Clinical outcomes and treatment necessity in patients with toxin-negative Clostridioides difficile stool samples.

PURPOSE: The clinical significance of negative toxin enzyme immunoassays (EIA) for Clostridioides difficile infections (CDIs) is unclear. Our study aimed to investigate the significance of toxin EIA-negative in the diagnosis and prognosis of CDI. METHODS: All stool specimens submitted for C. difficile toxin EIA testing were cultured to isolate C. difficile. In-house PCR for tcdA, tcdB, cdtA, and cdtB genes were performed using C. difficile isolates. Stool specimens were tested with C. difficile toxins A and B using EIA kit (RIDASCREEN Clostridium difficile toxin A/B, R-Biopharm AG, Darmstadt, Germany). Characteristics and subsequent CDI episodes of toxin EIA-negative and -positive patients were compared. RESULTS: Among 190 C. difficile PCR-positive patients, 83 (43.7%) were toxin EIA-negative. Multivariate analysis revealed independent associations toxin EIA-negative results and shorter hospital stays (OR = 0.98, 95% CI 0.96-0.99, p = 0.013) and less high-risk antibiotic exposure in the preceding month (OR = 0.38, 95% CI 0.16-0.94, p = 0.035). Toxin EIA-negative patients displayed a significantly lower white blood cell count rate (11.0 vs. 35.4%, p < 0.001). Among the 54 patients who were toxin EIA-negative and did not receive CDI treatment, three (5.6%) were diagnosed with CDI after 7-21 days without complication. CONCLUSION: Our study demonstrates that toxin EIA-negative patients had milder laboratory findings and no complications, despite not receiving treatment. Prolonged hospitalisation and exposure to high-risk antibiotics could potentially serve as markers for the development of toxin EIA-positive CDI.

Cyanobacterial Harmful Algal Mats (CyanoHAMs) in tropical rivers of central Mexico and their potential risks through toxin production.

Cyanobacteria inhabiting lotic environments have been poorly studied and characterized in Mexico, despite their potential risks from cyanotoxin production. This article aims to fill this knowledge gap by assessing the importance of benthic cyanobacteria as potential cyanotoxin producers in central Mexican rivers through: (i) the taxonomic identification of cyanobacteria found in these rivers, (ii) the environmental characterization of their habitats, and (iii) testing for the presence of toxin producing genes in the encountered taxa. Additionally, we introduce and discuss the use of the term "CyanoHAMs" for lotic water environments. Populations of cyanobacteria were collected from ten mountain rivers and identified using molecular techniques. Subsequently, these taxa were evaluated for genes producing anatoxins and microcystins via PCR. Through RDA analyses, the collected cyanobacteria were grouped into one of three categories based on their environmental preferences for the following: (1) waters with high ionic concentrations, (2) cold-temperate waters, or (3) waters with high nutrient enrichment. Populations from six locations were identified to genus level: Ancylothrix sp., Cyanoplacoma sp., and Oxynema sp. The latter was found to contain the gene that produces anatoxins and microcystins in siliceous rivers, while Oxynema tested positive for the gene that produces microcystins in calcareous rivers. Our results suggest that eutrophic environments are not necessarily required for toxin-producing cyanobacteria. Our records of Compactonostoc, Oxynema, and Ancylothrix represent the first for Mexico. Four taxa were identified to species level: Wilmottia aff. murrayi, Nostoc tlalocii, Nostoc montejanii, and Dichothrix aff. willei, with only the first testing positive using PCR for anatoxin and microcystin-producing genes in siliceous rivers. Due to the differences between benthic growths with respect to planktonic ones, we propose the adoption of the term Cyanobacterial Harmful Algal Mats (CyanoHAMs) as a more precise descriptor for future studies.

Prevalence of toxigenic Clostridium difficile in hospitalized patients in the southwestern province of Saudi Arabia: Confirmation using the GeneXpert analysis.

