Glutamate Dehydrogenase Assay Research Articles

A study to assess the prevalence and risk factors for Clostridioides difficile infection in patients with inflammatory bowel disease in a tertiary care hospital in Northern India

Objectives: The prevalence of Clostridioides difficile infection (CDI) is on rise among patients with inflammatory bowel disease (IBD). This study sought to describe the prevalence and risk factors of CDI in patients with IBD as compared to non-IBD controls. Materials and Methods: This was a prospective study conducted at a Department of Microbiology in collaboration with a Department of Gastroenterology. The patients with IBD and controls without IBD presenting with diarrhea were included in the study. The screening test for C. difficile infection was done by glutamate dehydrogenase (GDH) assay and toxin detection by enzyme-linked immunoassay (ELISA). Anaerobic culture for C. difficile was done on a selective cycloserine cefoxitin fructose agar and polymerase chain reaction (PCR) was done for Toxin A (TcdA) and Toxin B (TcdB) gene detection. C. difficile infection was confirmed if GDH and toxin ELISA or PCR were positive. Statistical Analysis: Data were analyzed with the Statistical Package for the Social Sciences version 20.0.The numerical variables were presented by means and standard deviations. Comparison of continuous variables was done using Student’s t-test. Categorical variables were analyzed by Chi square test. P<0.05 was considered to be statistically significant. Results: A total of 160 cases and 112 age- and gender-matched control were included in IBD group and nonIBD group, respectively. Only one culture was positive, 12 and six were positive for GDH ELISA and TcdA and TcdB ELISA, respectively, and 7 were positive by PCR for toxin genes. The factors found significantly associated with CDI were proton-pump inhibitors use (P = 0.001), levofloxacin (P =0.001), and azathioprine (P =0.042). Using PCR as a reference method for C. difficile toxin detection, the sensitivity, and specificity of GDH ELISA and ELISA for toxins were 100%, 96.8% and 85.7%, and 100%, respectively. Conclusions: The prevalence of CDI among patients with IBD has been found to be low, that is (only 4.4%) in this study population.

Diagnostic Guidance for C. difficile Infections.

Diagnosis of Clostridioides difficile infection (CDI) can be challenging. First of all, there has been debate on which of the two reference assays, cell cytotoxicity neutralization assay (CCNA) or toxigenic culture (TC), should be considered the gold standard for CDI detection. Although the CCNA suffers most from suboptimal storage conditions and subsequent toxin degradation, TC is reported to falsely increase CDI detection rates as it cannot differentiate CDI patients from patients asymptomatically colonised by toxigenic C. difficile. Several rapid assays are available for CDI detection and fall into three broad categories: (1) enzyme immunoassays for glutamate dehydrogenase, (2) enzyme immunoassays or single-molecule array assays for toxins A/B and (3) nucleic acid amplification tests detecting toxin genes. All three categories have their own limitations, being suboptimal specificity and/or sensitivity or the inability to discern colonised patients from CDI patients. In light of these limitations, multi-step algorithmic testing has been advocated by international guidelines (IDSA/SHEA and ESCMID) in order to optimize diagnostic accuracy. As a result, a survey performed in 2018-2019 in Europe revealed that most of all hospital sites reported using more than one test to diagnose CDI. CDI incidence rates are also influenced by sample selection criteria, as several studies have shown that if not all unformed stool samples are tested for CDI, many cases may be missed due to an absence of clinical suspicion. Since methods for diagnosing CDI remain imperfect, there has been a growing interest in alternative testing strategies like faecal microbiota biomarkers, immune modulating interleukins, cytokines and imaging methods. At the moment, these alternative methods might play an adjunctive role, but they are not suitable to replace conventional CDI testing strategies.

Rapid Quantification of C. difficile Glutamate Dehydrogenase and Toxin B (TcdB) with a NanoBiT Split-Luciferase Assay.

