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Mutagenicity testing of protein-containing and biological samples using the Ames/Salmonella plate incorporation test and the fluctuation test.

Mutagenicity testing of biological samples and proteins is complicated by the presence of histidine and histidine-related growth factors which may produce a false positive result in the Ames/Salmonella plate incorporation test. A bioassay method, utilizing an automated dispenser-photometer and Salmonella typhimurium strain TA1535 as the indicator bacteria, was used to estimate the presence of histidine-related growth factors in three enzyme solutions submitted for mutagenicity testing. One of the solutions was clearly positive in the Ames/Salmonella test and also contained the highest amount of L-histidine-HCl-equivalents. The two other solutions, with low or undetectable amounts of L-histidine-HCl-equivalents, gave equivocal and negative results, respectively, in the Ames/Salmonella test. Studies were also performed with strains TA98, TA100 and TA1535 to determine the amount of added L-histidine-HCl that would result in a 'positive' result in the Ames/Salmonella test. Because the minimum amount of L-histidine-HCl required to double the number of revertant colonies was 150 nmol/plate, and the maximum amount of L-histidine-HCl-equivalents supplied by the enzyme preparations was 40 nmol/plate at the highest tested dose, the mutagenicity test results of the enzyme solutions cannot be explained solely by histidine or related compounds. Smokers' and non-smokers' urines, concentrated with liquid extraction (CHCl3) and adsorbent (XAD-2 and XAD-2/Sep-Pak C18) techniques, were studied to reveal differences in efficiencies to extract histidine and histidine-related compounds in the urines. Amounts of 'histidine' in concentrates of urine were measured using the bioassay method and a chemical method employing derivatization with fluorescamine. The fluorescamine method also efficiently detected 3-methyl-L-histidine, a product of muscle metabolism excreted in urine, which was found to be unable to support auxotrophic growth in TA1535, leading to exaggerated estimations of the auxotrophic growth enhancing properties of urine extracts. The urine extracts, and pure L-histidine-HCl, were tested using a two-step fluctuation test to estimate auxotrophic growth factor effects in this type of test. Because of a strong dilution effect when adding the histidine-free selection medium, the fluctuation test employed in this study was not found to be particularly sensitive to growth factors. The results of this study indicate that use of a bioassay, employing the same indicator bacteria as the mutagenicity test themselves, is a reliable way to measure histidine-related growth factors in biological samples.(ABSTRACT TRUNCATED AT 400 WORDS)

Enzymes

Comparison of five methods, including the PDM Epsilometer test (E test), for antimicrobial susceptibility testing of Pseudomonas aeruginosa.

The antimicrobial susceptibilities of 100 clinical isolates of Pseudomonas aeruginosa to six antipseudomonal antibiotics were tested by five methods: the National Committee for Clinical Laboratory Standards (NCCLS) methods for broth microdilution, agar dilution, and agar disk diffusion; the Vitek Automicrobic System method (Vitek Systems, Hazelwood, Mo.); and the PDM Epsilometer test (E test) (AB Biodisk, Solna, Sweden). The E test results showed excellent correlation with agar dilution results, with over 90% agreement within 1 doubling dilution between the E test and reference agar dilution MICs for all antimicrobial agents tested. The E test results also showed good correlation with the results from the reference agar disk diffusion method, with 90 to 99% complete agreement and 100% essential agreement on categories for all antibiotics tested (essential agreement is the agreement obtained when minor discrepancies are ignored). Comparison of categories with the E test and broth microdilution methods, using the broth microdilution method as the reference method, gave only 59% complete agreement for gentamicin, with 28 minor discrepancies and 13 very major discrepancies. Some discrepancies were observed between results from the E test and broth methods for gentamicin, with the broth microdilution and Vitek methods giving higher MICs than the E test and other methods using agar. The most recent NCCLS guidelines for broth dilution testing have reduced the recommended levels of cation supplementation, which may enhance future agreement between results for the aminoglycosides and P. aeruginosa on broth and on agar. We found that the E test offers a simple, labor-efficient, and accurate method for MIC determination on an agar medium.

Adult

The micronucleus test as part of a short-term mutagenicity test program for the prediction of carcinogenicity evaluated by 143 agents tested.

