Evaluation of limulus amebocyte lysate and recombinant endotoxin alternative assays for an assessment of endotoxin detection specificity.

The United States Pharmacopeia (USP), European Pharmacopeia (EP), and Parenteral Drug Association (PDA) provide guidance on the validation of alternative microbiological methods (U.S. Pharmacopeia National Formulary 2019, Parenteral Drug Association Technical Report No. 33 2013, European Pharmacopoeia (Ph. Eur.) 2017). They define "specificity" as the ability to detect a range of microorganisms. In the context of alternative methods to the compendial Bacterial Endotoxin Test (BET) a range of endotoxins must be considered. This range should represent environmental endotoxins that present risks to pharmaceutical manufacturing processes, final products, and to the most important stakeholder: the patient. This study examines several alternative methods for the bacterial endotoxin detection test. It compares the official and harmonized BET test from two Limulus Amebocyte Lysate (LAL) suppliers to three commercially available recombinant Factor C (rFC) reagents that contain only one of the three enzymes in the horseshoe crab clotting cascade. The study also includes a recombinant reagent that has been developed to include all three of the enzymes involved in the LAL coagulation cascade, occurring in the presence of endotoxins. Pharmaceutically relevant water samples from various points in pharmaceutical water purification processes were used as a source of natural environmental endotoxins. While these water samples are not routinely tested for bacterial endotoxins, they do exist within manufacturing facilities and thus present risks to manufacturing operations (Sandle, 2019). A statistical analysis of 128 samples containing environmental endotoxin has shown that at the 5% level of significance, non-inferiority between the two compendial LAL methods was achieved. However, the non-inferiority claim could not be made with any of the recombinant reagents. The link between the BET and recombinant alternatives remains unresolved and, therefore, requires caution, continued development, and testing.

Sequential changes in hepatic mRNA abundance and serum concentration of serum amyloid A in cattle with acute inflammation caused by endotoxin.

The objective of the present study was to elucidate sequential changes in mRNA abundance of serum amyloid A (SAA) isotypes in endotoxin (ETX) challenge model cattle. Ten healthy cattle were separated to 2 groups: control and ETX groups. Cattle in the ETX group were challenged by 2.5 µg/kg of O111:B4 lipopolysaccharide in 4 ml of autologous serum. Blood samples were withdrawn at pre, 0.5, 1, 2, 4, 8, 12, 24, 48, 72 and 96 hr after ETX challenge. Plasma ETX activity, serum SAA concentrations, mRNA abundance of interleukin (IL)-6, SAA2 and SAA4 in the liver and polymorphonuclear leukocytes were measured. The plasma ETX activity in the ETX group increased at 0.5 hr after the ETX challenge. The serum SAA value remained higher between 12 and 72 hr after the ETX challenge than that of the control group. Hepatic IL-6 mRNA abundance in the ETX group increased at 2 hr after the ETX challenge. Hepatic SAA2 and SAA4 mRNA abundance significantly increased from 4 hr after administration, and remained significantly higher than those pre-values up to 12 and 24 hr, respectively. The abundance ratio of hepatic SAA2 was much higher than that of SAA4. The major isotype was SAA2 in liver tissue, and it is indicating systemic inflammation in cattle.

Sample Treatments That Solve Low Endotoxin Recovery Issues.

Dilution of samples with water (water dilution method) was not appropriate for measuring the endotoxin activity in solutions containing chelating agents and detergents-typical formulations showing low endotoxin recovery (LER). Dilution of the samples with 2 mM magnesium solution (magnesium dilution method) or addition of the samples directly to the Limulus amebocyte lysate (direct method) provided accurate endotoxin activity values in the samples. The difference between the water dilution method and the magnesium dilution method/direct method seemed to be caused by an endotoxin activity decrease during the water dilution method. Endotoxin activity was maintained for more than a month in LER solutions kept at 4°C when the activity was measured by the magnesium dilution method or the direct method. The magnesium concentration in the diluent for the magnesium dilution method should be greater than the concentration of the chelating agent in the sample. Magnesium dilution is the most appropriate dilution method for endotoxin measurement in LER solutions. LER effects were stronger in solutions with sodium citrate and polysorbate 20 than in solutions with phosphate buffer and polysorbate 80, respectively. Human serum albumin was also found to be an LER mitigating factor, and this suggests that protein might reduce LER effects.LAY ABSTRACT: Low endotoxin recovery (LER) is a phenomenon in which detectable endotoxin activity is reduced by biopharmaceutical product formulations containing a chelating agent and a detergent. The bacterial endotoxins test (BET) is a safety test used to detect endotoxin contamination of parenteral drugs and medical devices. Dilution of the samples with water is the most common method used to overcome interference by the samples with the BET. This study demonstrates that the water dilution method was not appropriate for measuring endotoxin activity in samples showing LER, and that the magnesium dilution method was the most appropriate method for this purpose. This study shows the conditions for the magnesium dilution method and some LER mitigating factors.

