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At least 19 recordsLinked to original sources
PubMed1998

Prevention of the influence of fibrin and alpha2-macroglobulin in the continuous measurement of the thrombin potential: implications for an endpoint determination of the optical density.

We proposed the endogenous thrombin potential (ETP) as an overall function test of the coagulation system. We recently introduced a routine test which requires defibrinated plasma. In order to develop an assay in which the ETP-value can be directly obtained by measuring the optical density, we investigated two methods to inhibit fibrinogen clottability and to inactivate alpha2-macroglobulin. The first method makes use of hydroxylamine to inactivate alpha2-macroglobulin and H-Gly-Pro-Arg-Pro-OH to inhibit fibrin polymerization. At pH 7.35, plasma incubated with 25 mM hydroxylamine and 1.5 mg/mL H-Gly-Pro-Arg-Pro-OH for 5 minutes at 37 degrees C resulted in a reduced endlevel of the amidolytic activity on small chromogenic substrates. The second method uses a metalloprotease purified from Crotalus basiliscus to remove alpha2-macroglobulin from plasma in combination with H-Gly-Pro-Arg-Pro-OH. Herein plasma is incubated with 3.5 LM protease during 15 minutes at 37 degrees C in the presence of 1 mg/mL polymerization inhibitor. The enzymatic method results in a zero endlevel of the amidolytic activity and this would imply that measurement of the ETP is reduced to an endpoint determination of the optical density. We show that the endpoint determination of the optical density correlates well with the calculated ETP in plasmas with different degrees of anticoagulation.

Biomarkers↗
PubMed2000

Neutralization assay for human group C rotaviruses using a reverse passive hemagglutination test for endpoint determination.

A novel neutralization assay for human group C rotavirus (CHRV) was developed by using a reverse passive hemagglutination (RPHA) test for endpoint determination. In this assay, the neutralization (N)-RPHA test, serial twofold dilutions of sera were mixed with a solution of CHRV that yielded an RPHA test titer of 8 at 3 days after infection. The mixtures were incubated at 37 degrees C for 1 h and were inoculated onto CaCo-2 cell monolayers in a 96-well microplate. Maintenance medium containing 100 microgram of pancreatin per ml was placed in each well. The plate was sealed with sticky plastic film and was incubated at 37 degrees C for 3 days under continuous rotation. Then, the RPHA test titer of each well was determined. The neutralization titer was expressed as the reciprocal of the maximum dilution of the serum that exhibited a fourfold (75%) or greater reduction in the RPHA test titer (8 to 2 or less). Seroconversion of neutralizing antibody was demonstrated by this method in four sets of paired serum specimens from patients with diarrheal disease caused by CHRV. The seroprevalence of CHRV in the general population in Okayama Prefecture was 26.8% by immunofluorescence and 25.5% by the N-RPHA test. The N-RPHA test described here is the first system used to assay for a neutralization antibody against CHRV and is applicable in both clinical and epidemiological settings.

Adolescent↗
PubMed1992

Innovative endpoint determination system for antifungal susceptibility testing of yeasts.

Fungal infections and antifungal resistance are increasingly recognized. Antifungal susceptibility testing remains unstandardized, and a particularly important problem is endpoint determination. In this paper we propose the yeast metabolic reduction of the tetrazolium salt 2,3-bis(2- methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetraz olium hydroxide (XTT) as a colorimetric endpoint which is quantitative and objective. Amphotericin B, fluorocytosine, and fluconazole dose-response curves were obtained, and a metabolic MIC could be determined by using precise criteria.

Antifungal Agents↗
PubMed1998

Development of reference procedures for broth microdilution antifungal susceptibility testing of yeasts with standardized endpoint determination.

Standard guidelines for the broth microdilution antifungal susceptibility testing of amphotericin B, flucytosine, fluconazole, miconazole and itraconazole are reported. These are a modification of the method developed by the National Committee for Clinical Laboratory Standards (NCCLS) on the following two points: standardization of the means of endpoint determination and the inclusion of miconazole and itraconazole in the testing. MIC was determined to be when the positive control had a turbidity of 0.2 at the 630 nm wavelength. The endpoint was 80% inhibition for azoles and 100% inhibition for other drugs. The method provided good reproducibility, and a wide range of MIC distribution was observed in all antifungal agents except amphotericin B.