To evaluate the usefulness of the micronucleus test as a short-term assay for the detection of carcinogens, the correlation between micronucleus test data for 143 chemicals and corresponding cancer data, has been analyzed. For comparison, analogous data from Ames's test have also been collected for the same chemicals. In a comparison of the micronucleus test and Ames's test it was found that they had about the same specificity (around 80%) and predictive value (around 90%), while there was a significant difference in sensitivity in favor of Ames's test. The difference in sensitivity could be partly explained by differences in metabolizing capacities of these two test systems. It is concluded that a more elaborate test procedure for the micronucleus test would increase that sensitivity of this test. The principal value of the micronucleus test lies in the fact that it is an in vivo method, which may pick up effects at the chromosomal level not covered by bacterial assays. This is emphasized by the finding that the combination of Ames's test and the micronucleus test did increase the sensitivity of the screening procedure for the prediction of carcinogenic effects.

Animals

Strain A mouse pulmonary tumor test results for chemicals previously tested in the National Cancer Institute carcinogenicity tests.

Sixty-five chemicals were coded and examined for their ability to induce lung tumors in strain A/St (laboratory A) or strain A/J (laboratory B) mice. Thirty-five chemicals were tested in laboratory A only, 6 in laboratory B only, and 24 in both laboratories. Two-year carcinogenicity test results as well as genotoxicity test data are available for most of these chemicals. There was poor interlaboratory agreement in strain A test results for the 24 chemicals tested in both laboratories. In addition, there was poor agreement between strain A test results from either laboratory and 2-year carcinogenicity test results or genotoxicity results. Possible explanations for these findings include selection of a large number of aromatic amines in the group of chemicals submitted for strain A testing, differences in strain A testing protocols and in statistical analysis of results from the two laboratories, low sensitivity of the strain A/St mice used in this particular study, and general problems inherent in comparing any relatively short-term animal tumor model with 2-year carcinogenicity tests. Since there is no absolute reference for carcinogenicity, no one test system is better than another. Carcinogenicity test data are relevant only to the test model employed.

Animals

Reliability of the anterior drawer test, the pivot shift test, and the Lachman test.

In 147 patients with arthroscopically proved chronic injuries of the anterior cruciate ligament, the anterior drawer test, the Lachman test, and the pivot shift test were done before operation under general or spinal anesthesia. Results of the anterior drawer test were positive in 79.6% of the patients, in 98.6% patients having the Lachman test, and in 89.8% of patients having the pivot shift test. In 19 cases (12.9%), arthroscopic examination showed reattachment of the proximally torn end of the anterior cruciate ligament to the posterior cruciate ligament. In these cases, results of the anterior drawer test were positive in 13 patients (68.4%), in 17 (89.5%) patients having the Lachman test, and in 12 patients (63.2%) having the pivot shift test. In 15 cases with a false negative pivot shift test, arthroscopy showed blockage of anterior subluxation of the lateral tibial condyle by a partially functioning portion of the anterior cruciate ligament, which was reattached to the posterior cruciate ligament in 7 cases. In 2 cases with a false negative Lachman test, there was firm reattachment of the torn end of the anterior cruciate ligament to the proximal portion of the posterior cruciate ligament combined with a bucket handle tear of the medial meniscus. The Lachman test was most sensitive in diagnosing chronic injuries of the anterior cruciate ligament. The pivot shift test was also sensitive but was influenced by many factors.

Adolescent

[Skin and laboratory tests: comparison of the epicutaneous patch test with the TTL and LIF tests in the diagnosis of medicamentous allergic contact dermatitis].

We investigated 158 patients from 15 to 82 years of age with clinically evident contact dermatitis, diagnosed at the Department for Allergic Diseases Investigations-Clinic for Dermatovenerological Diseases, Medical Faculty in Novi Sad, during the period of 1 year. We performed patch-epicutaneous test, lymphocyte transformation test (TTL) and leukocyte migration inhibition test (LIF). Among the 130 (82.2%) patients suffering from contact dermatitis, with the positive patch tests to commercial or standard battery epicutaneous allergens, 26 (20%) had at least one positive patch test to the medicament. In these cases contact allergic medicamentous dermatitis (SAMD) was proved by positive clinical and allergic investigations. In only one case, patch test was negative, with booth the TTL and LIF test positive. Among the medicaments TTL and LIF tested, antibiotics were the most frequent in 9 (34.61%) cases, analgetics were found in 6 (23.08%). Professional contact allergy to medicaments has been estimated in 5 patients (19.23%). According to the obtained results and statistic findings, relationships between the two variables-TTL and patch test, and LIF and patch test, were estimated due to the contact allergy to medicaments. Both of them were low and negative without statistic significance. Patch test remains to be the irreplaceable test with regard to CAMD.

Adolescent

Pancreolauryl test. Evaluation of a tubeless pancreatic function test in comparison with other indirect and direct tests for exocrine pancreatic function.

