Toxin ELISA Research Articles

P-207. Implementation of Two-Step C. difficile Testing to Reduce HO-CDI and Antibiotic Use with Analysis of Ordering and Treatment Practices

Abstract Background C. difficile infection (CDI) is an ongoing challenge for healthcare facilities. The mischaracterization of C. difficile colonization as active infection falsely elevates healthcare onset-CDI (HO-CDI) rates and increases antimicrobial overuse and healthcare costs. Two-step testing for CDI helps differentiate colonization from infection. We describe the change in HO-CDI rates and antimicrobial use following implementation of two-step C. difficile testing with analysis of order and treatment practices post-implementation.Figure 1:HO-CDI Rates Pre and Post-InterventionHO-CDI rates per 10,000 patient days pre and post-intervention. The vertical dotted black line indicates the date of intervention. Methods Two-step C. difficile testing (PCR followed by ELISA for toxin if PCR-positive) was implemented Sept. 5, 2023. HO-CDI rates were compared for 6 months pre and post intervention using MedCalc statistical software. Antimicrobial patient days per 1000 patient days for oral vancomycin and fidaxomicin was compared pre and post-intervention using Wilcoxen Rank Sum test. Analysis of all inpatients with a C. difficile order during the post-intervention period was performed including test result, demographics, diarrhea documentation, laxative use, C. difficile treatment and repeat testing.Figure 2:Antimicrobial Patient Days for PO Vancomycin and Fidaxomicin Pre and Post-InterventionAntimicrobial patient days per 1000 pre and post-intervention. The vertical dotted black line indicates the date of intervention. Results HO-CDI rates decreased from 6.44 to 2.89/10,000 patient days pre and post-intervention with an incidence rate ratio of 2.22, P< 0.0001 (Figure 1). Inpatient prescriptions for oral vancomycin and fidaxomicin decreased from 28.4 to 22.2 antimicrobial patient days per 1000 patient days, p=0.0295 (Figure 2). A total of 1836 tests were done in the post-intervention period with detailed analysis included in Table 1. Less than 20% of patients had ≥3 diarrhea stools documented and excluding orders on HD 1 or 2, stool documentation was present in 29% of patients. Analysis of the 66 HO-CDI cases was similar compared to all positive cases with 24 (36%) receiving laxatives and 21 (36%) with diarrhea documented.Table 1:Analysis of All Inpatient C. diff Orders Post-InterventionAnalysis of all inpatient C. diff orders post-intervention, where negative indicates PCR negative / toxin negative; discordant indicates PCR positive / toxin negative; and positive indicates PCR positive / toxin positive. Conclusion HO-CDI rates and C. difficile specific antimicrobial use were significantly decreased after implementation of two-step testing. Despite this decrease, over half of discordant result patients received C difficile treatment, indicating an opportunity for additional education. Optimization of diagnostic stewardship with regards to laxative use and appropriate stool documentation are areas of opportunity. Disclosures All Authors: No reported disclosures

Diagnosis of botulism in mammals aided by toxin ELISA and C and D gene RT-PCRs with an emphasis on farm animals.

Animal welfare and ethical considerations limit the use of the mouse lethality bioassay for the detection of botulinum toxins. This study assessed an ELISA and RT-PCRs to detect toxin types C and D in samples from mammals with flaccid paralysis or weakness. DNA/RNA tightly bound to the toxin molecules were used as surrogates for the presence of toxin. In total 391 diverse samples from 101 clinical cases from cattle and 9 other species were examined. Botulism was confirmed by ELISA in samples from 16 cases. Toxin type C or D was identified by RT-PCR in 10 cases, five of which were among the cases confirmed by ELISA. Among the 80 cases with negative samples, sampling was not comprehensive enough in 66 cases (botulism therefore not excluded) but was adequate in 14 cases (no indication of botulism). The last two categories included 16 cases with other laboratory findings explaining the clinical signs. The ELISA that detects botulinum C and D toxins was superior in identifying cases with positive samples compared to the RT-PCRs but the latter were able to identify the toxin type present when positive. Furthermore, the RT-PCRs are available to any laboratory with molecular testing capability. Both laboratory assays can screen a comprehensive range and a large number of samples which is crucial for a successful diagnosis of botulism.

Inhibiting Peptidoglycan Hydrolase Alleviates MRSA Pneumonia Through Autolysin-Mediated MDP-NOD2 Pathway.