Antifungal Agents↗
PubMed1995

Comparison of visual and spectrophotometric methods of MIC endpoint determinations by using broth microdilution methods to test five antifungal agents, including the new triazole D0870.

A study to compare three different methods for reading MIC endpoints tested by the broth microdilution modification of the National Committee for Clinical Laboratory Standards (Villanova, Pa.) reference method was conducted. MICs of amphotericin B, flucytosine, fluconazole, itraconazole, and a new triazole, D0870, were determined for five reference yeast strains and 100 clinical isolates of Candida spp. MICs were read visually according to National Committee for Clinical Laboratory Standards guidelines from microdilution trays that had been (VS) and had not been (V) shaken. MICs were also determined spectrophotometrically (SP) at 492 nm. SP endpoints were determined as the concentrations resulting in a > or = 50% inhibition of growth (flucytosine and azoles) and a > or = 90% inhibition of growth (amphotericin B) relative to control growth. The five reference strains were tested nine times each against all five antifungal agents, and the MIC results for each reading method were compared with a 3-log2 dilution reference range determined by the macrodilution (M27-P) method. Overall, 84 to 100% of the MICs determined by V, 93 to 100% of those determined by VS, and 89 to 100% of those determined by SP fell within the 3-log2 dilution reference range for each reference strain and antifungal agent. Reproducibility was 99% for V and SP and 98% for VS. Agreement among the three methods of reading ranged from 97 to 99%. Excellent agreement among reading methods was also observed for all antifungal agents when tested against 100 clinical isolates. Agreement between the standard V method (no agitation) and VS ranged from 99 to 100%, and that between V and SP ranged from 89 to 99%. The VS and SP reading methods provided more definitive endpoints than the V method, which does not involve shaking.

Antifungal Agents↗
PubMed1992

Antimicrobial susceptibility testing of Bilophila wadsworthia by using triphenyltetrazolium chloride to facilitate endpoint determination.

Initial susceptibility studies of Bilophila wadsworthia indicated significant resistance to several beta-lactam antibiotics, including imipenem and cefoxitin. NO beta-lactamase production was detected. However, some B. wadsworthia strains may grow as a heavy "haze" at up to the highest concentration of an antibiotic on standard antimicrobial agent-containing plates, and it is often difficult to determine the point at which conventional growth stops and haze growth begins. We investigated the nature of the haze growth of B. wadsworthia by using triphenyltetrazolium chloride (TTC) as an indicator of viability during antimicrobial susceptibility testing, by determining viability counts on antimicrobial agent-containing plates at various times, and by microscopically inspecting stained preparations of the growth on the control plate and the haze area. TTC MICs were determined by applying a TTC solution over the growth on plates inside the anaerobic chamber or within 5 min after exposure to air (aerobic TTC MICs). The haze growth reduced TTC in the chamber but not under aerobic conditions, whereas TTC was reduced by the conventional growth in both atmospheres. The aerobic TTC MICs correlated with the viability counts. Separated proteins resolved by polyacrylamide gel electrophoresis and isoelectric focusing showed TTC-reactive bands only when stained under anaerobic conditions, further demonstrating the sensitivity of TTC reduction to aerobic conditions. Microscopic examination of the haze growth indicated spheroplast formation. A new antibiogram for B. wadsworthia has been established by use of aerobic TTC endpoints; we believe that the lower MICs obtained with the TTC method are likely the ones that are clinically relevant and should be used in tests of B. wadsworthia. Also, we found that when the organisms were grown on pyruvate-containing medium, 87% of 56 strains tested were Beta-lactamase positive.

Anti-Bacterial Agents↗
PubMed1992

Paralytic shellfish poison (saxitoxin family) bioassays: automated endpoint determination and standardization of the in vitro tissue culture bioassay, and comparison with the standard mouse bioassay.

