Hospital admissions due to snake envenomation in the Republic of Cyprus: a 7-year retrospective review.

BACKGROUND: Snake envenomation is a major neglected tropical disease, lacking data in many countries including Cyprus, a Mediterranean island inhabited by the medically important blunt-nosed viper (Macrovipera lebetina). Reviewing the 2013-2019 period, we present first-time epidemiological snakebite data in the Republic of Cyprus. METHODS: We obtained data on snake envenomation-related hospital admissions from the Ministry of Health, and population and rainfall data from the Statistical Service of Cyprus and Department of Meteorology websites. Human-viper conflict information was acquired from interviews with 12 representatives of Cypriot institutions. RESULTS: Between 2013 and 2019, 288 snake envenomation cases were admitted to public hospitals, averaging 41 people annually. The minimum was 29 cases (2017) and the maximum was 58 (2015). Snake envenomation incidence increased from 4.55 per 100,000 population (2013) to 6.84 (2015), but remained low since 2017 (3.49 in 2019). Between 2000 and 2018, the deaths of one man (73 years), and indirectly, one woman (77 years), were related to snake envenomation. While 266 cases (92%) happened between April and October (the blunt-nosed viper activity period), most envenomations occurred in September (cumulative for 2013-2019), with 88 cases (31%). Snakebite incidence peaked in the 60-69 years age group (9.19 per 100,000 population), and was higher in males (6.85) than in females (2.82). Of all admitted patients, 242 (84%) were discharged within 4 days. Mean hospital stay duration was 2.65 days, with one case of 13 days. Most patients were admitted to the general hospitals in Paphos (51%), Limassol (30%) and Nicosia (11%), which provide secondary healthcare, with the last one providing tertiary healthcare. CONCLUSIONS: Snakebite-related deaths are very rare in the Republic of Cyprus. Most envenomation cases happened in late summer (September). Short hospital stays indicate mostly non-severe clinical courses. The hospital admission data suggest that snake envenomation risk is highest in Paphos district. The statistical data hint at males and middle- to older-aged people being at highest risk, whereas from our interview data we assume that outdoor workers are at higher risk than other occupational groups.

Investigation of serum survivin in dogs suffering from cancer: a multicenter study.

BACKGROUND: In contrast to human medicine, only a small number of serum tumor markers are established in veterinary medicine even though they are a non-invasive diagnostic tool. OBJECTIVES: This study examined whether survivin could be suitable as a potential canine serum tumor marker. METHODS: This study measured the serum survivin concentrations of dogs with mammary tumors (n = 33), squamous cell carcinoma (n = 9), soft-tissue sarcoma (n = 18) and multicentric lymphoma (n = 22), using a commercially available, competitive immunoassay kit (BlueGene). The serum survivin concentrations were compared with those of a healthy control group (n = 20) and a control group of dogs with non-neoplastic diseases (n = 17). RESULTS: Dogs with malignant tumors had serum survivin concentrations between 15 and 5,906 pg/mL (median, 72 pg/mL), those in the healthy group ranged from 7 to 99 pg/mL (median, 21 pg/mL) and those in the group of dogs suffering from non-neoplastic diseases from 15 to 93 pg/mL (median, 42 pg/mL). The differences in the survivin concentrations between the healthy dogs and dogs with malignant tumors and between the dogs with non-neoplastic diseases and those with malignant tumors were significant (p < 0.001 and p = 0.006, respectively). CONCLUSIONS: The serum survivin concentrations in dogs with malignant tumors, with some exceptions, are higher than in dogs with benign tumors and dogs that do not suffer from a malignancy. Therefore, survivin can provide information on the presence of malignant tumors and be used as a tumor marker in dogs.

Rapid and Sensitive SERS-Based Lateral Flow Test for SARS-CoV2-Specific IgM/IgG Antibodies.