Clostridium difficile (Clostridioides difficile) is a leading cause of nosocomial infections in hospitalized patients worldwide. Stool samples were collected from 112 inpatients admitted to different hospitals and were screened for C. difficile GDH + toxin A + B by immunoassay, and all positive samples by immunoassay were processed for molecular detection of C. difficile using the GeneXpert assay. C. difficile strains were detected in 12 (10.71%) out of 112 stool samples using the GDH + toxin A + B immunoassay method and toxigenic C. difficile was confirmed in 5 stool samples using the GeneXpert molecular assay. C. difficile strains were also detected in 7 (8.97%) out of 78 stool samples from intensive care unit patients, 3 (25%) out of 12 stool samples from internal medicine ward patients, 1 (11.11%) out of 9 stool samples from surgery ward patients, and 1 (10%) out of 10 stool samples from isolation ward patients using the GDH + toxin A + B immunoassay method and the toxigenic C. difficile strain was confirmed in 1, 2, 1, and 1 stool samples, respectively, using the GeneXpert molecular assay. Toxigenic C. difficile was confirmed in patients at 4 (51.14%) out of 7 hospitals. In the present study, we also analyzed the clinical information of patients with C. difficile-positive stool samples who were receiving one or more antibiotics during hospitalization. The binary toxin gene (cdt), the tcdC gene, and the C. difficile strain polymerase chain reaction (PCR) ribotype 027 were not detected using the GeneXpert molecular assay among 12 C. difficile-positive samples by immunoassay. This study should aid in the prevention of unnecessary empiric therapy and increase the understanding of the toxigenic C. difficile burden on the healthcare system in the southwestern province of Saudi Arabia.

Clostridioides difficile PCR Tcdb Cycle Threshold predicts toxin EIA positivity but not severity of infection.

BACKGROUND: Diagnosis of Clostridioides difficile Infection (CDI) entails compatible clinical presentation and laboratory findings. We evaluated real-time polymerase chain reaction (qPCR) cycle threshold (CT) as a predictor for disease severity and TcdB enzyme immunoassay (EIA) results. METHODS: Inpatients or emergency department patients who tested positive for tcdB gene by PCR were evaluated. Patients' stools underwent testing for GDH and TcdA/B by EIA. Medical health records were reviewed for demographic, clinical presentation, laboratory, treatment and outcome data. Severity of CDI was calculated using various severity score indexes. RESULTS: The median CT of cases was 32.05 ± 5.45. The optimal cut-off for predicting toxin EIA positivity and severe CDI based on chart review was 32.6 and 29.8, respectively, with the area under the receiver operator characteristics curve (AUC) of 0.74 and 0.60 respectively. CONCLUSION: CT value was an acceptable predictor for EIA toxin but less so for clinical severity. Our study potentially supports a diagnostic algorithm including CT value to reduce the number of EIA toxin assays performed.

Impact of a multipronged approach to reduce the incidence of Clostridioides difficile infections in hospitalized patients.

BACKGROUND: Effective approaches to reduce Clostridioides difficile infections (CDI) in hospitalized patients are needed. We report data from 3 years preceding and 3 years following interventions that proved successful, with detailed analysis of all cases the first year after implementation. METHODS: Interventions included a nursing protocol to identify cases present on admission by asking if the patient had 1 or more liquid stools in the last 24 hours, and a 2-step testing algorithm with samples positive by polymerase chain reaction (PCR) for the C. difficile toxin gene reflexing to an enzyme immunoassay (EIA) for the toxin antigen. RESULTS: Healthcare-associated infections due to CDI fell from ∼160 in each of the preceding 3 years to <65 in each of the subsequent 3 years (P < .001), while the ratio of observed-to-expected hospital-onset cases diminished to ∼0.50 (P < .02). In the first year, 395 samples were PCR(+), but only 118 (29.9%) of these were EIA(+). 55 (46.6%) of the PCR(+)/EIA(+) samples were from hospital day 1 or 2 and classified as present on admission. The mean time from stool collection to report of PCR results was ∼7.5 hours, and the EIA took on average only 68 additional minutes to be reported. CONCLUSIONS: The number of incident CDI cases can be dramatically decreased by implementing an admission screening question and a 2-step testing algorithm.

Monitoring Cyanobacterial Blooms during the COVID-19 Pandemic in Campania, Italy: The Case of Lake Avernus.