Methicillin-resistant Staphylococcus aureus (MRSA) is a cause of staph infection that is difficult to treat because of resistance to some antibiotics. A recent study indicated that diarylurea ZJ-2 is a novel antibacterial agent against multi-drug resistant Enterococcus faecium. In this work, we refined the bactericidal mechanism of ZJ-2 as a peptidoglycan (PG) hydrolase by affecting AtlA-mediated PG homeostasis. A wild-type strain (WT) and a mutant strain (ΔatlA) were used to investigate the effects of ZJ-2 on the cell wall, PG, and autolysin regulatory system by antimicrobial susceptibility testing, hemolytic toxin assay, microanalysis, autolysis assay, qRT-PCR, ELISA and mouse model of pneumonia. The results revealed that ZJ-2 down-regulated the expression of genes related to peptidoglycan hydrolase (PGH) (sprX, walR, atlA, and lytM), and reduced the levels of PG, muramyl dipeptide (MDP), cytokines, and hemolytic toxin, while ΔatlA interfered with the genes regulation and PG homeostasis. In the mouse MRSA pneumonia model, the same trend was observed in the nucleotide oligomerization domain protein 2 (NOD2) and relative proinflammatory factors. ZJ-2 may act as a novel inhibitor of PG hydrolyse, disrupting autolysin-mediated PG homeostasis, and reducing inflammation by down-regulating the MDP-NOD2 pathway.

754. A Two-step Testing Algorithm for Hospital-onset Clostridioides difficile Infection (CDI) Reduces Prescribing of C. difficile (CD) Therapy but Its Ability to Guide Treatment Decisions Remains Unclear

BackgroundDetermining true CDI versus CD colonization through CD testing is a continuing challenge. A previously introduced decision support tool at UVA Health significantly reduced inappropriate testing without adverse outcomes. More recently, our methodology changed from nucleic acid amplification test (NAAT) alone to an initial NAAT followed by ELISA for toxin to improve specificity. The purpose of this analysis was to assess provider interpretation of test results, using targeted CD therapy as a surrogate.MethodsThis single-center, retrospective study evaluated all patients with a positive NAAT (Cepheid Xpert® C. difficile) on day 4 or later of hospitalization following 2-step algorithm implementation from Feb 2020 through Feb 2021. Toxin negative (TOX-) test results (C. DIFF QUIK CHEK COMPLETE®) were accompanied by a comment that discordance may represent colonization or CDI and to consider ID consult. The proportion of toxin positive (TOX+) versus TOX- patients receiving ≥ 1 dose of CD therapy served as the primary outcome with partial courses considered < 10 days. Clinical outcomes were also compared.ResultsNinety patients with NAAT+ results were included, of whom 58 (64%) were TOX-. Thirty-two (100%) TOX+ (median days of therapy [IQR] = 14 [11-17]) versus 51 (88%) TOX- patients (median days of therapy [IQR] = 11 [7-14]) received CD therapy (p=0.04). Treatment decisions were guided by ID physicians for 32 (63%) TOX- patients; ID recommendations to discontinue CD therapy were followed in 2 out of 9 (22%) cases. TOX- patients received partial therapy due to patient death (n=5), presumptive colonization (n=3), and provider error (n=1). Of TOX- patients receiving partial or no treatment, there were no CDI-related adverse outcomes during the admission. CDI-related colectomy occurred in 2 (6%) and 1 (2%) TOX+ and TOX- patients, respectively. Five in-hospital deaths with CDI as a contributing factor occurred in the TOX+ group. ConclusionAdoption of a 2-step NAAT plus toxin testing algorithm for hospital-onset CDI reduced the frequency with which TOX- patients received CD therapy but the vast majority were still treated. Most providers considered a positive NAAT indicative of CDI, regardless of TOX status. Disclosures All Authors: No reported disclosures

Duration of protection against Bordetella pertussis infection elicited by whole-cell and acellular vaccine priming in Polish children and adolescents