C. difficile infection (CDI) is a leading healthcare-associated infection with a high morbidity and mortality and is a financial burden. No current standalone point-of-care test (POCT) is sufficient for the identification of true CDI over a disease-free carriage of C. difficile, so one is urgently required to ensure timely, appropriate treatment. Here, two types of binding proteins, Affimers and nanobodies, targeting two C. difficile biomarkers, glutamate dehydrogenase (GDH) and toxin B (TcdB), are combined in NanoBiT (NanoLuc Binary Technology) split-luciferase assays. The assays were optimized and their performance controlling parameters were examined. The 44 fM limit of detection (LoD), 4–5 log range and 1300-fold signal gain of the TcdB assay in buffer is the best observed for a NanoBiT assay to date. In the stool sample matrix, the GDH and TcdB assay sensitivity (LoD = 4.5 and 2 pM, respectively) and time to result (32 min) are similar to a current, commercial lateral flow POCT, but the NanoBit assay has no wash steps, detects clinically relevant TcdB over TcdA, and is quantitative. Development of the assay into a POCT may drive sensitivity further and offer an urgently needed ultrasensitive TcdB test for the rapid diagnosis of true CDI. The NanoBiTBiP (NanoBiT with Binding Proteins) system offers advantages over NanoBiT assays with antibodies as binding elements in terms of ease of production and assay performance. We expect this methodology and approach to be generally applicable to other biomarkers.

Comparative Evaluation of Three Immunoassays for the Simultaneous Detection of Clostridioides difficile Glutamate Dehydrogenase and Toxin A/B.

Background: In the medical laboratory, a step-by-step workflow for Clostridioides difficile infection (CDI) detection using glutamate dehydrogenase (GDH) and toxin A/B assays for initial screening, along with a nucleic acid amplification test (NAAT), has been recommended recently. In this study, we evaluated these three immunoassays for the simultaneous detection of GDH and Clostridioides difficile (CD) toxin A/B. Methods: A total of 304 stool samples were tested for the presence of GDH antigen and CD toxin A/B using VIDAS C. difficile GDH and toxin A/B (CDAB), RIDASCREEN C. difficile GDH and toxin A/B (RIDA), and C. DIFF QUIK CHEK COMPLETE according to the manufacturers’ recommendations. As complementary reference methods for GDH and toxin A/B detection in the three immunoassays, CD cultures using ChromID C. difficile agar and the Xpert C. difficile assay, respectively, were tested. Results: All three GDH assays showed overall substantial agreement with the CD culture. All three toxin A/B assays showed overall moderate agreement with the Xpert C. difficile assay. In comparison with consensus results, VIDAS GDH and QCC GDH showed almost perfect agreement, whereas RIDA GDH showed inferior but substantial agreement. All three toxin A/B assays showed almost perfect agreement. Conclusions: Since the QCC GDH and toxin A/B assay is relatively more sensitive and specific than the other two immunoassays for discriminating toxigenic or non-toxigenic CDI, QCC is very helpful for the simultaneous identification of GDH and CD toxin A/B in the initial step of the two-round workflow for diagnosing CDI.

Nationwide Survey for Current Status of Laboratory Diagnosis of Clostridioides difficile Infection in Korea.

BackgroundThe interest in Clostridioides difficile infection (CDI) has increased, and the choice of assays became wider since the first national survey in Korea on CDI diagnosis in 2015. We conducted a survey of the domestic CDI assays with more varied questions to understand the current situation in Korea.MethodsIn April 2018, about 50 questions on the current status of CDI assays and details on implementation and perceptions were written, and a survey questionnaire was administered to laboratory medicine specialists in 200 institutions.ResultsOne-hundred and fifty institutions responded to the questionnaire, of which 90 (60.0%) including one commercial laboratory, performed CDI assays. The toxin AB enzyme immunoassay (toxin AB EIA), nucleic acid amplification test (NAAT), and C. difficile culture, glutamate dehydrogenase assay, alone or in combination with other assays, were used in 75 (84.3%), 52 (58.4%), 35 (36.0%), and 23 (25.8%), respectively, and 65 (73.0%) institutions performed a combination of two or more assays. The sensitivity of toxin AB EIA was more negatively perceived, and that on specificity was more positively perceived. The perception of sensitivity and specificity of NAAT was mostly positive. Perception on the algorithm test projected it as useful but in need of countermeasures. Sixty-three (73.3%) institutions responded that they performed surveillance on CDI.ConclusionThis study provides useful evidence on the current status of CDI laboratory diagnosis in Korea as well as on items that require improvement and is thought to aid in standardizing and improving the CDI laboratory diagnosis in Korea.