As an immune response to COVID-19 infection, patients develop SARS-CoV-2-specific IgM/IgG antibodies. Here, we compare the performance of a conventional lateral flow assay (LFA) with a surface-enhanced Raman scattering (SERS)-based LFA test for the detection of SARS-CoV-2-specific IgM/IgG in sera of COVID-19 patients. Sensitive detection of IgM might enable early serological diagnosis of acute infections. Rapid detection in serum using a custom-built SERS reader is at least an order of magnitude more sensitive than the conventional LFAs with naked-eye detection. For absolute quantification and the determination of the limit of detection (LOD), a set of reference measurements using purified (total) IgM in buffer was performed. In this purified system, the sensitivity of SERS detection is even 7 orders of magnitude higher: the LOD for SERS was ca. 100 fg/mL compared to ca. 1 µg/mL for the naked-eye detection. This outlines the high potential of SERS-based LFAs in point-of-care testing once the interference of serum components with the gold conjugates and the nitrocellulose membrane is minimized.

Innovative and Highly Sensitive Detection of Clostridium perfringens Enterotoxin Based on Receptor Interaction and Monoclonal Antibodies.

Clostridium perfringens enterotoxin (CPE) regularly causes food poisoning and antibiotic-associated diarrhea; therefore, reliable toxin detection is crucial. To this aim, we explored stationary and mobile strategies to detect CPE either exclusively by monoclonal antibodies (mAbs) or, alternatively, by toxin-enrichment via the cellular receptor of CPE, claudin-4, and mAb detection. Among the newly generated mAbs, we identified nine CPE-specific mAbs targeting five distinct epitopes, among them mAbs recognizing CPE bound to claudin-4 or neutralizing CPE activity in vitro. In surface plasmon resonance experiments, all mAbs and claudin-4 revealed excellent affinities towards CPE, ranging from 0.05 to 2.3 nM. Integrated into sandwich enzyme-linked immunosorbent assays (ELISAs), the most sensitive mAb/mAb and claudin-4/mAb combinations achieved similar detection limits of 0.3 pg/mL and 1.0 pg/mL, respectively, specifically detecting recombinant CPE from spiked feces and native CPE from 30 different C. perfringens culture supernatants. The implementation of mAb- and receptor-based ELISAs into a mobile detection platform enabled the fast detection of CPE, which will be helpful in clinical laboratories to diagnose diarrhea of assumed bacterial origin. In conclusion, we successfully employed an endogenous receptor and novel high affinity mAbs for highly sensitive and specific CPE-detection. These tools will be useful for both basic and applied research.

Investigation of serum Ki-67 as a biomarker in tumor-bearing dogs.

Because of the limited number of tumor markers in veterinary medicine, there is need for identifying new markers. Ki-67 has been investigated as a tissue marker of malignant alterations. We hypothesized that Ki-67 would also be measurable in serum and should therefore be elevated in cases of malignancy. The purpose of this prospective study was to measure Ki-67 in clinically healthy dogs, dogs with nonmalignant diseases, and dogs with malignant tumors. Samples from 8 healthy dogs, 13 dogs with nonmalignant diseases, and 20 dogs with malignant tumors were collected. Ki-67 was measured using the commercially available canine-specific ELISA. Results demonstrated undetectable Ki-67 serum concentrations in healthy dogs. Dogs with nonmalignant diseases displayed low Ki-67 serum concentrations. In contrast, dogs with malignancies showed significantly increased serum Ki-67 concentrations compared with the healthy (p<0.001) or nonmalignant diseased dogs (p<0.001). The degree of malignancy had a positive influence on serum Ki-67 levels. In contrast, no influence of tumor size on Ki-67 serum concentration was observed (p>0.05). Comparing healthy dogs and tumor bearing dogs a sensitivity of 0.75 and a specificity of 1.0 can be calculated using a Ki-67 cut-off value of 5.5pg/mL. When dogs with a low degree of malignancy were compared with dogs of moderate-to-severe degree malignant tumors a sensitivity of 1.0 and a specificity of 1.0 can be observed at a Ki-67 cut-off value of 19.25pg/mL. In conclusion, our results demonstrate an association of malignancies with elevated Ki-67 serum concentrations in dogs.

Systemic colonization of clover (Trifolium repens) by Clostridium botulinum strain 2301.