BackgroundIn the context of reported resurgence of pertussis in the last decade, researchers hypothesized that acellular (aP) pertussis vaccines elicit a shorter-lived protection compared to whole-cell (wP) pertussis vaccines. However, in the studies seeking to demonstrate this hypothesis, exposure to each vaccine type was not concurrent, and contradictory epidemiologic modeling questioned its validity. The context of pertussis vaccination history in Poland, with both vaccine types used concurrently in comparable proportions, provided an opportunity to investigate this hypothesis. We sought to compare waning of protection by primary series vaccine type by measuring anti-pertussis toxin antibody concentrations as proxy for recent infection. Materials and methodsSerological samples from 2,745 children and adolescents aged ≥5 years and <16 years and with completed 5-dose pertussis vaccination series were tested by ELISA for pertussis toxin (PT) antibodies. Participants were stratified by type of priming vaccine (wP or aP). Vaccination timeliness and priming-specific trends in anti-PT antibody levels by time since last vaccine dose were analyzed. ResultsA total of 1,161 (42.5%) children received wP vaccines, and 1,314 (48.1%) received aP vaccines for their primary series and toddler booster. Overall, 53.57% of the subjects received doses 2–4 in a timely manner, while only 41.52% received all 5 doses at the recommended intervals. Using GMCs or seropositivity measures, both priming groups showed a re-increase in anti-PT antibody levels signing infection in recent years from 8 years after the school-entry booster onward. Comparisons did not show any significant differences between the two groups in the timing or intensity of this re-increase. ConclusionOur results clearly confirm that vaccine-elicited immunity against pertussis wanes among adolescents even after a complete infant, toddler and school-entry vaccination series. The timing and intensity of the waning of protection appear similar with whole-cell as with acellular pertussis vaccines.

Seroprevalence of an antibody against diphtheria, tetanus, and pertussis among the elderly in Khon Kaen, Thailand

BackgroundOwing to a declining birth rate and longer lifespan, the number of elderly people (≥ 60 years) in Thailand has grown rapidly. However, the elderly are at significant risk of infectious diseases because they have never been immunized, because they have not been completely immunized, or because their immunity has waned. Immunity against infectious diseases in the elderly is an important means of controlling diseases in the community. Our objective was to evaluate the seroprotective rate against diphtheria, tetanus, and pertussis in the elderly Thai population.MethodsIn total, 430 healthy individuals from the northeastern region of Thailand were enrolled in this study and stratified into five age groups: 60–65, 66–70, 71–75, 76–80, and > 80 years. Serum samples were collected and quantitatively analyzed for diphtheria, tetanus, and pertussis IgG antibody by using commercial ELISA kits. For anti-diphtheria toxoid and anti-tetanus toxoid ELISA, values < 0.01 IU/ml were interpreted as seronegative, and for anti-Bordetella pertussis toxin ELISA, values < 5 IU/ml were interpreted as seronegative; these definitions were in accord with previous studies.ResultsFor diphtheria toxoid Ab, the majority of the population had antibody levels > 0.01 IU/ml. For tetanus anti-toxoid Ab, the majority of the population had antibody levels of > 0.01 IU/ml, of which approximately 34% had durable antibody protection levels (DAPL) of ≥ 1 IU/ml. Meanwhile, nearly 45% of the population had an Ab level against pertussis lower than the protectivity level.ConclusionsIn total, 97.2%, 83.5%, and 55.8% of the population had a higher antibody level than the minimal protective level for diphtheria, tetanus, and pertussis, respectively. In order to prevent an outbreak of these diseases in the future, the elderly should be administered with Tdap revaccination to provide diphtheria herd immunity in the population; this will increase cocoon phenomenon for pertussis and protect the population from tetanus-prone injury.

Effect of Honey Supplementation on Clostridium Difficile Infection in Childhood Cancer

Background: the treatment of cancer is associated with nausea and vomiting, oral mucositis, constipation, xerostomia and diarrhea and weight loss, additionally; chemotherapeutic agents promote inflammatory changes in the gut, intestinal necrosis, and anaerobic conditions, allowing proliferation of Clostridium Difficile. Honey, as a natural honeybee product, has antioxidant, antimicrobial, immunomodulatory and anticancer effects. Honey can fight microbial infection by its immuno-activating, anti-inflammatory and prebiotic activity. Objectives: the aim of this study was to evaluate the effect of honey supplementation on frequency of Clostridium Difficile infection (CDI) and gastrointestinal complications in pediatric patients undergoing chemotherapy. Design: a cross sectional study conducted on 40 patients with malignancy recruited from Children's Hospital, Ain Shams University, Oncology Unit and Clinic, Cairo, Egypt in the period from December 2015 to December 2016. Patients were divided into two groups; group I (25 patients) received honey in the dose of 2gm/kg 3 times dailyfor 1month) while group II (15 patients) did not receive honey. All the studied patients were subjected to medical history and clinical examination, with special emphasis on gastrointestinal complication including oral mucositis, vomiting, diarrhea, constipation, and abdominal pain. Follow up was done for weight, height z score, gastrointestinal complications and any adverse events. Stool analysis, culture, C difficle toxin A, B by ELISA was done to all patients at baseline and repeated to patients receiving honey at week 4 of supplementation. Main outcome measure frequency of CDI, gastrointestinal complication, febrile neutropenia . Results: the frequency of C difficle was 8% (2), the first case was 9 years old patient with ALL (50%) and the other 11 years old patient with Burkitts lymphoma both were diagnosed by positive stool culture and positive stool ELISA for toxin A, B. gastrointestinal complications were significantly less and improved in the supplemented group and mean of hemoglobin significant increase in group 1. Conclusion: the frequency of CDI in children with cancer 8% diagnosed by stool culture and toxin A, B study in stool. Honey improved the oral mucositis and different GIT complications associated with chemotherapy.