Cyanobacteria are ubiquitous photosynthetic microorganisms considered as important contributors to the formation of Earth's atmosphere and to the process of nitrogen fixation. However, they are also frequently associated with toxic blooms, named cyanobacterial harmful algal blooms (cyanoHABs). This paper reports on an unusual out-of-season cyanoHAB and its dynamics during the COVID-19 pandemic, in Lake Avernus, South Italy. Fast detection strategy (FDS) was used to assess this phenomenon, through the integration of satellite imagery and biomolecular investigation of the environmental samples. Data obtained unveiled a widespread Microcystis sp. bloom in February 2020 (i.e., winter season in Italy), which completely disappeared at the end of the following COVID-19 lockdown, when almost all urban activities were suspended. Due to potential harmfulness of cyanoHABs, crude extracts from the "winter bloom" were evaluated for their cytotoxicity in two different human cell lines, namely normal dermal fibroblasts (NHDF) and breast adenocarcinoma cells (MCF-7). The chloroform extract was shown to exert the highest cytotoxic activity, which has been correlated to the presence of cyanotoxins, i.e., microcystins, micropeptins, anabaenopeptins, and aeruginopeptins, detected by molecular networking analysis of liquid chromatography tandem mass spectrometry (LC-MS/MS) data.

Cyanobacterial Toxins and Peptides in Lake Vegoritis, Greece.

Cyanotoxins (CTs) produced by cyanobacteria in surface freshwater are a major threat for public health and aquatic ecosystems. Cyanobacteria can also produce a wide variety of other understudied bioactive metabolites such as oligopeptides microginins (MGs), aeruginosins (AERs), aeruginosamides (AEGs) and anabaenopeptins (APs). This study reports on the co-occurrence of CTs and cyanopeptides (CPs) in Lake Vegoritis, Greece and presents their variant-specific profiles obtained during 3-years of monitoring (2018-2020). Fifteen CTs (cylindrospermopsin (CYN), anatoxin (ATX), nodularin (NOD), and 12 microcystins (MCs)) and ten CPs (3 APs, 4 MGs, 2 AERs and aeruginosamide (AEG A)) were targeted using an extended and validated LC-MS/MS protocol for the simultaneous determination of multi-class CTs and CPs. Results showed the presence of MCs (MC-LR, MC-RR, MC-YR, dmMC-LR, dmMC-RR, MC-HtyR, and MC-HilR) and CYN at concentrations of <1 µg/L, with MC-LR (79%) and CYN (71%) being the most frequently occurring. Anabaenopeptins B (AP B) and F (AP F) were detected in almost all samples and microginin T1 (MG T1) was the most abundant CP, reaching 47.0 µg/L. This is the first report of the co-occurrence of CTs and CPs in Lake Vegoritis, which is used for irrigation, fishing and recreational activities. The findings support the need for further investigations of the occurrence of CTs and the less studied cyanobacterial metabolites in lakes, to promote risk assessment with relevance to human exposure.

Trends in Clostridioides difficile diagnosis before and after a change in testing algorithm.

Clostridioides difficile (Clostridium difficile) (CD) infection remains a challenging diagnosis in hospitalized patients given the myriad of testing procedures and array of alternative causes for diarrhea. We identified 100 consecutive inpatients with positive CD testing in a single tertiary center before and after changing from nucleic acid amplification testing (NAAT) alone to a two-step algorithm involving Glutamate Dehydrogenase enzyme immunoassays (GDHEIA) followed by an enzyme immunoassay for CD toxins (EIA). Detailed clinical information was obtained retrospectively to assess for risk factors, clinical features, and treatment outcomes to correlate test results with clinical cases. We demonstrate that using a 2-step testing algorithm identifies patients with a consistent clinical illness for CD disease significantly more often than nucleic acid amplification testing alone without an increase in cases of severe CD disease. Our data suggest that NAAT alone results in an increase in unnecessary treatment of CD colonization.

¿La detección de toxinas de Clostridioides difficile es necesaria cuando se detecta la enzima glutamato deshidrogenasa? / Is Clostridioides difficile toxins detection necessary when the glutamate dehydrogenase enzyme is detected?