Relationship between bronchoalveolar lavage fluid and plasma endotoxin activity in calves with bronchopneumonia.

The aim of this study was to investigate the relationship between the endotoxin activity in plasma and that in bronchoalveolar lavage fluid (BALF) in bronchopneumonia. Thirty-three calves were included in this study (17 healthy calves and 16 calves with respiratory disease). In the calves with bronchopneumonia, the median endotoxin activity in plasma (0.437 EU/ml, P<0.001) and BALF (29.45 EU/ml, P<0.001) was significantly higher than in the control calves. Plasma endotoxin activity was significantly and positively correlated with that in BALF (r2=0.900, P<0.001). Based on the receiver operating characteristics curves, we propose a diagnostic cutoff point for plasma endotoxin activity (0.104 EU/ml, AUC=0.914, P<0.001, Se 81.3% and Sp 82.4%) for identification of bronchopneumonia in calves which could die within a week.

Factors Affecting Reduction of Reference Endotoxin Standard Activity Caused by Chelating Agent/Detergent Matrices: Kinetic Analysis of Low Endotoxin Recovery.

Reduction of reference standard endotoxin activity was kinetically analyzed under low endotoxin recovery conditions and was considered as an apparent first-order reaction. Temperature, pH, and salt concentrations affected the rates of reduction of reference standard endotoxin activity. Temperature appeared to be the most important factor affecting low endotoxin recovery. Components of low endotoxin recovery matrices, such as citrate and polysorbate 20, showed similar low endotoxin recovery effect at concentrations commonly used. Phosphate concentrations showed negative correlation against the half-life of reference standard endotoxin activity in solutions containing phosphate buffer and polysorbate 20. Activation energy for low endotoxin recovery with naturally occurring endotoxin was higher than that with reference standard endotoxin, and this explained one of the reasons for naturally occurring endotoxin resistance to low endotoxin recovery. Lower temperature, lower pH, and a higher salt concentration are preferable to avoid low endotoxin recovery in a hold-time study. This study provides useful data for anticipation of the severity of the low endotoxin recovery effect and future hold-time studies in the biopharmaceutical field.LAY ABSTRACT: Endotoxin derived from Gram-negative bacteria is potentially harmful when it is parenterally administrated. Therefore, injectables and medical devices are tested by the bacterial endotoxins test to detect contamination by endotoxin of those products. Low endotoxin recovery is a phenomenon of reduction of detectable standard endotoxin activity by certain matrices of biopharmaceutical products containing a chelating agent and a detergent, and it is a controversial topic because its mechanism and clinical risks are unknown. The author analyzed the kinetics of low endotoxin recovery to elucidate the mechanism of low endotoxin recovery and to propose conditions to avoid low endotoxin recovery.

Plasma endotoxin activity in Eastern grey kangaroos (Macropus giganteus) with lumpy jaw disease.

Progressive pyogranulomatous osteomyelitis involving the mandible or maxilla of captive macropods, referred to as "Lumpy jaw disease (LJD)", is one of the most significant causes of illness and death in captive macropods. The aim of the present study was to evaluate the relationship between the severity of LJD and plasma endotoxin activity in kangaroos. Plasma samples obtained from moderate (n=24) and severe LJD (n=12), and healthy kangaroos (n=46), were diluted 1:20 in endotoxin-free water and heated to 80°C for 10 min. Plasma endotoxin activity was measured using the Limulus amebocyte lysate (LAL)-kinetic turbidimetric (KT) assay. Plasma endotoxin activity was higher in kangaroos with severe LJD (0.199 ± 0.157 EU/ml) than in those with moderate LJD (0.051 ± 0.012 EU/ml, P<0.001) and healthy controls (0.057 ± 0.028 EU/ml, P<0.001). Our results suggest that the severity of LJD in captive macropods may be related to the plasma endotoxin activity.

Evaluation of a portable test system for assessing endotoxin activity in raw milk.

The aim of the present study was to compare endotoxin activities detected in raw milk samples obtained from cattle by a commercially available portable test system (PTS) and traditional microplate limulus amebocyte lysate (LAL)-based assay, which determined activities using a kinetic turbidimetric (KT) assay. Raw milk samples were obtained from 53 and 12 dairy cattle without and with clinical mastitis, respectively. Comparison between the KT and PTS was performed by the Friedman test. The Pearson product moment correlation coefficients were calculated to evaluate associations between any two continuous variables. Linear regression model analysis was also performed to obtain the equation describing the relationship between PTS and KT assay. The endotoxin activities detected in 200- or 400-fold diluted milk samples were similar between PTS and KT assay, whereas a significant difference was observed in 100-fold diluted milk (P<0.001). The results obtained from 200- (r(2)=0.778, P<0.001) and 400-fold diluted milk samples (r(2)=0.945, P<0.001) using PTS correlated with those using KT assay. The median milk endotoxin activities in Gram-positive and Gram-negative clinical mastitis cows were 0.655 and 11,523.5 EU/ml, respectively. The results of the present study suggest that PTS as a simple and easy test to assess endotoxin activity in raw milk is efficient, simple and reproducible.

