Real-Time Cytotoxicity Assay for Rapid and Sensitive Detection of Ricin from Complex Matrices

BACKGROUND: In the context of a potential bioterrorist attack sensitive and fast detection of functionally active toxins such as ricin from complex matrices is necessary to be able to start timely countermeasures. One of the functional detection methods currently available for ricin is the endpoint cytotoxicity assay, which suffers from a number of technical deficits. METHODOLOGY/FINDINGS: This work describes a novel online cytotoxicity assay for the detection of active ricin and Ricinus communis agglutinin, that is based on a real-time cell electronic sensing system and impedance measurement. Characteristic growth parameters of Vero cells were monitored online and used as standardized viability control. Upon incubation with toxin the cell status and the cytotoxic effect were visualized using a characteristic cell index-time profile. For ricin, tested in concentrations of 0.06 ng/mL or above, a concentration-dependent decrease of cell index correlating with cytotoxicity was recorded between 3.5 h and 60 h. For ricin, sensitive detection was determined after 24 h, with an IC50 of 0.4 ng/mL (for agglutinin, an IC50 of 30 ng/mL was observed). Using functionally blocking antibodies, the specificity for ricin and agglutinin was shown. For detection from complex matrices, ricin was spiked into several food matrices, and an IC50 ranging from 5.6 to 200 ng/mL was observed. Additionally, the assay proved to be useful in detecting active ricin in environmental sample materials, as shown for organic fertilizer containing R. communis material. CONCLUSIONS/SIGNIFICANCE: The cell-electrode impedance measurement provides a sensitive online detection method for biologically active cytotoxins such as ricin. As the cell status is monitored online, the assay can be standardized more efficiently than previous approaches based on endpoint measurement. More importantly, the real-time cytotoxicity assay provides a fast and easy tool to detect active ricin in complex sample matrices

Ten Thousand-Fold Higher than Acceptable Bacterial Loads Detected in Kenyan Hospital Environments: Targeted Approaches to Reduce Contamination Levels

Microbial monitoring of hospital surfaces can help identify target areas for improved infection prevention and control (IPCs). This study aimed to determine the levels and variations in the bacterial contamination of high-touch surfaces in five Kenyan hospitals and identify the contributing modifiable risk factors. A total of 559 high-touch surfaces in four departments identified as high risk of hospital-acquired infections were sampled and examined for bacterial levels of contamination using standard bacteriological culture methods. Bacteria were detected in 536/559 (95.9%) surfaces. The median bacterial load on all sampled surfaces was 6.0 × 104 CFU/cm2 (interquartile range (IQR); 8.0 × 103–1.0 × 106). Only 55/559 (9.8%) of the sampled surfaces had acceptable bacterial loads, <5 CFU/cm². Cleaning practices, such as daily washing of patient sheets, incident rate ratio (IRR) = 0.10 [95% CI: 0.04–0.24], providing hand wash stations, IRR = 0.25 [95% CI: 0.02–0.30], having running water, IRR = 0.19 [95% CI: 0.08–0.47] and soap for handwashing IRR = 0.21 [95% CI: 0.12–0.39] each significantly lowered bacterial loads. Transporting dirty linen in a designated container, IRR = 72.11 [95% CI: 20.22–257.14], increased bacterial loads. The study hospitals can best reduce the bacterial loads by improving waste-handling protocols, cleaning high-touch surfaces five times a day and providing soap at the handwash stations

Ten Thousand-Fold Higher than Acceptable Bacterial Loads Detected in Kenyan Hospital Environments: Targeted Approaches to Reduce Contamination Levels

Microbial monitoring of hospital surfaces can help identify target areas for improved infection prevention and control (IPCs). This study aimed to determine the levels and variations in the bacterial contamination of high-touch surfaces in five Kenyan hospitals and identify the contributing modifiable risk factors. A total of 559 high-touch surfaces in four departments identified as high risk of hospital-acquired infections were sampled and examined for bacterial levels of contamination using standard bacteriological culture methods. Bacteria were detected in 536/559 (95.9%) surfaces. The median bacterial load on all sampled surfaces was 6.0 × 104 CFU/cm2 (interquartile range (IQR); 8.0 × 103–1.0 × 106). Only 55/559 (9.8%) of the sampled surfaces had acceptable bacterial loads, &lt;5 CFU/cm². Cleaning practices, such as daily washing of patient sheets, incident rate ratio (IRR) = 0.10 [95% CI: 0.04–0.24], providing hand wash stations, IRR = 0.25 [95% CI: 0.02–0.30], having running water, IRR = 0.19 [95% CI: 0.08–0.47] and soap for handwashing IRR = 0.21 [95% CI: 0.12–0.39] each significantly lowered bacterial loads. Transporting dirty linen in a designated container, IRR = 72.11 [95% CI: 20.22–257.14], increased bacterial loads. The study hospitals can best reduce the bacterial loads by improving waste-handling protocols, cleaning high-touch surfaces five times a day and providing soap at the handwash stations.</jats:p