Optimization of the Design and Operating Conditions of an Amperometric Biosensor for Glutamate Concentration Measurements in the Blood Plasma

AbstractToday, the concentration of glutamate in the patient′s blood is an important indicator in medical diagnostics; therefore, it is necessary to have a simple, accurate, and fast tool to obtain the data. Here, a recently developed amperometric glutamate‐sensitive biosensor was optimized to improve its characteristics. The platinum disk electrode was used as a transducer. As a bioselective element we used the enzyme glutamate oxidase, covalently crosslinked with bovine serum albumin by glutaraldehyde. Circumstances of enzyme immobilization on the transducer‘s surface were optimized (enzyme and glutaraldehyde concentrations and immobilization duration). To test the ability of this biosensor to work in biological environments containing complex biological substances, the influence of the working solution was investigated (concentration of the working buffer, its temperature, presence of the protein in the analyzed sample). The linear range of biosensor was from 5 to 600 μM of glutamate and the sensitivity was 150–200 nA/mM. Measurements of glutamate concentrations in the blood plasma were performed by biosensor and glutamate dehydrogenase assay. The linear correlation between the methods was found in a range of 50.4–182.5 μM (R2=0.99). Thus, it has been shown that the developed biosensor makes it possible to measure the concentration of glutamate in blood plasma.

A 2-step algorithm combining glutamate dehydrogenase and nucleic acid amplification tests for the detection of Clostridioides difficile in stool specimens.

The optimized diagnosis algorithm of Clostridioides difficile infection (CDI) is worldwide concerns. The purpose of this study was to assess the toxigenic C. difficile test performance and propose an optimal laboratory workflow for the diagnosis of CDI in mild virulent epidemic areas. Diarrhea samples collected from patients were analyzed by glutamate dehydrogenase (GDH), toxin AB (CDAB), and nucleic acid amplification test (NAAT). We assessed the performance of GDH, the GDH-CDAB algorithm, and the GDH-NAAT algorithm using toxigenic culture (TC) as a reference method. In this study, 186 diarrhea samples were collected. The numbers of TC-positive and TC-negative samples were 39 and 147, respectively. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and kappa of the GDH assay were 100%, 80.3%, 57.4%, 100%, and 0.63; of the GDH-CDAB algorithm were 48.7%, 97.3%, 82.6%, 87.7%, and 0.54; and of the GDH-NAAT algorithm were 74.4%, 100%, 100%, 93.6%, and 0.82, respectively. The GDH-NAAT algorithm has great concordance with TC in detecting toxigenic C. difficile (kappa = 0.82), while the sensitivity of the GDH-CDAB algorithm was too low to meet the demand of CDI diagnosis clinically. GDH-NAAT algorithm is recommended for the detection of toxigenic C. difficile with high specificity, increased sensitivity, and cost-effective.

Severe Clostridium difficile infections in intensive care units: Diverse clinical presentations.