In recent years, cases of botulism in cattle and other farm animals and also in farmers increased dramatically. It was proposed, that these cases could be affiliated with the spreading of compost or other organic manures contaminated with Clostridium botulinum spores on farm land. Thus, soils and fodder plants and finally farm animals could be contaminated. Therefore, the colonization behavior and interaction of the botulinum neurotoxin (BoNT D) producing C. botulinum strain 2301 and the non-toxin producing Clostridium sporogenes strain 1739 were investigated on clover (Trifolium repens) in a field experiment as well as in phytochamber experiments applying axenic and additionally soil based systems under controlled conditions. Plants were harvested and divided into root and shoot parts for further DNA isolation and polymerase chain reaction (PCR) assays; subsamples were fixed for fluorescence in situ hybridization analysis in combination with confocal laser scanning microscopy. In addition, we observed significant differences in the growth behavior of clover plants when inoculated with clostridial spores, indicating a plant growth promoting effect. Inoculated plants showed an increased growth index (shoot size, wet and dry weight) and an enlarged root system induced by the systemic colonization of clover by C. botulinum strain 2301. To target C. botulinum and C. sporogenes, 16S rDNA directed primers were used and to specifically detect C. botulinum, BoNT D toxin genes targeted primers, using a multiplex PCR approach, were applied. Our results demonstrate an effective colonization of roots and shoots of clover by C. botulinum strain 2301 and C. sporogenes strain 1739. Detailed analysis of colonization behavior showed that C. botulinum can occur as individual cells, in cell clusters and in microcolonies within the rhizosphere, lateral roots and within the roots tissue of clover.

An enzyme-linked immunosorbent assay for detection of botulinum toxin-antibodies.

INTRODUCTION: Antibodies against botulinum neurotoxin (BNT-AB) can be detected by the mouse protection assay (MPA), the hemidiaphragm assay (HDA), and by enzyme-linked immunosorbent assays (ELISA). Both MPA and HDA require sacrifice of experimental animals, and they are technically delicate and labor intensive. We introduce a specially developed ELISA for detection of BNT-A-AB and evaluate it against the HDA. METHODS: Thirty serum samples were tested by HDA and by the new ELISA. Results were compared, and receiver operating characteristic analyses were used to optimize ELISA parameter constellation to obtain either maximal overall accuracy, maximal test sensitivity, or maximal test specificity. When the ELISA is optimized for sensitivity, a sensitivity of 100% and a specificity of 55% can be reached. When it is optimized for specificity, a specificity of 100% and a sensitivity of 90% can be obtained. RESULTS: We present an ELISA for BNT-AB detection that can be-for the first time-customized for special purposes. Adjusted for optimal sensitivity, it reaches the best sensitivity of all BNT-AB tests available. CONCLUSIONS: Using the new ELISA together with the HDA as a confirmation test allows testing for BNT-AB in large numbers of patients receiving BT drugs in an economical, fast, and more animal-friendly way.

Effects of insecticidal crystal proteins (Cry proteins) produced by genetically modified maize (Bt maize) on the nematode Caenorhabditis elegans.

The genetically modified maize MON89034 × MON88017 expresses different crystal (Cry) proteins with pesticidal activity against the European corn borer (Cry1.105; Cry2Ab2) and the Western corn root worm (Cry3Bb1). Non-target organisms, such as soil nematodes, might be exposed to the Cry proteins that enter the soil in course of crop growing. Therefore, the risk of those proteins for nematodes was assessed by testing their toxic effects on Caenorhabditis elegans. All three insecticidal Cry proteins showed dose-dependent inhibitory effects on C. elegans reproduction (EC50: 0.12-0.38 µmol L(-1)), however, at concentrations that were far above the expected soil concentrations. Moreover, a reduced toxicity was observed when Cry proteins were added jointly. A C. elegans mutant strain deficient for receptors for the nematicidal Cry5B was also resistant against Cry1.105 and Cry2Ab2, suggesting that these Cry proteins bound to the same or similar receptors as nematicidal Cry proteins and thereby affect the reproduction of C. elegans.

Neurotransmitter vesicle release from human model neurons (NT2) is sensitive to botulinum toxin A.