Mouse neuroblastoma cells swell and eventually lyse upon exposure to veratridine, which, when added together with ouabain, enhances sodium ion influx. In the presence of saxitoxin (STX), which blocks sodium channels, the action of the other two compounds is inhibited and the cells remain morphologically normal. A tissue culture bioassay using mouse neuroblastoma cells, developed by Kogure and colleagues, takes advantage of these principles; in this bioassay, the fraction of the cells protected from the actions of ouabain and veratridine is in direct proportion to the concentration of STX and its analogues. We have modified this bioassay, improving its convenience and speed by eliminating the need to count individual cells to determine the saxitoxin equivalents, and instead have employed a microplate reader for automated determinations of absorbances of crystal violet from stained neuroblastoma cells. When these changes and other minor technical modifications were tested in the tissue culture bioassay systematically, we found the lower detection limit to be around 10 ng STX equivalents (eq) per ml of extract ( = 2.0 micrograms STX eq/100 g shellfish tissue). Our version of the tissue culture bioassay was compared with the standard mouse bioassay using 10 acid extracts of dinoflagellates (Alexandrium excavata and A. fundyense) and 47 AOAC extracts of shellfish tissues. The tissue culture bioassay provided results virtually identical to those obtained with the mouse bioassay (r > 0.96), and moreover, was considerably more sensitive. The results gained from high performance liquid chromatographic (HPLC) analysis of 12 of the same extracts were less consistent when compared with the results from both bioassay methods. The automated tissue culture (neuroblastoma cell) bioassay may be a valid alternative to live animal testing for paralytic shellfish poisoning.

Animals↗
PubMed1999

Influence of incubation time, inoculum size, and glucose concentrations on spectrophotometric endpoint determinations for amphotericin B, fluconazole, and itraconazole.

We addressed the influence of the incubation time (24 h versus 48 h), starting inoculum size (standard inoculum size, approximately 10(3) CFU/ml, versus large inoculum size, approximately 10(4) CFU/ml), and supplementation with 2% glucose of RPMI 1640 medium on the spectrophotometric determination of the MICs of amphotericin B, fluconazole, and itraconazole. We compared the MICs determined spectrophotometrically with those determined by the standard broth macrodilution method (National Committee for Clinical Laboratory Standards approved guideline M27-A). The agreement between the results of the spectrophotometric and standard methods for amphotericin B testing was 100%; this agreement was independent of the inoculum size and incubation time. On the other hand, the agreement for the results for fluconazole testing and itraconazole testing was dependent on the inoculum size and incubation time. With large inoculum size, excellent agreement can be achieved at 24 h. With standard inoculum size, acceptable agreement can be achieved only at 48 h. In contrast to previous observations, the addition of 2% glucose did not have any significant impact on the growth density at 24 h, nor did it improve the agreement with the standard method. Furthermore, supplemental glucose might falsely elevate the MIC at 48 h.

Amphotericin B↗
PubMed1990

Measurements of antifungal levels in corneal tissue: a simplified bioassay for amphotericin B.

Measuring precise antifungal levels in the cornea with broth-dilution bioassays is difficult, as standard techniques involving visual determination of endpoints are hindered by corneal debris. To increase the precision of the measurement, we modified the sample preparation for bioassay of rabbit corneas treated with subconjunctival amphotericin B. Endpoint determination and variance were compared for a freshly thawed corneal suspension and the supernatant after 24 h equilibration; bioassay of the corneal suspension after 24 h equilibration served as an additional control. All endpoints were read visually in a masked fashion and were verified by culture. The three methods gave comparable endpoint values with equivalent degrees of variance. Amphotericin B levels were consistent by both visual and culture determination; however, endpoints were clearly visible and easier to read for the supernatant. Visual determination of the endpoints for the supernatant following 24 h equilibration simplified and ensured the precision of the bioassay technique.

Amphotericin B↗
PubMed1998

Development of a dot blot neutralizing assay for HHV-6 and HHV-7 using specific monoclonal antibodies.

To elucidate further immune responses to human herpesviruses 6 and 7 (HHV-6 and -7), a neutralizing antibody assay was established for these viruses using a dot blot method. Three monoclonal antibodies against HHV-6 and 12 monoclonal antibodies against HHV-7 were developed and characterized by radio-immunoprecipitation. One monoclonal antibody which recognizes the 135 kDa late polypeptide of HHV-6 and several which recognize the 125 kDa late polypeptide of HHV-7 were selected to monitor virus growth by a dot blot antigen-detection method. The dot blot method was then used for the assay of HHV-6 and -7 neutralizing antibodies in human serum samples. The neutralization endpoints determined by the dot blot were comparable to those determined by immunofluorescence (IF). The neutralizing antibody titers appeared to correlate with the antibody titers determined by the indirect IF antibody test. The dot blot neutralization assay is easy to perform, is highly reproducible and objective when compared with the conventional methods based on cytopathology or IF for determining neutralization endpoints.