Clostridium difficile infections (CDIs) cause significant mortality and morbidity. Critically ill patients are susceptible to CDIs and tend to have severe CDIs, and their clinical presentations are not merely diarrhea. From September 2017 to March 2018, the adults with CDIs in the ICUs were included. Fecal specimens with positive results of glutamate dehydrogenase assay were cultured for C. difficile, and toxinotyping and ribotyping for available C. difficile isolates were done. The CDI cases were categorized into the diarrheal group and ileus group. Difficult-to-treat cases with the presentations of life-threatening complications (bowel perforation or bacteremia), toxic megacolon, and refractory diarrhea, were analyzed. Totally 23 cases, including 6 cases of ileus and 17 of diarrhea, were included. Overall, the incidence of CDI in the ICUs was 10.7 cases per 10,000 patient-days. The ileus group tended to have more severe presentation, shorter ICU stay, higher ICU mortality, and receive initial intravenous metronidazole therapy. Severe and fulminant CDIs accounted for 65.2% (15 cases). The ICU mortality rate was 39.1%, but only one death was directly related to CDI (4.3%). Of nine (39.1%) difficult-to-treat cases, there was only one isolate of RT611 with tcdC deletion and cdtA/cdtB from a case with toxic megacolon. No hypervirulent isolates of RT027 or 078 were detected. Severe CDIs in the ICU were not rare. Clinicians should be aware of abdominal symptoms and signs other than diarrhea, such as ileus, to make timely diagnosis and management of CDI.

Comparative Study of Clostridium difficile Clinical Detection Methods in Patients with Diarrhoea.

Objectives The aim of this study was to evaluate the clinical application of three methods for detecting Clostridium difficile in fecal samples. Methods One hundred and fifty fecal specimens were collected and tested for C. difficile using three methods: (1) the toxigenic culture (TC); (2) the VIDAS enzyme immunoassay (EIA): the VIDAS glutamate dehydrogenase (GDH) assay and toxin A/B assay were used to detect GDH antigen and A/B toxin; and (3) the GeneXpert PCR assay. The toxigenic culture was used as a reference to evaluate the performance of the VIDAS EIA and the GeneXpert PCR assay. Results Of 150 specimens, 26 carried both A and B toxin genes, and none of the samples were positive for the binary toxin gene. Toxin-producing C. difficile using three methods: (1) the toxigenic culture (TC); (2) the VIDAS enzyme immunoassay (EIA): the VIDAS glutamate dehydrogenase (GDH) assay and toxin A/B assay were used to detect GDH antigen and A/B toxin; and (3) the GeneXpert PCR assay. The toxigenic culture was used as a reference to evaluate the performance of the VIDAS EIA and the GeneXpert PCR assay. C. difficile using three methods: (1) the toxigenic culture (TC); (2) the VIDAS enzyme immunoassay (EIA): the VIDAS glutamate dehydrogenase (GDH) assay and toxin A/B assay were used to detect GDH antigen and A/B toxin; and (3) the GeneXpert PCR assay. The toxigenic culture was used as a reference to evaluate the performance of the VIDAS EIA and the GeneXpert PCR assay. Conclusion The VIDAS GDH assay is useful for initial screening of C. difficile using three methods: (1) the toxigenic culture (TC); (2) the VIDAS enzyme immunoassay (EIA): the VIDAS glutamate dehydrogenase (GDH) assay and toxin A/B assay were used to detect GDH antigen and A/B toxin; and (3) the GeneXpert PCR assay. The toxigenic culture was used as a reference to evaluate the performance of the VIDAS EIA and the GeneXpert PCR assay.

Using PCR in place of GDH as the first line assay in a two-step CDI testing algorithm – evidence of help not hindrance from samples processed at clinical microbiology laboratories in Wales