Resumen Introducción: Clostridioides difficile causa diarrea y colitis pseudomembranosa. Su diagnóstico se realiza con la detección de glutamato-deshidrogenasa (GDH) o las toxinas A y B y se confirma con pruebas de amplificación de ácidos nucleicos. Objetivo: Definir si la determinación de GDH es redundante a la de las toxinas. Métodos: Estudio observacional retrospectivo de muestras fecales de pacientes con sospecha de infección por Clostridioides difficile. Las toxinas y GDH se determinaron mediante inmunocromatografía. Se realizó una simulación bayesiana con los cocientes de probabilidad; se consideró significativo un valor de p < 0.05. Resultados: Se analizaron 329 resultados de GDH y toxinas A y B. Se encontró una prevalencia de infección de Clostridioides difficile de 18.2 %. La sensibilidad y especificidad de la prueba de GDH fue de 0.90 y 0.89, respectivamente. El cociente de probabilidad positivo fue de 8.9 y el negativo, de 0.11. Conclusiones: Un resultado negativo de GDH disminuye considerablemente la probabilidad de infección, pero no la descarta. La detección de toxinas de Clostridioides difficile puede ser necesaria en instituciones donde la amplificación de ácidos nucleicos no es económica o accesible.

Abstract Introduction: Clostridioides difficile causes diarrhea and pseudomembranous colitis. Its diagnosis is made with glutamate dehydrogenase (GDH) or toxins A and B detection and is confirmed with nucleic acid amplification tests. Objective: To define if GDH determination is redundant to that of toxins. Methods: Retrospective, observational study in diarrheal stools of patients with suspected Clostridioides difficile infection. Toxins and GDH were determined by immunochromatography. Bayesian simulation was performed with likelihood ratios; a p-value < 0.05 was regarded as significant. Results: 329 GDH and toxin A and B results were analyzed. Clostridioides difficile infection prevalence was 18.2 %. Sensitivity and specificity of the GDH test were 0.90 and 0.89, respectively. Positive likelihood ratio was 8.9, and negative was 0.11. Conclusions: A negative GDH result considerably reduces the probability of infection but does not rule it out. Clostridioides difficile toxins detection may be necessary in institutions where nucleic acid amplification is not affordable or accessible.

Clostridioides difficile laboratory diagnostic techniques: a comparative approach of rapid and molecular methods.

Clostridioides difficile infection is a public health problem because of it is easily spread; with harmful consequences, it is essential to reduce hospital costs and prevent its dissemination by having a precise diagnosis. The gold standard for its diagnosis is polymerase chain reaction (PCR); however, the technique is not available for all laboratories due to the high cost. New approaches using non-molecular tests to detect C. difficile and toxin A/B production has been proposed to improve cost benefits. The objective of this study is to compare molecular methods (PCR) and rapid methods (immunochromatographic test and enzymatic immunoassay). A series of tests comprising these diagnostic techniques was performed with 50 patients with a clinical diagnosis for Clostridioides difficile on GeneXpert® devices test; a calculation of the sensitivity was executed, followed by a comparison of the efficiency of all techniques. Greater sensitivity was observed in the PCR-based methods (BD MAX™ and BioFire FilmArray®) and the GDH-based assays (RIDASCREEN® and Alere Techlab®). The proposed algorithm represents minor monetary disadvantages but a significant temporal optimization of 10%. Future studies concerning both positive and negative results could be advantageous because of the possibility of calculating more method concordance indexes, such as the specificity and Kappa index, in addition to being able to indicate a monetary profit if the proposed algorithm was applied due to the nonproceeding PCR cases.

The reference method influence on the sensitivity of the Clostridium difficile enzyme immunoassays: A meta analysis.