Animals↗
PubMed1995

Comparison of two alternative microdilution procedures with the National Committee for Clinical Laboratory Standards reference macrodilution method M27-P for in vitro testing of fluconazole-resistant and -susceptible isolates of Candida albicans.

The National Committee for Clinical Laboratory Standards has proposed a reference broth macrodilution method for in vitro antifungal susceptibility testing of yeasts (the M27-P method). This method is cumbersome and time-consuming and includes MIC endpoint determination by the visual and subjective inspection of growth inhibition after 48 h of incubation. Two alternative microdilution procedures for MIC endpoint determination, a spectrophotometric MIC endpoint test that evaluates 80% growth inhibition by the drug and a colorimetric method with an oxidation-reduction indicator (Alamar Blue), were compared with the M27-P method for fluconazole susceptibility testing of 45 susceptible and resistant isolates of Candida albicans. The spectrophotometric method was performed with RPMI 1640 medium with 2% glucose, and the other two tests were performed with plain RPMI 1640 medium. All tests were incubated at 35 degrees C. Excellent agreement was demonstrated between the M27-P method and both 24-h microdilution tests (97.7%) as well as between the two microdilution tests (95.5%). Also, there was agreement in the detection in vivo of fluconazole resistance by the three methods. These preliminary data indicate that both microdilution methods may serve as less subjective alternatives to the M27-P method for the determination of fluconazole MIC endpoints.

AIDS-Related Opportunistic Infections↗
PubMed1998

A statistically defined endpoint titer determination method for immunoassays.

Results of immunoassays for which no positive standards are available are often expressed as endpoint titers. The endpoint titer is defined as the reciprocal of the highest analyte dilution that gives a reading above the cutoff. Unfortunately, there is no generally accepted rule for the determination of these cutoff values. In enzyme-linked immunosorbent assays (ELISA) a value two or three times the mean background or negative control reading is sometimes used. Other investigators set the cutoff arbitrarily at a certain absorbance value. These procedures do not provide statistically meaningful information about the risk of overtitration or false low titers. We have solved this problem by devising a practical method for establishing a statistically valid cutoff. The procedure involves calculating the upper prediction limit using the Student t-distribution. The mathematical formula which defines the upper prediction limit is expressed as the standard deviation multiplied by a factor which is based on the number of negative controls and the confidence level (1 - alpha). Appropriate factors are provided for 2 to 30 negative controls and for confidence levels ranging from 95% to 99.9%. Our new method is more reliable than other nonstatistical procedures yet does not require sophisticated computation. It can be applied to a variety of immunoassays provided that negative controls are available.

Enzyme-Linked Immunosorbent Assay↗
PubMed1978

Improvement of the micromethod for the limulus lysate test.

Frauch's micro-slide method was improved to facilitate the endpoint-determination of the Limulus test. Two precise observations, by inverted phase contrast microscopy and with a staining procedure, were newly performed as additions to the slide test. The staining procedure was proposed as an improved method for the Limulus test since it is simple and convenient. In the staining method, bromophenol blue (BPB) solution was used as the staining solution. A negative (-), a strong positive (++) and a weak positive reaction (+) were characterized by a "ring" formation, a "cloud-like" spread of gel and a "spot" in the "cloud" respectively. Since the distinction between (-) and (+) reactions was obvious in the proposed method, determination of the endpoint was easier than in the ordinary tube and Frauch's method. The sensitivity of the present method was equal to or higher than that of other methods. Inverted phase contrast microscopy was utilized to confirm the findings obtained by the staining method. The volume of the lysate used in this method was as little as 1/10 of that used in the tube method.

Blood Platelets↗
PubMed2001

Standardization of antifungal susceptibility variables for a semiautomated methodology.