Mo1845 Is Colonization With Non-Toxigenic Clostridium difficile Organism Protective Against Toxigenic Strains?

Background: Clostridium difficile infection (CDI) requires production of toxins A and/or B to manifest clinically but not all strains produce these specific virulence factors. Nontoxigenic C. difficile strains (NT-CD) cause asymptomatic colonization of gastrointestinal tract. Studies using hamster model have demonstrated that colonization with NT-CD decreases risk of subsequent infection with toxigenic strains (T-CD). The aim of this study was to investigate if use of two-step stool toxin assay can determine if colonization with NT-CD protects against CDI.Methods:CDI testing involves a two-step stool assay: Glutamate dehydrogenase (GDH) and toxin ELISA followed by PCR. GDH detects the presence of both T-CD and NT-CD. The ELISA and PCR tests detect presence of toxins A and B. Two groups of patients tested for clinically suspected CDI were identified GDH negative / toxin negative (G-T-) (No CDI or colonization) and GDH positive / toxin negative and PCR negative (G+T) (Colonization with NT-CD). Only patients with high risk for CDI (Age > 50 years, ≥ 2 hospitalizations and recent antibiotic use) were included. Patients with prior history of CDI or current infection (GDH positive and toxin or PCR positive) were excluded. Based on previous studies, CDI rate in patients colonized with NT-CD is about 1% as compared to 15% in those not colonized. Chart review was performed retrospectively from October 2013 to include a total of 100 subjects in each group (80% power and 95% Confidence intervals). Incidence of CDI in one year follow up period after initial negative toxin assay was assessed. Chi-square test for binomial data and 2 tailed t-test for continuous data were used. Results: A total of 200 patients (Age = 68.7 ± 11.8 years, 57.5% women) were included. 9 CDI occurred in G+Tgroup compared to 7 CDI in G-Tgroup (p=0.79). The recurrence rates were 22.2% (2 cases) in G+T-group and 14.2% (1 case) in G-T-group (p=0.69). Hospitalization rates were similar between two groups (3.45 ± 2.1 days in G+T-and 3.65 ± 2 days in G-T-group, p=0.49). The average length of stay was 7.39 ± 4 days in G+Tcompared to 7.73 ± 4.7 days in G-T-group (p=0.58). Discussion: Our study indicates that colonization with NT-CD does not protect against future CDI or reduce recurrence of CDI. This is contradictory to previous animal studies supporting protective effects of NT-CD. The explanation for this discordance could be that protective effect of NT-CD may be related to specific strains of NT-CD. Moreover unlike in animal studies, human microbiome may be impacted by several additional factors like exposure to antibiotics and frequent hospitalizations, leading to acquisition of toxigenic strains. It would be interesting to study in future if fecal microbiota transplantation works by favoring colonization with specific NT-CD strains to prevent recurrent CDI .

Whole-Genome Sequencing for Risk Assessment of Long-term Shiga Toxin–producingEscherichia coli

Whole-Genome Sequencing for Risk Assessment of Long-term Shiga Toxin–producing<i>Escherichia coli</i>

Impact of solar irradiation on cholera toxin secretion by different strains of Vibrio cholerae

Cholera toxin is the aetiological agent of cholera – a deadly waterborne disease acquired through the consumption of untreated water contaminated with CTXФ bacteriophage harbouring strains of V. cholerae. Solar disinfection is a re-emerging technique that relies on the ultraviolet component of sunlight to inactivate the growth of Vibrio cholerae in water, rendering the water microbiologically safe for consumption. However, studies have shown that DNA damaging agents, such as ultraviolet light, induce the replication of the CTXФ bacteriophage with subsequent expression of the cholera toxin. In this study we investigated the impact of solar irradiation on the secretion of cholera toxin by toxigenic strains of V. cholerae in water. The cholera toxin ELISA assay, qualitative and quantitative real-time PCR as well as growth on solid media were used to determine cholera toxin secretion, DNA integrity and growth of the bacteria after 7 h and 31 h of solar irradiation. Solar irradiation in water reduced the integrity of DNA, inactivated the growth of V. cholerae and, most importantly, prevented the secretion of detectable levels of cholera toxin. This finding is encouraging for resource-poor communities that may rely on solar disinfection to alleviate the burden of cholera-related fatalities.