Background Guidance recommends the use of a two-stage algorithm for diagnosis of Clostridioides difficile infection (CDI) utilising either PCR or a glutamate dehydrogenase (GDH) assay as the first line screen. There is ongoing debate as to whether PCR is a hindrance due to fear of over-diagnosis. Methods Between December 2017 and March 2019 over 65,000 tests for CDI were undertaken in Wales. PCR (Serosep EntericBio C. difficile assay) was employed as the first line test for 36% of samples with the remainder tested using a GDH assay (Techlab C. diff ChekTM -60). Positive samples were tested using a toxin EIA (Techlab Tox A/B Quik Chekò). Culture (alcohol shock & CCEY; Oxoid) and capillary electrophoresis PCR ribotyping were undertaken at the UK Anaerobe Reference Unit (UKARU). Results The proportion of samples testing positive using PCR and GDH was 5.2% and 8.3% with a toxin EIA positive percentage of 30% and 25% respectively. Of these samples 73% (n=2543) of GDH and 80% (n=974) of PCR positive samples were referred for culture and ribotyping. No C. difficile was isolated from 15% of GDH and 10.5% of PCR positive specimens. Non-toxigenic strains were isolated from 6.6% of GDH and 0.2% of PCR positive samples and approximately equal proportions of both GDH and PCR positive samples were ribotyped (60% vs. 63% of 2125 samples). Conclusions There was no evidence of increased ascertainment of CDI using PCR. In fact, culture and ribotyping demonstrated an improved specificity for PCR that is helpful for accurate CDI diagnosis.

Laboratory Diagnosis of Clostridium difficile Infection in Korea: The First National Survey.

In May 2015, we conducted a voluntary online survey on laboratory diagnostic assays for Clostridium difficile infection (CDI) across clinical microbiology laboratories in Korea. Responses were obtained from 66 laboratories, including 61 hospitals and five commercial laboratories. Among them, nine laboratories reported having not conducted CDI assays. The toxin AB enzyme immunoassay (toxin AB EIA), nucleic acid amplification test (NAAT), and C. difficile culture, alone or in combination with other assays, were used in 51 (89.5%), 37 (64.9%), and 37 (64.9%) of the remaining 57 laboratories, respectively, and 23 (40.4%) of the laboratories performed all three assays. Only one laboratory used the glutamate dehydrogenase assay. Nine laboratories used the toxin AB EIA as a stand-alone assay. The median (range) of examined specimens in one month for the toxin AB EIA, NAAT, and C. difficile culture was 160 (50–2,060), 70 (7–720), and 130 (9–750), respectively. These findings serve as valuable basic data regarding the current status of laboratory diagnosis of CDI in Korea, offering guidance for improved implementation.

An amperometric glutamate biosensor for monitoring glutamate release from brain nerve terminals and in blood plasma

An excess of the excitatory neurotransmitter, glutamate, in the synaptic cleft during hypoxia/ischemia provokes development of neurotoxicity and originates from the reversal of Na+-dependent glutamate transporters located in the plasma membrane of presynaptic brain nerve terminals. Here, we have optimized an electrochemical glutamate biosensor using glutamate oxidase and developed a biosensor-based methodological approach for analysis of rates of tonic, exocytotic and transporter-mediated glutamate release from isolated rat brain nerve terminals (synaptosomes). Changes in the extracellular glutamate concentrations from 11.5 ± 0.9 to 11.7 ± 0.9 μΜ for 6 min reflected a low tonic release of endogenous glutamate from nerve terminals. Depolarization-induced exocytotic release of endogenous glutamate was equal to 7.5 ± 1.0 μΜ and transporter reversal was 8.0 ± 1.0 μΜ for 6 min. The biosensor data correlated well with the results obtained using radiolabelled L-[14C]glutamate, spectrofluorimetric glutamate dehydrogenase and amino acid analyzer assays. The blood plasma glutamate concentration was also tested, and reliability of the biosensor measurements was confirmed by glutamate dehydrogenase assay. Therefore, the biosensor-based approach for accurate monitoring rates of tonic, exocytotic and transporter-mediated release of glutamate in nerve terminals was developed and its adequacy was confirmed by independent analytical methods. The biosensor measurements provided precise data on changes in the concentrations of endogenous glutamate in nerve terminals in response to stimulation. We consider that the glutamate biosensor-based approach can be applied in clinics for neuromonitoring glutamate-related parameters in brain samples, liquids and blood plasma in stroke, brain trauma, therapeutic hypothermia treatment, etc., and also in laboratory work to record glutamate release and uptake kinetics in nerve terminals.