Recently, the methodology that will serve as a basis of the standard for antifungal susceptibility testing of fermentative yeasts of the European Committee on Antibiotic Susceptibility Testing has been described. This procedure employs a spectrophotometric method for both inoculum adjustment and endpoint determination. However, the utilization of a spectrophotometer requires studies for standardization. The present work analyzes the following parameters: (i) accuracy of inoculum preparation, (ii) correlation between optical density and CFU per milliliter, (iii) influence of the wavelength on the endpoint determination, and (iv) influence of the dimethyl sulfoxide concentration on the growth kinetics. The main results can be summarized as follows: (i) inoculum preparation following the methodology recommended by the National Committee for Clinical Laboratory Standards is an exact procedure; (ii) the relationship between optical density and CFU per milliliter is linear (coefficient of determination, r(2) = 0.84); (iii) MICs obtained by means of spectrophotometric readings at different wavelengths are identical (for amphotericin B, an intraclass correlation coefficient of 0.98 was obtained; for fluconazole, the intraclass correlation coefficient was 1); and (iv) a 2% concentration of dimethyl sulfoxide produces a significantly slower and lower growth curve of Candida spp. than other concentrations.

Amphotericin B↗
PubMed1999

Use of spectrophotometric reading for in vitro antifungal susceptibility testing of Aspergillus spp.

A comparative study of visual and spectrophotometric MIC endpoint determinations for antifungal susceptibility testing of Aspergillus species was performed. A broth microdilution method adapted from the National Committee for Clinical Laboratory Standards (NCCLS) was used for susceptibility testing of 180 clinical isolates of Aspergillus species against amphotericin B and itraconazole. MICs were determined visually and spectrophotometrically at 490 nm after 24, 48, and 72 h of incubation, and MIC pairs were compared. The agreement between the two methods was 99% for amphotericin B and ranged from 95 to 98% for itraconazole. It is concluded that spectrophotometric MIC endpoint determination is a valuable alternative to the visual reference method for susceptibility testing of Aspergillus species.

Amphotericin B↗
PubMed1992

In vitro antimicrobial susceptibility testing of Borrelia burgdorferi: a microdilution MIC method and time-kill studies.

The susceptibility of Borrelia burgdorferi, the causative agent of Lyme borreliosis, to various antimicrobial agents varies widely among published studies. These differences are probably due in part to variations in susceptibility testing techniques and growth endpoint determinations. We developed a microdilution method for determining the MICs of antibiotics against B. burgdorferi. The method incorporated BSK II medium, a final inoculum of 10(6) cells per ml, and a 72-h incubation period and was found to be simple and highly reproducible. A variety of antibiotics and strains of B. burgdorferi and one strain of Borrelia hermsii were examined by this method. MICs of penicillin, ceftriaxone, and erythromycin for the B31 strain of B. burgdorferi were 0.06, 0.03, and 0.03 microgram/ml, respectively. We compared the MICs obtained by the microdilution method with those obtained by a macrodilution method using similar criteria for endpoint determinations and found the values obtained by both methods to be in close agreement. To further investigate the bactericidal activities of penicillin, ceftriaxone, and erythromycin against strain B31, we used subsurface plating to determine MBCs and we also performed time-kill studies. The MBCs of penicillin, ceftriaxone, and erythromycin were 0.125, 0.03, and 0.06 micrograms/ml, respectively. Time-kill curves demonstrated a greater than or equal to 3-log10-unit killing after 72 h with penicillin, ceftriaxone, and erythromycin; ceftriaxone provided the greatest reduction in CFU. The described methods offer a more standardized and objective approach to susceptibility testing of B. burgdorferi.

Anti-Bacterial Agents↗
PubMed1996

Endpoints for determining mineral element requirements; an introduction.

There are numerous measures of essential mineral element status, including growth rate, tissue and physiological fluid concentrations, enzyme concentrations and activities, chemical balance and mobilizable stores. For the adult human, blood and its specific nutrient concentrations provide a useful but frequently inadequate index. The first limiting biochemical system should provide the most valid index, but in many cases it is unknown or not readily measured. Chemical balance and mobilizable stores provide valid measures but are difficult to determine. Two indices are infinitely more valuable than one and should be determined if possible. More research is needed to establish valid indicators of nutritional status for mineral elements.

Adult↗