Plasma endotoxin activity in kangaroos with oral necrobacillosis (lumpy jaw disease) using an automated handheld testing system.

The aim of the present study was to evaluate the reliability and effectiveness of directly determining endotoxin activity in plasma samples from kangaroos with lumpy jaw disease (LJD, n=15) and healthy controls (n=12). Prior to the present study, the ability of the commercially available automated handheld portable test system (PTS(TM)) to detect endotoxin activity in kangaroo plasma was compared with that of the traditional LAL-kinetic turbidimetric (KT) assay. Plasma samples, which were obtained from endotoxin-challenged cattle, were diluted 1:20 in endotoxin-free water and heated to 80°C for 10 min. The performance of the PTS(TM) was not significantly different from that of the traditional LAL-based assay. The data obtained using PTS(TM) correlated with those using KT (r(2)=0.963, P<0.001). These findings indicated that the PTS(TM) is applicable as a simplified system to assess endotoxin activity in macropods. In the present study, we demonstrated the diagnostic value of plasma endotoxin activity in kangaroos with systemic inflammation caused by oral necrobacillosis and identified plasma endotoxin activity as a sensitive marker of systemic inflammation in kangaroos with LJD. Based on ROC curves, we proposed a diagnostic cut-off point for endotoxin activity of >0.22 EU/ml for the identification of LJD. Our results indicate that the assessment of plasma endotoxin activity is a promising diagnostic tool for determining the outcome of LJD in captive macropods.

Detecting endotoxin activity in bovine serum using an automated testing system.

The aim of the present study was to compare the ability of the commercially available portable test system (PTS(TM)) to detect endotoxin activity in bovine serum, with that of the traditional LAL-kinetic turbidimetric (KT) and chromogenic (KC) assays. Prior to testing, serum samples, which were obtained from endotoxin-challenged cattle, were diluted 1:20 in endotoxin-free water and heated to 80°C for 10 min. The performance of the PTS(TM) was not significantly different from that of the traditional LAL-based assays. The results using PTS(TM) correlated with those using KT (r(2)=0.963, P<0.001) or KC assays (r(2)=0.982, P<0.001). Based on these findings, the PTS(TM) could be applied as a simplified system to assess endotoxin activity in bovine serum.

Hyperoxic condition prevents bacterial translocation and elevation of plasma microorganism components during hemorrhagic shock.

To evaluate the influence of hyperoxic conditions on bacterial translocation (BT) and microorganism components during hemorrhagic shock, rats were divided into a group breathing 100% oxygen and a group breathing room air. The groups were then subjected to hemorrhagic shock. Systemic blood and mesenteric lymph nodes were cultured for BT, and systemic plasma concentrations of microorganism components were measured by the silkworm larvae plasma (SLP) test and the endotoxin test. Hyperoxic conditions prevented both BT and plasma SLP-reactive substance (peptidoglycan and beta-glucan) elevation during hemorrhagic shock. Our findings suggest that hyperoxic treatment might improve host conditions during hemorrhagic shock.

Diagnostic and predictive value of the silkworm larvae plasma test for postoperative infection following gastrointestinal surgery.

OBJECTIVE: To determine whether the silkworm larvae plasma (SLP) test is a reliable diagnostic marker of infection in patients with infectious complications following gastrointestinal surgery. DESIGN: Prospective study. SETTING: Department of Surgery, University Hospital, Shiga University of Medical Science. PATIENTS: One hundred and twelve adult patients undergoing gastrointestinal surgery INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Thirty-nine of 112 patients developed infectious complications (infected group). Seven patients with sepsis (severely infected group) and 32 patients without sepsis (minor infected group) were observed. The operation time, blood loss, and age were significantly greater in both infected groups than in the noninfected group. The systemic inflammatory response syndrome score on postoperative day (POD) 1 and POD7 was highest in the severely infected group. The increase in C-reactive protein on POD3 and POD7 was significantly higher in both infected groups than in the noninfected group. White blood cell counts on POD7 were elevated significantly higher in the severely infected group than in the other groups. Immediately after surgery, SLP activity significantly increased compared with presurgery in all groups and was significantly higher in the minor and severely infected groups than in the noninfected group. The increased SLP activity returned to preoperative levels in the minor and noninfected groups; however, SLP activity in the severely infected groups remained high throughout the observational period. The most significant factor and time point that predicted infectious complications were the SLP test on POD1; sensitivity 66.7%, specificity 90.4%, positive and negative predictive values 78.8% and 83.5%. The area under the receiver operating characteristic curve for the SLP test was 0.813 +/- 0.046. CONCLUSIONS: The SLP test appears to be a useful marker of diagnosis and prediction of infectious complications following gastrointestinal surgery. Moreover, the SLP test may be able to evaluate not only the existence but also the severity of infection in surgical patients.