Botulinum neurotoxins (BoNTs) internalize into nerve terminals and block the release of neurotransmitters into the synapse. BoNTs are widely used as a therapeutic agent for treatment of movement disorders and recently gained more attention as a biological weapon. Consequently, there is strong interest to develop a cell-based assay platform to screen the toxicity and bioactivity of the BoNTs. In this study, we present an in vitro screening assay for BoNT/A based on differentiated human embryonal carcinoma stem (NT2) cells. The human NT2 cells fully differentiated into mature neurons that display immunoreactivity to cytoskeletal markers (ßIII-tubulin and MAP2) and presynaptic proteins (synapsin and synaptotagmin I). We showed that the human NT2 cells undergo a process of exo-endocytotic synaptic vesicle recycling upon depolarization with high K(+) buffer. By employing an antibody directed against light chain of BoNT/A, we detected internalized toxin as a punctate staining along the neurites of the NT2 neurons. Using well-established methods of synaptic vesicle exocytosis assay (luminal synaptotagmin I and FM1-43 imaging) we show that pre-incubation with BoNT/A resulted in a blockade of vesicle release from human NT2 neurons in a dose-dependent manner. Moreover, this blocking effect of BoNT/A was abolished by pre-adsorbing the toxin with neutralizing antibody. In a proof of principle, we demonstrate that our cell culture assay for vesicle release is sensitive to BoNT/A and the activity of BoNT/A can be blocked by specific neutralizing antibodies. Overall our data suggest that human NT2 neurons are suitable for large scale screening of botulinum bioactivity.

Pentaplexed quantitative real-time PCR assay for the simultaneous detection and quantification of botulinum neurotoxin-producing clostridia in food and clinical samples.

Botulinum neurotoxins are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven distinct serotypes (A to G) known to cause botulism in animals and humans. In this study, a multiplexed quantitative real-time PCR assay for the simultaneous detection of the human pathogenic C. botulinum serotypes A, B, E, and F was developed. Based on the TaqMan chemistry, we used five individual primer-probe sets within one PCR, combining both minor groove binder- and locked nucleic acid-containing probes. Each hydrolysis probe was individually labeled with distinguishable fluorochromes, thus enabling discrimination between the serotypes A, B, E, and F. To avoid false-negative results, we designed an internal amplification control, which was simultaneously amplified with the four target genes, thus yielding a pentaplexed PCR approach with 95% detection probabilities between 7 and 287 genome equivalents per PCR. In addition, we developed six individual singleplex real-time PCR assays based on the TaqMan chemistry for the detection of the C. botulinum serotypes A, B, C, D, E, and F. Upon analysis of 42 C. botulinum and 57 non-C. botulinum strains, the singleplex and multiplex PCR assays showed an excellent specificity. Using spiked food samples we were able to detect between 10(3) and 10(5) CFU/ml, respectively. Furthermore, we were able to detect C. botulinum in samples from several cases of botulism in Germany. Overall, the pentaplexed assay showed high sensitivity and specificity and allowed for the simultaneous screening and differentiation of specimens for C. botulinum A, B, E, and F.

The case of botulinum toxin in milk: experimental data.

Botulinum neurotoxin (BoNT) is the most toxic substance known to man and the causative agent of botulism. Due to its high toxicity and the availability of the producing organism Clostridium botulinum, BoNT is regarded as a potential biological warfare agent. Because of the mild pasteurization process, as well as rapid product distribution and consumption, the milk supply chain has long been considered a potential target of a bioterrorist attack. Since, to our knowledge, no empirical data on the inactivation of BoNT in milk during pasteurization are available at this time, we investigated the activities of BoNT type A (BoNT/A) and BoNT/B, as well as their respective complexes, during a laboratory-scale pasteurization process. When we monitored milk alkaline phosphatase activity, which is an industry-accepted parameter of successfully completed pasteurization, our method proved comparable to the industrial process. After heating raw milk spiked with a set amount of BoNT/A or BoNT/B or one of their respective complexes, the structural integrity of the toxin was determined by enzyme-linked immunosorbent assay (ELISA) and its functional activity by mouse bioassay. We demonstrated that standard pasteurization at 72 degrees C for 15 s inactivates at least 99.99% of BoNT/A and BoNT/B and at least 99.5% of their respective complexes. Our results suggest that if BoNTs or their complexes were deliberately released into the milk supply chain, standard pasteurization conditions would reduce their activity much more dramatically than originally anticipated and thus lower the threat level of the widely discussed "BoNT in milk" scenario.