Recovery and detection of botulinum toxin type A from drinking water

s Toxins 2011 / Toxicon 68 (2013) 60–123 94 Double anchorage to the membrane and intact interchain disulfide bond are required for the lowpH-induced entry of tetanus and botulinum neurotoxins into neurons M. Pirazzini , O. Rossetto , P. Bolognese , C.C. Shone , C. Montecucco a Dipartimento di Scienze Biomediche and Istituto CNR di Neuroscienze, Universita di Padova, Padova, Italy Health Protection Agency, Porton Down, Salisbury, UK E-mail address: marcopiraz@yahoo.it (M. Pirazzini). Purpose of study: To study themembrane translocation of the clostridial neurotoxins in neurons. Methods used: We have developed a protocol based on an experiment first performed with diphtheria toxin. Conditions were found that by-pass the SV endocytosis step and induce the entry of the L chain directly from the plasma membrane. The neurotoxin, bound to the plasma membrane in the cold, was exposed to a warm, low pH extracellular medium, and the entry of the L chain was monitored by measuring its specific metalloprotease activity with a ratiometric method. Summary of results: We found that the neurotoxin has tobebound to themembraneviaat least twoanchorage sites in order for a productive low-pH-induced structural change to take place. In addition, this process can only occur if the single inter-chain disulfide bond is intact. The pH dependence of the conformational change of tetanus neurotoxin and botulinum neurotoxins B, C and D is similar and take places in the same slightly acidic range, which comprises that present inside synaptic vesicles. Furthermore, using PROPKA3.0 software, we found that tetanus and botulinum neurotoxins share a pool of conserved acid residues that are predicted to be protonated in the pH range 4.5-6. Conclusions: Tetanus and botulinum B, C and D neurotoxins undergo to a conformational change in a very similar pH range (4.5-6), which leads to light chain internalization. These neurotoxins share a pool of acidic residues which could be involved in the initial steps of the pHdependent conformational change. http://dx.doi.org/10.1016/j.toxicon.2012.07.099 Recovery and detection of botulinum toxin type A from drinking water B.H. Raphael , M. Lautenschlager , A. Kahler , S. Pai , B.A. Parks , S. Kalb , S. Maslanka , S. Shah , M. Magnuson , V. Hill a Division of Foodborne, Waterborne, and Environmental Diseases, National Center of Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA Division of Laboratory Science, National Center of Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, USA National Homeland Security Research Center, U.S. Environmental Protection Agency, Cincinnati, OH, USA E-mail address: braphael@cdc.gov (B.H. Raphael). Purpose of study: Botulinum neurotoxin (BoNT) represents a possible biothreat agent for drinking water systems. However, a standard method for monitoring drinking water potentially contaminated with BoNT is not currently available to response laboratories. Methods used: Both the botulinum toxin ELISA and Endopep-MS (a mass spectrometric-based endopeptidase method) were adapted to quantify complex BoNT/A in deionized water and in chlorine-demand-free (CDF) water. Summary of results: The method detection limit (MDL) of the ELISA and theMSmethods in CDF water was 500 and 50 pg/ml of BoNT/A complex toxin, respectively. While adapting these methods to municipal tap water samples, residual chlorine in such samples was neutralized with 50 mg/L sodium thiosulfate prior to toxin spiking because free chlorine rapidly (<5 minutes) inactivates BoNT. Prior to testing by either ELISA or MS, municipal tap water samples from 5 U.S. states having distinct water quality properties were processed using ultrafiltration (UF) to concentrate the toxin by up to 200-fold. In order to evaluate the efficiency of toxin concentration using UF, 100 L tap water samples were spiked with 5 ug BoNT/A, which represents a final concentration equivalent to the MDL of the Endopep-MS method (i.e., 50 pg/ml). Recovery efficiencies of the UF method for BoNT/A complex, quantified by ELISA, ranged from 11%-36%, while efficiencies quantified by MS ranged from 26%-55%. Conclusions: Both the botulinum toxin ELISA and the Endopep-MS methods were effective for the quantification of BoNT in municipal tap water samples of varying properties. Additionally, UF was capable of significantly increasing the BoNT/A concentration in tap water samples for analysis by either ELISA or EndopepMS. http://dx.doi.org/10.1016/j.toxicon.2012.07.100 Simultaneous detection and differentiation of botulinum toxin serotypes A and B by internallyquenched fluorogenic peptides