Elevation of plasma peptidoglycan and peripheral blood neutrophil activation during hemorrhagic shock: plasma peptidoglycan reflects bacterial translocation and may affect neutrophil activation.

OBJECTIVE: To investigate the relations among bacterial transloation, plasma peptidoglycan elevation, and peripheral blood neutrophil activation during hemorrhagic shock. DESIGN: Prospective, randomized, unblinded animal study. SETTING: Surgical research laboratories of Shiga University of Medical Science. SUBJECTS: Male, specific pathogen-free Sprague-Dawley rats. INTERVENTIONS: The rats were randomly divided into three groups: a conventional group with normal intestinal flora (NF), an antibiotic (streptomycin and penicillin G) decontaminated group (AD), and a sham shock group with normal intestinal flora. The NF and AD groups were subjected to hemorrhagic shock (mean arterial pressure 30 mm Hg, for 30 to 90 mins). Rats were killed at 30, 60, and 90 mins after shock induction. Systemic blood and mesenteric lymph nodes (MLNs) were cultured for the determination of bacterial translocation (BT). Systemic plasma peptidoglycan and endotoxin concentrations were measured. To evaluate peripheral blood neutrophil activation, phagocytosis and hydrogen peroxide generation were assayed by flow cytometry. MEASUREMENTS AND MAIN RESULTS: In the NF group, BT to MLNs was significantly increased from 30 mins after shock induction. Blood culture and plasma endotoxin were positive at 90 mins but there were no significant differences. Assayed plasma peptidoglycan was significantly increased at 90 mins. Phagocytosis and hydrogen peroxide generation were significantly increased. Assayed plasma peptidoglycan concentrations showed significant positive correlations with the magnitude of BT to MLNs (r2 = .54) and hydrogen peroxide generation (r2 = .22) in individual animals. Furthermore, BT and these parameters were significantly suppressed in the AD group. CONCLUSIONS: First, we concluded that assayed plasma peptidoglycan reflects BT induced by hemorrhage because the increase in assayed plasma peptidoglycan was suppressed, as was BT, by antibiotic decontamination. Second, peripheral blood neutrophil activation was also suppressed when BT was prevented. We concluded BT to be involved in neutrophil activation. Our findings suggest hydrogen peroxide generation by neutrophils to be involved in plasma peptidoglycan elevation.

Evaluation of the bacterial endotoxin test for quantification of endotoxin contamination of porcine vaccines.

We investigated the application of the bacterial endotoxin test for the quantification of the endotoxin contamination of various commercial porcine vaccines. In endotoxin-spiked samples, Freund's complete adjuvant and aluminum hydroxide gel adjuvant failed to interfere with the results of the endotoxin test, and both recovery ratios were within the permissible range mentioned in the Japanese Pharmacopoeia. At the various dilutions tested, none of the adjuvants in commercial porcine vaccines caused noteworthy interference in the test. In addition, none of the 39 samples of porcine vaccines approved in Japan induced an interfering effect in the endotoxin test. Our findings suggest that the bacterial endotoxin test using endotoxin-specific Limulus amoebocyte lysate (LAL) can detect endotoxin contamination in commercial porcine vaccines containing either oil or aluminum adjuvants.

A silkworm larvae plasma test for detecting peptidoglycan in cerebrospinal fluid is useful for the diagnosis of bacterial meningitis.

The silkworm larvae plasma (SLP) test has been established based on a cascade reaction triggered by either peptidoglycan or (1, 3)-beta-D-glucan to produce melanin. We applied this test to the diagnosis of bacterial meningitis. Cerebrospinal fluid (CSF) obtained from patients with bacterial meningitis due to gram-positive bacteria, gram-negative bacteria, or fungi, showed positive reactions to the test. In contrast, CSF from patients with viral meningitis or noninfectious illnesses gave negative reactions. Therefore, this test seems to be useful for diagnosis of bacterial and fungal meningitis. When this test was used together with two types of limulus tests, an endotoxin-specific test, and a conventional test, meningitis was further characterized as gram-positive, gram-negative or fungal meningitis. The SLP test requires a computerized instrument for quantitative colorimetric measurement. A qualitative alternative of this test also can be accomplished by visually observing the darkening color. Thus, this method can be applied for simple and rapid diagnosis of meningitis.