Deproteinization is Necessary for the Accurate Determination of Ammonia Levels by Glutamate Dehydrogenase Assay in Blood Plasma From Subjects With Liver Injury.

To determine the effect of presence of high concentrations of nicotinamide adenine dinucleotide (NADH)- and nicotinamide adenine dinucleotide phosphate (NADPH)-consuming enzymes on the accuracy of glutamate dehydrogenase (GLDH) assay for ammonia. We measured ammonia concentrations using GLDH and NADH or NADPH in blood-plasma specimens and specimens deproteinized by sulfosalicylic acid from CCl4-treated or control rats. The nonspecific oxidation of NADH and NADPH was measured in mixtures without GLDH. We observed a gradual decrease (~0.5%) in absorbance in the plasma of controls after the addition of NADH but not after adding NADPH. The decrease in absorbance in plasma of CCl4-treated animals was 13.2% and 5.2% after the addition of NADH and NADPH, respectively. The decrease in absorbance was not detected in deproteinized specimens. The values of ammonia concentration were higher in the plasma specimens compared with the deproteinized ones. Deproteinization is necessary for accurate measurement of ammonia using GLDH assay in the blood plasma of subjects with liver injury.

Trends in the use of laboratory tests for the diagnosis of Clostridium difficile infection and association with incidence rates in Quebec, Canada, 2010-2014

Several Clostridium difficile infection (CDI) surveillance programs do not specify laboratory strategies to use. We investigated the evolution in testing strategies used across Quebec, Canada, and its association with incidence rates. Cross-sectional study of 95 hospitals by surveys conducted in 2010 and in 2013-2014. The association between testing strategies and institutional CDI incidence rates was analyzed via multivariate Poisson regressions. The most common assays in 2014 were toxin A/B enzyme immunoassays (EIAs) (61 institutions, 64%), glutamate dehydrogenase (GDH) EIAs (51 institutions, 53.7%), and nucleic acid amplification tests (NAATs) (34 institutions, 35.8%). The most frequent algorithm was a single-step NAAT (20 institutions, 21%). Between 2010 and 2014, 35 institutions (37%) modified their algorithm. Institutions detecting toxigenic C difficile instead of C difficile toxin increased from 14 to 37 (P < .001). Institutions detecting toxigenic C difficile had higher CDI rates (7.9 vs 6.6 per 10,000 patient days; P = .01). Institutions using single-step NAATs, GDH plus toxigenic cultures, and GDH plus cytotoxicity assays had higher CDI rates than those using an EIA-based algorithm (P < .05). Laboratory detection of CDI has changed since 2010. There is an association between diagnostic algorithms and CDI incidence. Mitigation strategies are warranted.

Prevalence and Multilocus Genotyping Analysis of Cryptosporidium and Giardia Isolates from Dogs in Chiang Mai, Thailand.

The occurrence and zoonotic potential of Cryptosporidium spp. and Giardia duodenalis isolated from dogs in Chiang Mai, Thailand were determined. Fecal samples were collected from 109 dogs between July and August 2008. Cryptosporidium spp. infection was determined by immunofluorescent assay (IFA), PCR assays that amplify Cryptosporidium heat-shock protein 70 kDa (hsp70), and two PCR assays that amplify a small subunit-ribosomal RNA (SSU-rRNA). Giardia duodenalis infection was identified using zinc sulfate centrifugal flotation, IFA, and four PCR assays that amplify the Giardia glutamate dehydrogenase (gdh), beta-giardin (bg), and generic and dog-specific assays of triosephosphate isomerase (tpi) genes. Overall prevalence of Cryptosporidium spp. and G. duodenalis was 31.2% and 45.9%, respectively. Sequence analysis of 22 Cryptosporidium-positive samples and 21 Giardia-positive samples revealed the presence of C. canis in 15, and C. parvum in 7, G. duodenalis Assemblage C in 8, D in 11, and mixed of C and D in 2 dogs. Dogs in Chiang Mai were commonly exposed to Cryptosporidium spp. and G. duodenalis. Cryptosporidium parvum can be isolated from the feces of dogs, and all G. duodenalis assemblages were dog-specific. Dogs could be a reservoir for a zoonotic Cryptosporidium infection in humans, but further studies will be required to determine the clinical and zoonotic importance.