The use of enzyme immunoassays to screen for toxins A and B produced by Clostridium difficile is a common procedure in algorithms designed for its detection. Moreover, the absence of a unique test capable of providing reliable results at low cost motivates a great discussion about which algorithm is the best. Thus, several studies have evaluated the performance of these enzyme immunoassays. However, all fail to provide sufficient explanations for the different behaviours observed in different studies that evaluate the same index test against a common reference method. Our main goal was to find out which factors affect the sensitivity of these assays, since the specificity is very close to 1. In this research, we verified that sensitivity increases with the prevalence rate and with the proportion of reported cases of onset diarrhea. Therefore, its use is advisable for high prevalence rates (e.g. in an epidemic setting). As far as reference methods are concerned, nucleic acid amplification tests can be used as a reference method, with a performance similar to the well-accepted toxigenic culture. The method chosen for toxigenicity screening in a toxigenic culture also seems to affect the evaluation performance of tests and should be better studied in the future.

Predictors of Clostridioides difficile Infection-Related Complications and Treatment Patterns among Nucleic Acid Amplification Test-Positive/Toxin Enzyme Immunoassay-Negative Patients.

The addition of toxin enzyme immunoassay (EIA) to nucleic acid amplification tests, including PCR, creates challenges in the diagnosis and management of Clostridioides difficile infection (CDI). There are limited data in large cohorts, with discordant results, that is, PCR-positive/EIA-negative (PCR+/EIA-) results. We conducted a retrospective cohort study on all PCR+/EIA- adult inpatients and assessed CDI-related complications and clinical failure. We identified 240 individuals. Twenty-three (9.6%) patients experienced a CDI-related complication, including 2 cases of megacolon, 1 colectomy, and 22 intensive care unit (ICU) admissions. In multivariable logistic regression analyses, baseline severe disease by Infectious Diseases Society of America (IDSA) criteria (odds ratio [OR], 5.84; 95% confidence interval [CI], 1.88 to 18.1; P = 0.002), baseline fulminant colitis (OR, 84.7; 95% CI, 14.3 to 500; P < 0.001), fever of >38.5°C (OR, 4.61; 95% CI, 1.42 to 15.0; P = 0.011), and proton pump inhibitor (PPI) use (OR, 3.50; 95% CI, 1.19 to 10.3; P = 0.023) were associated with increased odds of CDI-related complications. For 67 PCR+/EIA- patients who did not receive complete treatment, clinical failure was observed in 10 (15%) patients. A comparison of PCR+/EIA- patients who received complete treatment to all 112 PCR+/EIA+ patients showed no differences in CDI-related complications (11% and 13% for PCR+/EIA- and PCR+/EIA+ patients, respectively), 60-day all-cause mortality (17% and 18% for PCR+/EIA- and PCR+/EIA+ patients, respectively), or recurrent CDI (7% and 9% for PCR+/EIA- and PCR+/EIA+ patients, respectively). Predictors of CDI-attributable complications among PCR+/EIA- patients include baseline severe disease by IDSA criteria, baseline fulminant colitis, and fever of >38.5°C. Identifying the subgroup of PCR+/EIA- patients who could have true disease, and therefore allowing them to be targeted for treatment, is critical.

Toxins and mobile antimicrobial resistance genes in Bacillus probiotics constitute a potential risk for One Health.

Probiotic microbes conferring health benefits to the hosts have attracted great attention. However, the safety of probiotics is not guaranteed, although the increasing widespread use of probiotics with excellent overall safety records. Here, we performed a systematic evaluation of the safety of commercial Bacillus probiotics intended for usage in humans, animals, plants, aquaculture and environment in China. Nearly half of the 65 isolated Bacillus spp. strains from these commercial probiotic products were capable of producing hazardous toxins. Infections with the representative isolates could cause sepsis, intestinal inflammation and liver injury in different mouse models. Additionally, these isolates harbor multiple antimicrobial resistance genes coupled with mobile genetic elements. Collectively, the capability for producing various toxins and harboring mobile antimicrobial resistance genes in Bacillus probiotics indicates a potential risk for One Health.

Risk factors for Clostridioides difficile infection in hospitalized patients and associated mortality in Japan: a multi-centre prospective cohort study.