The sensitivity and specificity of the pancreolauryl test was evaluated in comparison with the NBT-PABA test, the estimation of fecal chymotrypsin and fat, and the secretin-pancreozymin test in 168 patients with and without pancreatic disease. The overall sensitivity rate was as follows: pancreolauryl test 90%, NBT-PABA test 86%, fecal chymotrypsin 66%. In patients with pancreatic steatorrhea the sensitivity of the pancreolauryl test was 100%, the NBT-PABA test 97%, and the fecal chymotrypsin estimation 92%. The specificity of these tests was: pancreolauryl test 97.6%, fecal chymotrypsin 87%, and NBT-PABA test 81.8%. The pancreolauryl test may be recommended as a noninvasive easy-to-perform tubeless pancreatic function test with a sufficiently high sensitivity and specificity.

4-Aminobenzoic Acid

Non-isotopic immunoassay drug tests in racing horses: a review of their application to pre- and post-race testing, drug quantitation, and human drug testing.

We have introduced large scale non-isotopic immunoassay testing into pre- and post-race drug testing in racehorses. The technologies utilized are Particle Concentration Fluorescence Immuno Assay (PCFIA) and the one-step Enzyme Linked Immuno Sorbent Assay (ELISA). These technologies are rapid, inexpensive, and highly effective. On introduction into post-race testing in the Western United States, these ELISA tests exposed several previously undetected patterns of drug abuse. The drugs detected were buprenorphine, oxymorphone, mazindol, sufentanil and cocaine. This led to the suspension of a large number of trainers and exposed the high false negative rate of thin layer chromatography (TLC) based testing. More recently, we have introduced both PCFIA and ELISA assays into pre- and post-race testing in Illinois. Within days, our pre-race PCFIA tests detected signs of acepromazine abuse. Directed searches of post-race urines from these horses showed evidence for acepromazine metabolites in the urine of these horses. Examination of frozen samples from associated horses yielded about 70 ELISA "positives" for acepromazine. To date, about 25 of these ELISA "positives" have been confirmed by mass spectrometry. We have also raised antibodies to phenylbutazone and furosemide to enable rapid and inexpensive quantitation of these permitted medications. Furosemide is a particular problem since its use requires a pre-race detention barn. For furosemide, we have developed a regulatory schedule based on our immunoassay test that allows elimination of the detention barn. For phenylbutazone, we have developed a similar immunoassay that allows rapid and inexpensive quantitation of this drug in blood. To enable racing authorities to test jockeys and other racetrack personnel, we have adapted PCFIA technology to human drug testing, and a full range of very sensitive tests for human drugs of abuse is available. These immunoassays are sufficiently sensitive to control abuse of the most potent drugs available to horsemen. In principle, an immunoassay can be raised to any drug within about six months, and made available worldwide at competitive rates. It appears clear that these non-isotopic immunoassays provide racing with the only technological basis that is sufficiently sensitive to detect the most potent abused drugs pre- and post-race, and has the flexibility to be readily adaptable to different drugs. Because of the high false negative rate generated by TLC, credible pre- and post-race testing programs cannot be based on TLC alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Acepromazine

Stapedius reflex test, brainstem audiometry and opto-vestibular tests in diagnosis of acoustic neurinomas. A comparison of test sensitivity in patients with moderate hearing loss.

The stapedius reflex test, brainstem audiometry and the opto-vestibular tests for identifying acoustic neurinomas (AN) were evaluated and compared in a study of 21 patients with radiologically or surgically verified AN and a pure tone average not exceeding 60 dB HL. The stapedius reflex test results were interpreted according to the criteria developed at this clinic. The stimuli for the auditory brainstem response (ABR) were 2 kHz haversine waves and 4 kHz square waves. The vestibular examination consisted of a caloric test and the recording of eye-tracking and gaze nystagmus. In the cases studied the stapedius reflex test gave 1 false-negative result and ABR none. The ENG gave 3 false-negatives whereas the results of speech discrimination tests were misleading in no fewer than 1/3 of the cases. The results of the different tests were directly correlated but correlation coefficients did not exceed 0.65. Tumours larger than 15-20 mm showed a different test pattern than those below that size: stapedius reflex response, ABR and caloric response were eliminated and all of these patients had abnormal optomotor function. It is suggested that an optimal routine test procedure should consist of a pure tone audiogram, supplemented by ABR or the stapedius reflex test. Opto-vestibular tests may be of value in a preliminary estimation of tumour size.

Audiometry

A comparison between two exercise tests on cycle; a computerized test versus the Astrand test.