Evaluation of two novel chemiluminescence immunoassays for the detection of Clostridium difficile glutamate dehydrogenase and toxin A&B

A novel immunoassay for Clostridium difficile glutamate dehydrogenase (GDH) and toxin A&B (LIAISON, DiaSorin) was compared to another GDH assay (Alere), PCR and toxigenic culture. The GDH-DiaSorin is slightly more sensitive than the GDH-Alere. Sensitivity of the Toxin-Diasorin test is in accordance to the sensitivity of other immunoassays in literature.

Mono-sulfonated tetrazolium salt based NAD(P)H detection reagents suitable for dehydrogenase and real-time cell viability assays

Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and is important for several biological processes. For GDH inhibitor screening, we developed a novel mono-sulfonated tetrazolium salt (EZMTT), which can be synthesized using H2O2 oxidation and purified easily on silica gel in large quantities. The EZMTT detection method showed linear dose responses to NAD(P)H, dehydrogenase concentration and cell numbers. In E. coli GDH assay, the EZMTT method showed excellent assay reproducibility with a Z factor of 0.9 and caused no false positives in the presence of antioxidants (such as BME). Using the EZMTT-formazan-NAD(P)H system, we showed that EGCG is a potent E. coli GDH inhibitor (IC50 45 nM) and identified that Ebselen, a multifunctional thioredoxin reductase inhibitor, inactivated E. coli GDH (IC50 213 nM). In cell-based assays at 0.5 mM tetrazolium concentration, EZMTT showed essentially no toxicity after a 3-day incubation, whereas 40% of inhibition was observed for WST-8. In conclusion, EZMTT is a novel tetrazolium salt which provides improved features that are suitable for dehydrogenases and real-time cell-based high-throughput screening (HTS).

Evaluation of the VIDAS glutamate dehydrogenase assay for the detection of Clostridium difficile

We evaluated the performance of the VIDAS GDH assay for the detection of Clostridium difficile. In total, 350 fecal specimens collected from patients clinically suspected of having CDI were analyzed by C. difficile culture and enzyme-linked fluorescent immunoassay (VIDAS GDH); the results were compared with those of toxigenic C. difficile culture (TC), PCR (Xpert C. difficile assay), and toxin AB EIA (VIDAS CDAB). The numbers of culture-positive and culture-negative samples were 108 and 242, respectively. The concordance between the GDH assay and C. difficile culture was 90.3%. With PCR, 12 more samples were found to be positive in GDH-positive/C. difficile culture-negative specimens. Thus, the concordance between GDH assay and C. difficile culture/PCR was 93.7%. The sensitivity, specificity, positive predictive value, and negative predictive value of the VIDAS GDH assay were 97.2%, 87.2%, 77.2%, and 98.6%, respectively, based on the C. difficile culture, and 97.5%, 91.7%, 86.0%, and 98.6%, respectively, based on C. difficile culture/PCR. Positivity rates of the GDH assay were partially associated with those of semi-quantitative C. difficile cultures, which were maximized in grade 3 (>100 colony-forming unit [CFU]) compared with grade 1 (<10 CFU).We evaluated the two-step or three-step algorithm using GDH assay as a first step. No toxin EIA-positive case was found among GDH-negative samples, and 60.8% (48/79) were TC- and/or PCR-positive among the GDH-positive/toxin EIA-negative samples. Thus, approximately 25% of the 350 samples required a confirmatory test (TC or PCR) in the GDH-toxin EIA algorithm, whereas only 2.3% of the total samples in GDH-PCR algorithm was discrepant and required another confirmatory test like TC.