Ultrafiltration improves ELISA and Endopep MS analysis of botulinum neurotoxin type A in drinking water

The objective of this study was to adapt and evaluate two in vitro botulinum neurotoxin (BoNT) detection methods, including the Botulinum Toxin ELISA and the Endopep MS (a mass spectrometric-based endopeptidase method), for use with drinking water samples. The method detection limits (MDL) of the ELISA and Endopep MS were 260pg/mL and 21pg/mL of BoNT/A complex toxin, respectively. Since toxin could be present in water samples at highly dilute concentrations, large volume (100-L) samples of municipal tap water from five US municipalities having distinct water compositions were dechlorinated, spiked with 5μg BoNT/A, and subjected to tangential-flow ultrafiltration (UF) using hollow fiber dialyzers. The recovery efficiency of BoNT/A using UF and quantified by ELISA ranged from 11% to 36% while efficiencies quantified by MS ranged from 26% to 55%. BoNT/A was shown to be stable in dechlorinated municipal tap water stored at 4°C for up to four weeks. In addition, toxin present in UF-concentrated water samples was also shown to be stable at 4°C for up to four weeks, allowing holding of samples prior to analysis. Finally, UF was used to concentrate a level of toxin (7pg/mL) which is below the MDL for direct analysis by both ELISA and Endopep MS. Following UF, toxin was detectable in these samples using both in vitro analysis methods. These data demonstrate that UF-concentration of toxin from large volume water samples followed by use of existing analytical methods for detection of BoNT/A can be used in support of a monitoring program for contaminants in drinking water.

In vivo lysogenization of a Clostridium difficile bacteriophage ФCD119

Clostridium difficile is a nosocomial pathogen identified as the cause of antibiotic-associated diarrhea and colitis. In this study, we have documented the lysogeny of a C. difficile bacteriophage in hamsters during C. difficile infection. The lysogens isolated from the hamsters were toxin typed and their phage integration site was confirmed by PCR. Through toxin ELISA it was found that the toxin production in the in vivo isolated lysogens was affected due to ФCD119 lysogenization as in the case of in vitro isolated ФCD119 lysogens. Together our findings indicate that a baceriophage can lysogenize its C. difficile host even during the infection process and highlights the importance of lysogeny of C. difficile phages as an evolutionary adaptation for survival.

Recurrence of extensive injection site reactions following DTPa or dTpa vaccine in children 4–6 years old

Objectives The aim of this study was to compare the immunogenicity and reactogenicity of a lower dose diphtheria, tetanus and pertussis vaccine (dTpa) with the recommended vaccine (DTPa) given as a fifth dose to 4–6-year old children who previously experienced an extensive injection site reaction (ISR). Material and methods Children aged 4–6 years who had experienced an extensive ISR following a 4th dose of DTPa were recruited and randomly assigned to receive either the recommended DTPa or the lower dose dTpa vaccine. Parents recorded local reactions and systemic events for 15 days following vaccination. Immunogenicity was assessed pre and post vaccination by ELISA for diphtheria (D), tetanus (T), pertussis toxin (PT), filamentous haemagglutinin (FHA), and pertactin (PRN). Results A total of 53 participants were vaccinated. There was a 72% recurrence rate of ISR, with a trend ( p = 0.055) towards fewer ISR in the dTpa (61.5%) compared with the DTPa group (85.2%). There was no difference in reports of pain or irritability between groups. All participants had seroprotective levels of antibody to D and T and seroresponse to each of the 3 pertussis antigens following vaccination with higher GMCs in DTPa vs dTpa group. There was no increase in antibody avidity observed post vaccination, regardless of vaccine given. Conclusion Recurrence of ISR with the 5th dose of diphtheria, tetanus and pertussis vaccination in children who have previously experienced an extensive ISR is high. Vaccination with a dTpa vaccine may reduce the risk of fifth dose ISR.