Two submaximal cycle ergometer test methods, the Astrand nomogram test and a computerized two-point extrapolation test (Cat Eye ergociser, commercially available), were compared in order to determine agreement and repeatability of estimates of maximum oxygen consumption (VO2max). Twenty healthy women, divided into two groups of ten according to their age (mean 35.3 and mean 46.9), performed test-retest with each method. In both methods the VO2max was estimated from workload and the corresponding heart rate. The correlation between the VO2max using the two methods was high (r = 0.85, P < 0.001). Some of the estimates derived from the computerized test had large errors, which reduced the agreement between the tests. The variation (2SD), expressed in per cent of mean VO2max was 19% for the Astrand test and 34% for the computerized test. Furthermore, the computerized test underestimated the VO2max with approximately 5 ml kg-1 min-1 compared with the Astrand test. Due to this underestimation and the greater variation of the VO2max in the computerized test, it is not recommended to use the two methods interchangeably in clinical practice.

Adult

Diagnosis of reaginic allergy with house dust, animal dander and pollen allergens in adult patients. III. Case histories and combinations of case histories, skin tests and the radioallergosorbent test, RAST, compared with provocation tests.

Case histories alone and combined with the results of skin tests and/or the radioallergosorbent test (RAST) were compared with the results of provocation tests in 397 patients with asthma or allergic rhinitis. Case histories often turned out to be doubtful. The agreement between negative histories and negative provocation tests was 77% and that between positive histories and positive provocation tests 64%. When case histories were supported by concordant skin test or RAST results, the agreement with provocation tests increased to about 90%. When a scoring system for the combinations of different diagnostic methods was employed, a 100% agreement with provocation tests was obtained for cases with high scores. It is concluded that by using the case history in conjunction with RAST and/or skin test results, a reliable diagnosis can be achieved, eliminating the necessity of provocation tests.

Allergens

[An experimental study of the hand test situation: effects of test definition during instruction on hand test performance].

The purpose of this study was to examine how Hand Test performance is affected by test definition included with task instructions. The Hand Test was administered to male college students equally divided into three groups which differed in terms of the type of test definition included with the task instruction as follows: (1) Emotionally disturbed test definition (ED Group); (2) Imagery test definition (IM Group); and, (3) Standard instruction with no test definition (N Group). The MMPI was subsequently administered to all subjects with the following results. The ED Group revealed higher DES and lower ACQ scores than the N Group. The IM Group obtained higher DES and TEN scores and lower ACQ and ICP (CRIP) scores than the N Group. The ED Group exhibited positive correlations between PASS, ACQ and ICP (CRIP) scores and psychopathological MMPI scales while the IM Group revealed negative correlations between ACQ, PASS, TEN, ICP (CRIP), FEAR and DES scores and psychopathological MMPI scales. These results indicated in general that Hand Test performance is sensitive to the test situation and that this factor should be taken into account when Hand Test responses are interpreted.

Adolescent

Comparing normal hearing and hearing-impaired subject's performance on the Northwestern Auditory Test Number 6, California Consonant Test, and Pascoe's High-Frequency Word Test.

The Northwestern Auditory Test No. 6, the California Consonant Test, and Pascoe's High-Frequency Test were presented to two groups of listeners. One group consisted of 12 patients with normal hearing up to 2 kHz accompanied by a high-frequency loss. The second group consisted of persons with normal hearing. There were significant differences between the two groups of listeners in overall discrimination scores, among the four intensity levels (10, 20, 30, 40 dB) and the three speech discrimination tests. When the data from the hearing-impaired subjects were analyzed, it was found that these individuals obtained higher scores on the NU 6 material at 20, 30, and 40 dB SL when compared with the California Consonant Test and Pascoe's test. The results from the Newmann-Kuel multicomparison test indicated no statistical significance between the results of the California test and Pascoe's test; however, the distribution of scores at 40 dB was different. Results of this study suggested that the NU 6 test was not sufficiently sensitive to detect consonant confusions in individuals with high-frequency hearing loss.

Adult

Specificity of the indirect haemagglutination test with Plasmodium falciparum test cells. Comparison of indirect haemagglutination and fluorescent antibody tests on African sera.

The specificity of the indirect haemagglutination (IHA) test with Plasmodium falciparum placental antigen-sensitized test cells was examined with sera from healthy blood donors and from patients with diseases other than malaria. Only 1 nonspecific antibody reaction was seen in more than 700 tests. A comparison of IHA titres and indirect fluorescent antibody (IFA) titres with IgG and IgM conjugates on 503 sera from inhabitants of Mto Wa Mbu in Tanzania showed in successive age groups an increasing number of seropositive reactors with both tests. The increase in the proportion of positive reactions and in the mean titre levels started earlier but was more gradual in the IFA test than in the IHA test. Parasite carriers had higher antibody levels than people without an apparent parasitaemia. Parasite carriers in the younger age groups especially were more frequently seronegative in the IHA test than in the IFA test with anti-IgG conjugates. The reactivity of the IHA test with P. falciparum (Palo Alto/Aotus) antigen was higher than that with P. falciparum placental antigen and was thus closer to that of the IFA test with IgG conjugates.