Excitotoxic potential of exogenous ferritin and apoferritin: Changes in ambient level of glutamate and synaptic vesicle acidification in brain nerve terminals

Ferritin, an iron storage protein, is present in the serum and cerebrospinal fluid, has receptors on the cell surface, able to penetrate the brain-blood barrier, can be secreted from the cells, and leaks from destroyed cell in insult and brain trauma. The effect of exogenous ferritin on the key characteristic of glutamatergic neurotransmission was assessed in rat brain nerve terminals (synaptosomes). Exogenous ferritin (80 μg/ml, iron content 0.7%) significantly increased the ambient level of L-[(14)C]glutamate (0.200±0.015 versus 0.368±0.016 nmol/mg of protein) and endogenous glutamate (fluorimetric glutamate dehydrogenase assay) in the nerve terminals. This increase was not a result of augmentation of tonic release because the velocity of tonic release of L-[(14)C]glutamate was not changed significantly in ferritin-treated synaptosomes as compared to the control. Ferritin caused a decrease in synaptic vesicle acidification that was shown using fluorescent dye acridine orange. Iron-dependence of the effects of ferritin was analyzed with apoferritin (0.0025% residual iron). Apoferritin weakly affected the proton electrochemical gradient of synaptic vesicles but increased the ambient level and decreased the initial velocity of uptake of L-[(14)C]glutamate by synaptosomes, nevertheless these effects were ~30% lesser than those caused by ferritin. Exogenous ferritin can provoke the development of excitotoxicity increasing the ambient level of glutamate and lowering synaptic vesicle acidification and glutamate uptake in the nerve terminals, however these effects are not completely iron-dependent. Thus, in the CNS exogenous ferritin can act as a modulator of glutamate homeostasis in iron-dependent and iron-independent manner.

Glutamate release from platelets: Exocytosis versus glutamate transporter reversal

Platelets express neuronal and glial glutamate transporters EAAT 1–3 in the plasma membrane and vesicular glutamate transporters VGLUT 1,2 in the membrane of secretory granules. This study is focused on the assessment of non-exocytotic glutamate release, that is, the unstimulated release, heteroexchange and glutamate transporter reversal in platelets. Using the glutamate dehydrogenase assay, the absence of unstimulated release of endogenous glutamate from platelets was demonstrated, even after inhibition of glutamate transporters and cytoplasmic enzyme glutamine synthetase by dl-threo-β-benzyloxyaspartate and methionine sulfoximine, respectively. Depolarization of the plasma membrane by exposure to elevated [K+] did not induce the release of glutamate from platelets that was shown using the glutamate dehydrogenase assay and radiolabeled l-[14C]glutamate. Glutamate efflux by means of heteroexchange with transportable inhibitor of glutamate transporters dl-threo-β-hydroxyaspartate (dl-THA) was not observed. Furthermore, the protonophore cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) and inhibitor of V-type H+-ATPase bafilomycin A1 also failed to stimulate the release of glutamate from platelets. However, exocytotic release of glutamate from secretory granules in response to thrombin stimulation was not prevented by elevated [K+], dl-THA, FCCP and bafilomycin A1.In contrast to nerve terminals, platelets cannot release glutamate in a non-exocytotic manner. Heteroexchange, transporter-mediated and unstimulated release of glutamate are not inherent to platelets. Therefore, platelets may be used as a peripheral marker/model for the analysis of glutamate uptake by brain nerve terminals only (direct function of transporters), whereas the mechanisms of glutamate release are different in platelets and nerve terminals. Glutamate is released by platelets exclusively by means of exocytosis. Also, reverse function of vesicular glutamate transporters of platelets is rather ambiguous.