoculation, after incubation of the inoculated gastric fluids, upon pathogen resuspension in duodenal fluids and after incubation of the inoculated duodenal fluids. L. monocytogenes CFU changes were assessed by colony counting, as well as reverse transcription quantitative PCR by using inlB as a target. Gene transcription was assessed by reverse transcription quantitative PCR. In 56% of the cases, reduction of the pathogen CFU occurred immediately after exposure to gastric aspirate. Upregulation of hly and inlC was observed in 52 and 58% of the cases, respectively. On the contrary, no upregulation or downregulation was noticed regarding sigB, prfA, plcA, plcB, inlA, inlB, inlJ, inlP, and lmo2672. In addition, sigB and plcA transcription was positively and negatively associated, respectively, with an increase of the pH value, and inlA transcription was negatively associated with the presence of gastric atrophy. Finally, a positive correlation between the transcriptomic responses of plcB, inlA, inlB, inlC, inlJ, inlP, and lmo2672 was detected. This study revealed that the CFU of the pathogen was negatively affected after exposure to human gastroduodenal aspirates, as well as significant correlations between the characteristics of the aspirates with the virulence potential of the pathogen.

Genetic Analysis of the Listeria Pathogenicity Island 1 of Listeria monocytogenes 1/2a and 4b Isolates.

The aim of the present study was to apply descriptive, phylogenetic, recombination, and selection analyses on alignments of the Listeria Pathogenicity Island 1 (LIPI-1) of 1/2a and 4b Listeria monocytogenes isolates of different origin in order to gain insights into the evolution of this virulence gene cluster. For that purpose, a total of 19 L. monocytogenes isolates (9 meat isolates, serotype 1/2a; 5 meat isolates, serotype 4b; 5 strawberry isolates, serotype 4b) that have been previously separated at strain level were subjected to sequencing of their LIPI-1. Descriptive analysis revealed extensive nucleotide diversity mostly in the intragenic regions. The actA gene of 1/2a and 4b meat isolates and the hly gene of the 4b strawberry isolates exhibited the higher diversity; limited diversity was observed in prfA and plcA genes of the 4b isolates and mpl gene of the 1/2a isolates. Phylogenetic analysis of the complete island resulted in two major clusters that were consistent with serotype assignment of the isolates. Moreover, effective discrimination between serotypes was obtained by plcA, plcB, mpl, actA and the intergenic regions plcA-prfA and plcA-hly. In all cases but plcB and plcA-prfA 4b isolates were also differentiated according to their source of isolation as well. Selection analysis revealed that the island consisted of randomly evolving DNA with the exception of prfA gene of 1/2a isolates and actA gene of 4b meat isolates for which purifying selection or population expansion was indicated. Finally, no statistically significant evidence for recombination has been observed.

Expression of Listeria monocytogenes key virulence genes during growth in liquid medium, on rocket and melon at 4, 10 and 30 °C.

The aim of the present study was to assess the expression of key virulence genes, during growth of a Listeria monocytogenes isolate in liquid medium, on melon and rocket at different temperatures and time. For that purpose, BHI broth, rocket and melon were inoculated at 7.0-7.5 log CFU mL(-1) or g(-1)and stored at 4, 10 and 30 °C. Sampling took place upon inoculation and after 0.5, 6 and 24 h of incubation. The RNA was stabilized and the expression of hly, plcA, plcB, sigB, inlA, inlB, inlC, inlJ, lmo2672 and lmo2470 was assessed by RT-qPCR. The results obtained were summarized into two observations; the first one referring to the interactive effect of incubation temperature and type of substrate and the second one to the effect of time on gene expression. Regarding the latter, nearly all genes were regulated upon inoculation and exhibited differential expression in the subsequent sampling times indicating the existence of additional regulatory mechanisms yet to be explored.

Estimation of Listeria monocytogenes and Escherichia coli O157:H7 prevalence and levels in naturally contaminated rocket and cucumber samples by deterministic and stochastic approaches.