BACKGROUND: Although population characteristics and antimicrobial prescribing practices suggest that the hospitalized population in Japan is at high risk of Clostridioides difficile infection (CDI), the epidemiology of CDI in Japan is poorly understood. AIM: This prospective cohort study aimed to investigate the epidemiology of CDI at 12 hospitals in Japan. METHODS: Patients with clinically significant diarrhoea (CSD) were enrolled. Stool specimens were tested for C. difficile by toxin A and/or B enzyme immunoassay (EIA) in the hospital laboratories, and a toxigenic culture and nucleic acid amplification tests were performed at a central laboratory. The risk factors of CDI and the impact of CDI on mortality were investigated. FINDINGS: In total, 566 patients with CSD were included in the analyses. A total of 152 patients received the diagnosis of CDI by Toxin A/B EIA, toxigenic culture, or nucleic acid amplification test. Factors associated with CDI included low albumin (adjusted odds ratio (aOR): 1.56; 95% confidence interval (CI): 1.03-2.34) and length of hospital stay before stool collection >18 days (aOR: 1.73; 95% CI: 1.09-2.75). CDI was associated with an increased mortality on univariate analysis (OR: 1.6, 95% CI: 1.0-2.6) but was not associated with an increased risk of mortality on multivariable analysis. CONCLUSION: Risk factors for CDI in Japan were similar to those identified in the USA and Europe. However, CDI was not associated with an increased risk of mortality in this population of patients with CSD.

High Agreement Between an Ultrasensitive Clostridioides difficile Toxin Assay and a C. difficile Laboratory Algorithm Utilizing GDH-and-Toxin Enzyme Immunoassays and Cytotoxin Testing.

The Singulex Clarity C. diff toxins A/B (Clarity) assay is an automated, ultrasensitive immunoassay for the detection of Clostridioides difficile toxins in stool. In this study, the performance of the Clarity assay was compared to that of a multistep algorithm using an enzyme immunoassay (EIA) for detection of glutamate dehydrogenase (GDH) and toxins A and B arbitrated by a semiquantitative cell cytotoxicity neutralization assay (CCNA). The performance of the assay was evaluated using 211 residual deidentified stool samples tested with a GDH-and-toxin EIA (C. Diff Quik Chek Complete; Techlab), with GDH-and-toxin discordant samples tested with CCNA. The stool samples were stored at -80°C before being tested with the Clarity assay. For samples discordant between Clarity and the standard-of-care algorithm, the samples were tested with PCR (Xpert C. difficile; Cepheid), and chart review was performed. The testing algorithm resulted in 34 GDH+/toxin+, 53 GDH-/toxin-, and 124 GDH+/toxin- samples, of which 39 were CCNA+ and 85 were CCNA- Clarity had 96.2% negative agreement with GDH-/toxin- samples, 100% positive agreement with GDH+/toxin+ samples, and 95.3% agreement with GDH+/toxin-/CCNA- samples. The Clarity result was invalid for one sample. Clarity agreed with 61.5% of GDH+/toxin-/CCNA+ samples, 90.0% of GDH+/toxin-/CCNA+ (high-positive) samples, and 31.6% of GDH+/toxin-/CCNA+ (low-positive) samples. The Singulex Clarity C. diff toxins A/B assay demonstrated high agreement with a testing algorithm utilizing a GDH-and-toxin EIA and CCNA. This novel automated assay may offer an accurate, stand-alone solution for C. difficile infection (CDI) diagnostics, and further prospective clinical studies are merited.

Functional characterization of the type I toxin Lpt from Lactobacillus rhamnosus by fluorescence and atomic force microscopy.

Lpt is a 29 amino acid long type I toxin identified in the plasmid DNA of wild Lactobacillus rhamnosus strains isolated from food. We previously reported that transcription of the encoding gene was upregulated under nutritional starvation conditions mimicking cheese ripening environment. The heterologous expression of the Lpt peptide in E. coli resulted in cell growth inhibition, nucleoid condensation and compromised integrity of the cell membrane. Fusion of the Lpt peptide with the fluorescent protein mCherry allowed to visualize the accumulation of the peptide into the membrane, while mutagenesis experiments showed that either the insertion of a negatively charged amino acid into the hydrophobic α-helix or deletion of the hydrophilic C-terminal region, leads to a non-toxic peptide. AFM imaging of Lpt expressing E. coli cells has revealed the presence of surface defects that are compatible with the loss of portions of the outer membrane bilayer. This observation provides support for the so-called "carpet" model, by which the Lpt peptide is supposed to destabilize the phospholipid packing through a detergent-like mechanism leading to the removal of small patches of bilayer through micellization.