Utility of Botulinum Toxin ELISA A, B, E, F kits for clinical laboratory investigations of human botulism

The Botulinum Toxin ELISA effectively provided presumptive identification of toxin in 1381 investigation samples including clinical specimens, suspect foods, and cultures. Additionally, the ELISA detected all toxins produced by a panel of stock strains representing known subtypes and was negative for non-botulinum toxin producing Clostridium and enteric pathogens. ELISA results were reproducible both within the same kit (CV < 9%) and among different production lots (CV < 23%). Fifty-five of 57 laboratories correctly identified unknown samples in a multi-laboratory study. The ELISA provides a rapid, robust in vitro screening method which will reduce animal dependence during laboratory investigations of botulism.

Etiology of Acute Gastroenteritis in Three Sentinel General Practices, Austria 2007

We studied the etiology of acute gastroenteritis in a village with a total population of approximately 6,000. This is the first study in Austria that has investigated a broad range of pathogens recovered from an unselected population of patients who had consulted general practitioners because of gastroenteritis. In 2007, all patients who visited one of three local general practitioners for acute gastroenteritis were invited to provide stool specimens to be tested for Salmonella, Shigella, Campylobacter, enterohemorrhagic Escherichia coli (EHEC) (mTSB enrichment [R-Biopharm] followed by toxin ELISA plus culture), enteropathogenic E. coli (EPEC), Yersinia, Vibrio cholerae, Clostridium difficile (toxin plus culture), rotavirus plus adenovirus (RIDA) Quick Rotavirus/Adenovirus Combi test), Giardia duodenalis plus Cryptosporidium parvum (RIDA) Quick Cryptosporidium/Giardia Combi test), astrovirus (ELISA), and norovirus (reverse-transcriptase PCR). Stool specimens were provided by 306 patients (161 female) with acute diarrhea. The ages of the patients ranged from 1 to 89 years (mean 37, median 36). Pathogens were detected in 71 (23.2%) patients, with incidence peaks in February and June. Norovirus accounted for 36.0% of positive results, C. difficile for 18.7%, rotavirus for 17.3%, Campylobacter for 9.3%, Salmonella for 6.6%, adenovirus for 5.4%, G. duodenalis and C. parvum for 2.7% each, and Yersinia enterocolitica for 1.3%. No cases of shigellosis or infection with EHEC, EPEC, or astrovirus were diagnosed. Viruses accounted for 58.7% of the 75 positive results, bacteria for 36.0%, and parasites for 5.3%. Our study underlines a dominant role of norovirus and toxigenic C. difficile as etiologic agents of acute gastroenteritis among the patients of general practitioners.

Effect of sub-MIC concentrations of metronidazole, vancomycin, clindamycin and linezolid on toxin gene transcription and production in Clostridium difficile

Clostridium difficile is the major cause of hospital-acquired infectious diarrhoea. Several antimicrobials are known to induce and promote C. difficile-associated diarrhoea (CDAD). The impact of metronidazole (MTR), vancomycin (VAN), clindamycin (CLI) and linezolid (LZD) on growth, toxin gene transcription and toxin production in C. difficile was investigated. Four C. difficile strains were grown with and without sub-MIC concentrations of MTR, VAN, CLI and LZD (0.5x MIC) and growth was measured by colony counts. Toxin production was detected using ELISA (for toxin A) and a cytotoxicity assay (for toxin B) in culture supernatants and also in sonicated cells. Real-time PCR was used to measure transcription of the toxin A and B genes. The aim of this work was to combine analysis of toxin A and B production by ELISA or cell culture assay with transcriptomic analysis. The four strains showed similar growth and different levels of toxin production in the absence of antibiotics. An antibiotic-free control showed toxin production at a late stage when the plateau phase of bacterial growth was reached, whereas antibiotic-exposed strains showed earlier toxin production. All of the antibiotics used except CLI increased the transcription rate of toxin genes. The findings of this study show that sub-MIC concentrations of antibiotics can cause changes in gene transcription of the major virulence factors of C. difficile. This study describes a new method for transcriptomic analysis of toxin genes in C. difficile.