Challenging the problem of clostridial identification with matrix-assisted laser desorption and ionization-time-of-flight mass spectrometry (MALDI-TOF MS).

Diverse techniques were applied to effect the identification and classification of isolated clostridial strains. Nevertheless, the correct identification of clostridial strains remains a laborious, time-consuming task which entails a not inconsiderable degree of expertise. In addition to this, traditional methods based on the metabolic properties of the bacteria require rigorously standardized media and growth conditions to assure the attainment of reproducible results. Although DNA-based methods, like the PCR of a species specific gene, are known to yield precise and reproducible results, their degree of effectivity is circumscribed by the fact that even the incidence of a toxin encoding gene is not necessarily linked to nor consequently indicative of the presence of an infectious disease. Moreover, most of these methods postulate an initial assumption concerning the expected bacterial species involved before the choice of PCR primer for use can be made. Consequently, the scope of these methods is restricted to that of targeted analyses. The 16S rDNA sequencing which is assumed to be the gold standard for bacterial classification having the unequivocal advantage of being capable of determining even uncultivable bacteria is nonetheless a time-consuming and costly technique. In the present study we describe the utilization of matrix-assisted laser desorption and ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for whole cell fingerprinting in combination with a dedicated bioinformatic software tool to distinguish between various clostridial species. Total 64 clostridial strains of 31 different species each displayed a mass spectrum unique to the strain involved, to the effect that it was also possible not only to differentiate between the strains examined, but also to establish to which species the individual strains belonged to. Starting with a single colony it was possible to correctly identify a Clostridium species within minutes. It was even possible to identify species which are normally difficult to differentiate by traditional methods, such as C. chauvoei and C. septicum. With the results obtained we were able to assemble a dendrogram of the Clostridium species which showed considerable similarities to dendrograms based upon 16S rDNA sequencing data. To conclude, our findings indicate that, inasmuch as the MALDI-TOF MS technology employed is based on a high-quality reference database, it may serve as an effective tool for the swift and reliable identification and classification of Clostridia.

Evaluation of lateral flow assays for the detection of botulinum neurotoxin type A and their application in laboratory diagnosis of botulism.

Four lateral flow assays (LFAs) were evaluated for the detection of purified botulinum neurotoxin A, toxin complex, and unpurified culture supernatant. They included the BioThreat (Tetracore, Rockville, MD), SMART (New Horizons Diagnostics, Columbia, MD), BADD (ADVNT Biotechnologies, Phoenix, AZ), and RAMP (Response Biomedical, Burnaby, BC, Canada) assays. BioThreat and SMART did not detect the purified toxin. The best sensitivity was achieved with the RAMP test (50 ng mL(-1)). BioThreat and SMART measured as low as 10 ng mL(-1) of the toxin complex. Specificity data differed among the tests. BADD gave false-positive signals with uninoculated bacterial culture medium. BioThreat and RAMP were further evaluated with clinical sample matrices (serum, gastric, and rectum contents from pigs). Because of matrix effects and a generally low positive response, the assays are unsuitable for the direct detection of the toxin. However, the LFAs can be a helpful tool in screening bacterial cultures for toxigenic Clostridium botulinum, if further validated according to the laboratory needs.

Persistence and mobility of a Clostridium botulinum spore population introduced to soil with spiked compost.