Environmental contamination across multiple hospital departments with multidrug-resistant bacteria pose an elevated risk of healthcare-associated infections in Kenyan hospitals

Abstract Background Healthcare-associated infections (HAIs) are often caused by multidrug-resistant (MDR) bacteria contaminating hospital environments which can cause outbreaks as well as sporadic transmission. Methods This study systematically sampled and utilized standard bacteriological culture methods to determine the numbers and types of MDR Enterococcus faecalis/faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species, and Escherichia coli (ESKAPEE) from high-touch environments of five Kenyan hospitals; level 6 and 5 hospitals (A, B, and C), and level 4 hospitals (D and E), in 2018. Six hundred and seventeen high-touch surfaces across six hospital departments; surgical, general, maternity, newborn, outpatient and pediatric were sampled. Results 78/617 (12.6%) of the sampled high-touch surfaces were contaminated with MDR ESKAPEE; A. baumannii, 23/617 (3.7%), K. pneumoniae, 22/617 (3.6%), Enterobacter species, 19/617 (3.1%), methicillin resistant S. aureus (MRSA), 5/617 (0.8%), E. coli, 5/617 (0.8%), P. aeruginosa, 2/617 (0.3%), and E. faecalis and faecium, 2/617 (0.3%). Items found in patient areas, such as beddings, newborn incubators, baby cots, and sinks were the most frequently contaminated. Level 6 and 5 hospitals, B, 21/122 (17.2%), A, 21/122 (17.2%), and C, 18/136 (13.2%), were more frequently contaminated with MDR ESKAPEE than level 4 hospitals; D, 6/101 (5.9%), and E, 8/131 (6.1%). All the sampled hospital departments were contaminated with MDR ESKAPEE, with high levels observed in newborn, surgical and maternity. All the A. baumannii, Enterobacter species, and K. pneumoniae isolates were non-susceptible to piperacillin, ceftriaxone and cefepime. 22/23 (95.6%) of the A. baumannii isolates were non-susceptible to meropenem. In addition, 5 K. pneumoniae isolates were resistant to all the antibiotics tested except for colistin. Conclusion The presence of MDR ESKAPEE across all the hospitals demonstrated gaps in infection prevention practices (IPCs) that should be addressed. Non-susceptibility to last-line antibiotics such as meropenem threatens the ability to treat infections. </jats:sec

Comparative evaluation of fluorescent in situ hybridization and Giemsa microscopy with quantitative real-time PCR technique in detecting malaria parasites in a holoendemic region of Kenya

Abstract Background Early and accurate diagnosis of malaria is important in treatment as well as in the clinical evaluation of drugs and vaccines. Evaluation of Giemsa-stained smears remains the gold standard for malaria diagnosis, although diagnostic errors and potential bias estimates of protective efficacy have been reported in practice. Plasmodium genus fluorescent in situ hybridization (P-Genus FISH) is a microscopy-based method that uses fluorescent labelled oligonucleotide probes targeted to pathogen specific ribosomal RNA fragments to detect malaria parasites in whole blood. This study sought to evaluate the diagnostic performance of P-Genus FISH alongside Giemsa microscopy compared to quantitative reverse transcription polymerase chain reaction (qRT-PCR) in a clinical setting. Method Five hundred study participants were recruited prospectively and screened for Plasmodium parasites by P-Genus FISH assay, and Giemsa microscopy. The microscopic methods were performed by two trained personnel and were blinded, and if the results were discordant a third reading was performed as a tie breaker. The diagnostic performance of both methods was evaluated against qRT-PCR as a more sensitive method. Results The number of Plasmodium positive cases was 26.8% by P-Genus FISH, 33.2% by Giemsa microscopy, and 51.2% by qRT-PCR. The three methods had 46.8% concordant results with 61 positive cases and 173 negative cases. Compared to qRT-PCR the sensitivity and specificity of P-Genus FISH assay was 29.3 and 75.8%, respectively, while microscopy had 58.2 and 93.0% respectively. Microscopy had a higher positive and negative predictive values (89.8 and 68.0% respectively) compared to P-Genus FISH (56.0 and 50.5%). In overall, microscopy had a good measure of agreement (76%, k = 0.51) compared to P-Genus FISH (52%, k = 0.05). Conclusion The diagnostic performance of P-Genus FISH was shown to be inferior to Giemsa microscopy in the clinical samples. This hinders the possible application of the method in the field despite the many advantages of the method especially diagnosis of low parasite density infections. The P-Genus assay has great potential but application of the method in clinical setting would rely on extensive training of microscopist and continuous proficiency testing

Shiga toxin pathogenesis : kidney complications and renal failure

The kidneys are the major organs affected in diarrhea-associated hemolytic uremic syndrome (D(+)HUS). The pathophysiology of renal disease in D(+)HUS is largely the result of the interaction between bacterial virulence factors such as Shiga toxin and lipopolysaccharide and host cells in the kidney and in the blood circulation. This chapter describes in detail the current knowledge of how these bacterial toxins may lead to kidney disease and renal failure. The toxin receptors expressed by specific blood and resident renal cell types are also discussed as are the actions of the toxins on these cells