Adolescent

[The diagnostic value of a new whole blood histamine release test using paper disc-coupled antigens--results compared with intracutaneous test, RAST, and eye test].

We have developed a novel histamine release test (HR) using whole blood and antigen-coupled RAST paper discs, for the screening of allergens rather in a short time with a small amount of blood. Histamine was determined by a RIA kit. In order to evaluate the diagnostic value, the results of the test were compared with those of RAST, intracutaneous tests, and eye tests in 45 allergic patients. HR correlates better with RAST than intracutaneous test in almost all antigens. The closest positive correlation with the other tests was seen in mite allergen, followed by pollen, foods and mould spores in that order. When the HR was positive for house dust, 100% of the patients were also positive for the eye test, which is reported to closely correlate with bronchial provocation tests. HR seemed to be a useful test not only for the screening but also for the determination of pathogenic allergens.

Adolescent

Pituitary function tests: comparison of ACTH and 11-deoxy-cortisol responses in the metyrapone test and with the insulin hypoglycemia test.

PURPOSE: To compare the sensitivity of ACTH and 11-deoxy-cortisol (comp. S) responses in the short metyrapone test and the latter with the insulin hypoglycemia test. METHODS: Retrospective evaluation of 115 short metyrapone tests and comparison of 18 pairs of metyrapone and insulin tests. 20 healthy controls and 95 patients with confirmed pituitary disease were studied. All hormones were measured by sensitive radioimmunoassays. RESULTS: In patients with pituitary disease not requiring hydrocortisone substitution (n = 70), the ACTH response in the metyrapone test was subnormal in 47 cases (< 33 pmol/L), the comp. S response (< 200 nmol/L) in 21 cases only. Comparison of the relationship between ACTH and comp. S with an ACTH-cortisol dose-response curve obtained in normal subjects shows that subnormal ACTH responses after metyrapone in the range between 13 and 33 pmol/L still generate normal comp. S responses. The results of the metyrapone test correlated significantly with those of the insulin test. CONCLUSIONS: Measuring plasma ACTH in the scope of the metyrapone test makes the test more sensitive to detect secondary adrenal insufficiency than with steroid measurements alone. Results of the metyrapone test correlate significantly with the cortisol response to insulin hypoglycemia.

Adrenocorticotropic Hormone

[The peritoneal sperm migration test (PSM-test) following fractional artificial insemination during laparoscopic fertility testing].

The peritoneal sperm migration test following fractional artificial insemination during diagnostic laparoscopy was carried out in the context of fertility investigation in 78 women age 25 to age 40. 35 cases had a positive PSM test, 43 had a negative PSM test. The results were correlated to the phase of menstrual cycle, the semen analysis and the tubal patency test. The test showed the best results when it was done in the preovulatory phase in conjunction with a normal semen analysis and a normal tubal patency test. In 2 cases with severe oligospermia and in two cases with bilateral tubal occlusion the test was still positive. The test is of clinical importance and can be done without increased risk during any laparoscopy for infertility. Valuable conclusion about the migration of sperms through the uterus and the tubes in the individual couple can be obtained. This is of prognostic importance. The test shows in addition to mechanical tubal patency by chromopertubation the functional or physiologic tubal permeability. Negative PSM tests are relatively frequent and are caused by tubal, uterine or sperm factors.

Adult

[Evaluation of two quantitative tests for salivary secretion--the chewing gum test and the Saxon test in normal subjects and in patients with Sjögren's syndrome].

A cooperative multicenter study was performed to evaluate two salivary secretion methods-the chewing gum test and the Saxon test by a crossover method. Twenty healthy volunteers and 27 patients with Sjögren's syndrome were examined. In healthy controls the amount of saliva collected per minute was larger in the Saxon test. Both tests revealed a significant decrease of salivary secretion in patients with Sjögren's syndrome, but the amount of saliva per minute was also larger in Saxon test. By repeated trials of both tests a notable increase of saliva was always observed at the second trial. Since this phenomenon may be due to the learning effect of the tests, one should perform at least one practice before starting formal tests. Saxon test could be performed in a patient having dental prostheses who was unable to masticate chewing gum. The Saxon test, thus, may be an efficient method, to evaluate the salivary secretory function.

Adult