In a recent study it could be shown that compost samples can contain Clostridium botulinum. It was investigated if C. botulinum introduced with compost into botulinum-free soil can persist and be translocated within the soil. Compost was spiked with two C. botulinum type D spore concentrations (10(3) and 10(5) spores g(-1)) and the composts were spread on an experimental site. Over a period of 939 days, samples were taken from the upper (0-5 cm) and the lower (10-30 cm) soil horizons. Physical and chemical as well as microbiological variables were measured. Clostridium botulinum spores were quantified in a culture MPN-PCR assay. On day 757 the last positive sample was obtained in the plots with the lower spore concentration (10(3) g(-1)). The bacteria were never detected in the samples taken from the lower horizons of these plots. Clostridium botulinum persisted over the whole investigation period in the plots which were treated with compost spiked with 10(5) spores g(-1). The concentrations found were between 20 and 20,000 spores g(-1) soil. The bacteria were vertically translocated and could be found in the lower soil horizons (20-2000 spores g(-1) soil) starting 70 days after the compost was spread.

Immunomagnetic beads assay for the detection of botulinum neurotoxin types C and D.

An immunomagnetic beads assay for the simultaneous quantification of botulinum neurotoxin types C and D was developed. Specific monoclonal antibodies against the heavy chain of the toxin and affinity-purified biotinylated polyclonal antibodies (pAbs) were used. The antibodies were preincubated with the sample. The complex being formed was then captured by magnetic beads coated with antimouse IgG. Streptavidin-poly-horseradish peroxidase, a signal amplifier, bound to the biotinylated pAb. A maximum sensitivity of approximately 0.3 minimal lethal doses for mice per milliliter was achieved with culture supernatants of both toxin types.

Sensitive detection of botulinum neurotoxin types C and D with an immunoaffinity chromatographic column test.

A sensitive and specific immunoassay for the simultaneous detection of Clostridium botulinum type C (BoNT/C) and type D neurotoxin was developed. Goat anti-mouse immunoglobulin G was bound to polyethylene disks in a small disposable column used for this assay. The sample was preincubated together with monoclonal antibodies specific for the heavy chain of BoNT/C and D and affinity-purified, biotinylated polyclonal antibodies against these neurotoxins. This complex was captured on the assay disk. Streptavidin-poly-horseradish peroxidase was used as a conjugate, and a precipitating substrate allowed the direct semiquantitative readout of the assay, if necessary. For a more accurate quantitative detection, the substrate can be eluted and measured in a photometer. Depending on the preincubation time, a sensitivity of 1 mouse lethal dose ml(-1) was achieved in culture supernatants.

A new scaleable method for the purification of botulinum neurotoxin type E.

Botulinum neurotoxins belong to the most toxic substances in nature. Well-known as a food poisoning agent for almost two centuries, the beneficial aspects of this bacterial metabolite were rediscovered about 30 years ago. These toxins, which are produced by the anaerobic bacterium Clostridium botulinum are nowadays used to treat a variety of neuro-muscular disorders. The increased demand requires techniques for the production and purification of these toxins on an industrial scale. The method described herein is based on filtration and chromatography procedures only. Precipitation, centrifugation and dialysis steps were consequently excluded to develop a protocol, which can easily be scaled up from the laboratory purification to industrial needs. About 4 mg of BoNT/E were purified from a 10-L batch culture corresponding to an overall recovery of approximately 14%.

[Diagnosis of botulism since 1995. Report of test results]. / Botulismusdiagnose seit 1995. Ein Erfahrungsbericht.

Since 1970 our laboratory is specialized in diagnosis of clostridial diseases, including Clostridium botulinum and botulism. Since 1995, samples from more than 900 suspected botulinal cases were received, mainly in cattle, horses and men. 524 outbreaks were diagnosed as clearly positive by toxin neutralisation; 83 cases remained inconclusive with the toxin neutralisation. The geographical distribution of the positive cases in Germany is demonstrated for cattle and horses. Dispatch and treatment of specimens and interpretation of results are discussed.

[Development of an in vitro assay for the detection of Botulinum Neurotoxins]

Botulism is a severe, often lethal intoxication, which affects man and animal. It is caused by the neurotoxins (BoNT) of Clostridium botulinum, an anaerobic spore-former. At present the toxin types A-G have been identified. The extreme toxicity of these substances still make the animal testing inevitable. No alternative method could reach the sensitivity of the mouse bioassay. Based on the investigations presented, a serological test shall be developed, which concentrates the sample and detects the toxin in one step. Preliminary results of an in vitro test detecting BoNT/D are discussed.