The aims of the present study were to determine the prevalence and levels of Listeria monocytogenes and Escherichia coli O157:H7 in rocket and cucumber samples by deterministic (estimation of a single value) and stochastic (estimation of a range of values) approaches. In parallel, the chromogenic media commonly used for the recovery of these microorganisms were evaluated and compared, and the efficiency of an enzyme-linked immunosorbent assay (ELISA)-based protocol was validated. L. monocytogenes and E. coli O157:H7 were detected and enumerated using agar Listeria according to Ottaviani and Agosti plus RAPID' L. mono medium and Fluorocult plus sorbitol MacConkey medium with cefixime and tellurite in parallel, respectively. Identity was confirmed with biochemical and molecular tests and the ELISA. Performance indices of the media and the prevalence of both pathogens were estimated using Bayesian inference. In rocket, prevalence of both L. monocytogenes and E. coli O157:H7 was estimated at 7% (7 of 100 samples). In cucumber, prevalence was 6% (6 of 100 samples) and 3% (3 of 100 samples) for L. monocytogenes and E. coli O157:H7, respectively. The levels derived from the presence-absence data using Bayesian modeling were estimated at 0.12 CFU/25 g (0.06 to 0.20) and 0.09 CFU/25 g (0.04 to 0.170) for L. monocytogenes in rocket and cucumber samples, respectively. The corresponding values for E. coli O157:H7 were 0.59 CFU/25 g (0.43 to 0.78) and 1.78 CFU/25 g (1.38 to 2.24), respectively. The sensitivity and specificity of the culture media differed for rocket and cucumber samples. The ELISA technique had a high level of cross-reactivity. Parallel testing with at least two culture media was required to achieve a reliable result for L. monocytogenes or E. coli O157:H7 prevalence in rocket and cucumber samples.

Effect of Lemongrass Essential Oil Vapors on Microbial Dynamics and Listeria monocytogenes Survival on Rocket and Melon Stored under Different Packaging Conditions and Temperatures.

The aim of the present study was to examine the effect of lemongrass essential oil vapors on the dynamics of surface microbiota and L. monocytogenes growth on rocket and melon under different packaging conditions and storage temperature. For that purpose, rocket and melon were placed on Expanded Polystyrene (EPS) trays, sprayed with L. monocytogenes to a population of 4.5-5.0 log CFU·g(-1), packaged using microperforated Oriented Polypropylene (OPP) film in either air or Microperforated Active Modified Atmosphere (MAMA) (initial atmosphere 5% O2, 10% CO2) including a Whatman paper containing the essential oil, without contact with the product, and stored at 0, 5, 10, and 15 °C. Application of lemongrass exhibited a bactericidal effect on enterococci and a fungistatic effect on yeast-mould populations but only during air storage of rocket. The former took place at all temperatures and the latter only at 10 and 15 °C. No effect on shelf life of both products was recorded. However, an important effect on the sensorial properties was observed; during the first 4-5 days of storage both products were organoleptically unacceptable. Regarding MAMA packaging, it affected only Pseudomonas spp. population resulting in a reduction of 1-2 log CFU·g(-1) in both products.

Listeria monocytogenes serotype prevalence and biodiversity in diverse food products.

The aim of this study was to assess serotype prevalence and biodiversity of Listeria monocytogenes strains isolated from diverse food products, i.e., minced pork, fruits, and vegetables. Three hundred twenty-six samples previously purchased from supermarkets and street markets within the Athens area were studied for L. monocytogenes prevalence. A total of 121 strains were isolated from the 36 samples that were positive for L. monocytogenes. Serotyping was performed with multiplex PCR, and biodiversity was assessed with random amplified polymorphic DNA (RAPD) PCR analysis using M13, UBC155, and HLWL85 as primers and with repetitive element palindromic (rep) PCR analysis using (GTG)5 as the primer. The majority (17 of 22) of the contaminated minced pork samples contained strains identified as serotype 1/2a, either alone or in combination with strains belonging to serotypes 1/2b, 4a, 4c, or 4ab. However, all L. monocytogenes isolates from fruits and vegetables belonged to serotype 4b. Rep-PCR provided better differentiation of the isolates than did RAPD PCR and resulted in discrimination of the isolates into a larger number of unique profiles. Complete differentiation was achieved only with the combination of these subtyping techniques.