High co-expression of TNF-α and CARDS toxin is a good predictor for refractory Mycoplasma pneumoniae pneumonia.

BACKGROUND: Early distinction between refractory M. pneumoniae pneumonia (RMPP) and non-RMPP (NRMPP) is still difficult. The community-acquired respiratory distress syndrome (CARDS) toxin can induce inflammatory and histopathological phenotypes associated with M. pneumoniae infection. This study aimed to investigate the clinical significance of CARDS toxin and pro-inflammatory cytokines in children with RMPP and to explore whether CARDS toxin can induce TNF-α expression. METHODS: Levels of CARDS toxin and cytokines in BALF from control and children with MPP were determined by real-time PCR and ELISA, respectively. A receiver-operating characteristic (ROC) analysis was performed to assess the diagnostic values of CARDS toxin, TNF-α, and IL-6 in RMPP. The recombinant CARDS toxin was constructed and prepared at different concentrations for stimulation of RAW264.7 cells. After co-culture with CARDS toxin, cytokines were detected by ELISA and the mRNA levels were measured by real-time PCR. Effects of CARDS toxin and TNF-α on inflammatory cell infiltration and mucus secretion in mouse lungs were also evaluated. RESULTS: Levels of CARDS toxin, TNF-α and IL-6 in bronchoalveolar lavage fluid (BALF) were significantly higher in RMPP cases compared with NRMPP cases. Furthermore, TNF-α had better diagnostic ability for differentiation of RMPP with AUC of 0.824 and Youden index of 0.692 compared with CARDS toxin and IL-6. Moreover, CARDS toxin was positively correlated with TNF-α level in MPP cases. In vitro assay revealed that CARDS toxin induced RAW264.7 macrophages to secrete TNF-α. Further in vivo assay showed that TNF-α deletion partially abrogated the CARDS toxin-mediated induction of inflammatory cell infiltration and mucus secretion in mouse lungs. CONCLUSIONS: The high co-expression of TNF-α and CARDS toxin in BALF is a good diagnostic biomarker for differentiating children with RMPP and NRMPP.

Diversity, Cyanotoxin Production, and Bioactivities of Cyanobacteria Isolated from Freshwaters of Greece.

Cyanobacteria are a diverse group of photosynthetic Gram-negative bacteria that produce an array of secondary compounds with selective bioactivity against a broad spectrum of organisms and cell lines. In this study, 29 strains isolated from freshwaters in Greece were classified using a polyphasic approach and assigned to Chroococcales, Synechococcales, and Nostocales, representing 11 genera and 17 taxa. There were good agreements between 16S ribosomal RNA (rRNA)-cpcBA-internal genetic spacer (IGS) characterization and morphological features, except for the Jaaginema-Limnothrix group which appears intermixed and needs further elucidation. Methanol extracts of the strains were analyzed for cyanotoxin production and tested against pathogenic bacteria species and several cancer cell lines. We report for the first time a Nostoc oryzae strain isolated from rice fields capable of producing microcystins (MCs) and a Chlorogloeopsis fritschii strain isolated from the plankton of a lake, suggesting that this species may also occur in freshwater temperate habitats. Strains with very high or identical 16S rRNA gene sequences displayed different antibacterial and cytotoxic activities. Extracts from Synechococcus cf. nidulans showed the most potent antibacterial activity against Staphylococcus aureus, whereas Jaaginema sp. strains exhibited potent cytotoxic activities against human colorectal adenocarcinoma and hepatocellular carcinoma cells. Jaaginema Thessaloniki Aristotle University Microalgae and Cyanobacteria (TAU-MAC) 0110 and 0210 strains caused pronounced changes in the actin network and triggered the formation of numerous lipid droplets in hepatocellular carcinoma and green monkey kidney cells, suggesting oxidative stress and/or mitochondrial damage leading to apoptosis.