ISApl 1, a novel insertion element of Actinobacillus pleuropneumoniae, prevents ApxIV-based serological detection of serotype 7 strain AP76

Actinobacillus pleuropneumoniae, a gram-negative rod of the Pasteurellaceae family, causes pleuropneumonia in pigs. Establishing A. pleuropneumoniae free herds is difficult due to the occurrence of persistently infected animals. The ApxIV toxin is expressed by A. pleuropneumoniae in vivo and an ELISA based on the toxin is used to detect infection and to differentiate between infected and vaccinated animals. In this study, we have identified a 1070 bp insertion element of the IS 30 family, designated ISApl 1, in the A. pleuropneumoniae serotype 7 strain AP76. ISApl 1 contains a 924 bp ORF encoding a transposase, which is flanked by 27 bp inverted repeats showing six mismatches. We investigated the occurrence of ISApl 1 in other A. pleuropneumoniae strains, and its possible interference with virulence associated factors. Four insertion sites were identified in AP76: within the apxIVA toxin ORF, within a putative autotransporter adhesin ORF, upstream of a capsular polysaccharide biosynthesis gene cluster, and downstream of a beta-lactamase gene. ISApl 1 is also present in some serotype 7 field isolates, but not in reference or field strains of other serotypes. In A. pleuropneumoniae AP76, the transposase gene is transcribed in vitro. The insertion in the apxIVA toxin gene remains stable after animal passage. Since this insertion should disrupt toxin expression, we tested 7 pigs infected with AP76 at day 21 post-infection. All were negative in the ApxIV ELISA but four out of seven were positive in an ApxII toxin ELISA. These results show that insertion elements can affect the detection of A. pleuropneumoniae infected animals.

Comparison of Two Enzyme Immunoassays for Clostridium difficile Toxin A

Clostridium difficile is one of the most important pathogens responsible for nosocomial diarrhea. The disease is mediated by two toxins, designated as A and B; therefore, identification of the toxins is important for diagnosis. However, culture or cytotoxin assay are not easily done because of tedious procedures. Instead, toxin A immunoassay is widely used. We evaluated two different enzyme immunoassays (EIA) for C. difficile toxin A and compared them with culture and PCR results. A total of 65 stool specimens were examined for toxin A using enzyme linked fluorescent immunoassay (ELFA, VIDAS CD II, Bio-Merieux, France) and enzyme linked immunosolvent assay (ELISA, C.DIFFICILE TOX A II, TECHLAB, USA ) and were also cultured for C. difficile using cycloserine cefoxitine fructose agar. We amplified toxin A and B genes using primers NK9-NK 11 and NK104-NK105, respectively, in 23 C. difficile isolates. The concordance rate between ELFA and ELISA was 76.9%. The sensitivity and specificity of the ELFA and ELISA based on the culture and PCR results for toxin A gene were 84.6%/98.1% and 84.6%/67.3%. Positive and negative predictive values were 91%/96.2% in VIDAS and 78.0%/ 94.6% in TECHLAB. The positive rates of toxin B genes were 100%, 83.3% and 50% in toxin A positive, variant and negative strains, respectively. The sensitivities of the ELFA and ELISA for toxin A were the same, but specificity and positive predictive value of the ELFA were higher than those of the ELISA. PCR or EIA method detecting both toxin A and toxin B is strongly recommended, because the variant strains (toxin A negative and toxin B positive) of C. difficile may be more prevalent than were anticipated in Korea.

Effect of the interval between shearing and dipping on the spread of Corynebacterium pseudotuberculosis infection in sheep.

To determine whether the spread of Corynebacterium pseudotuberculosis infection to sheep in dips could be controlled by increasing the time between shearing and dipping. A controlled treatment trial where only the time between shearing and dipping was varied. ANIMALS AND PROCEDURE: One hundred and ninety-five sheep were found to be negative for C. pseudotuberculosis exposure by assay of CLA toxin antibody, were divided into four treatment groups. Each was shorn at either 0, 2, 4 or 8 weeks before dipping in a solution containing C. pseudotuberculosis. Blood samples were taken 6 weeks after dipping and sheep were slaughtered 12 weeks after dipping. A fifth smaller group of 14 sheep shorn 26 weeks before dipping, was also exposed to C. pseudotuberculosis and was slaughtered with the other sheep. The occurrence of caseous lymphadenitis abscesses did not differ between groups or with sheep shorn 26 weeks before dipping. The proportion of sheep that seroconverted to the C. pseudotuberculosis toxin and cell wall ELISA was larger in sheep dipped immediately after shearing than in sheep in the other groups. Delaying dipping until 8 weeks after shearing did not decrease the C. pseudotuberculosis infection rate due to dipping. Sheep dipped immediately after shearing developed higher concentrations of antibody to C. pseudotuberculosis than sheep when dipping occurred between 2 and 8 weeks and later after shearing.