Evaluation of Miniature Test for Bacteriuria Using Dehydrated Media and Nitrite Pads

A new dip-inoculum method for detecting bacteriuria which utilizes dehydrated media pads and a nitrite pad attached to a small plastic strip was evaluated in hospitalized patients. Discrepant interpretations were made by independent observers in 9.3% of the specimens with > 10(5) colonies per ml. The media pads failed to support growth of yeast and gave variable results with Staphylococcus epidermidis and non-group D streptococci. False-negative culture results commonly occurred if the patients were receiving antibiotics. The nitrite test occasionally remained positive for brief periods after the elimination of bacteriuria by antibiotics. Conditions and drugs (especially phenazopyridine) which discolor urine interfered with reading both the culture and nitrite tests. Although not suitable for hospital use, or for monitoring therapy, the test strip is probably as reliable as the calibrated loop-streak plate culture for office screening.     

Studies on Media for Enumerating Enterococci in Frozen Vegetables

A study was made of the relative sensitivity and specificity of presumptive and confirmatory media for the most probable number enumeration of enterococci in frozen vegetables. Azide dextrose broth yielded the highest numbers of confirmable enterococci and its sensitivity was shown to be comparable to nonselective media. The use of ethyl violet azide broth as a confirmatory medium resulted in a significant number of false positive tests. Growth in broth containing 6.5% sodium chloride incubated at 45 C for 2 days was found to be a more specific confirmatory test for enterococci.     

Topiramate as a Cause of False Positive in the Overnight 1-Mg Dexamethasone Suppression Test

ObjectiveTo describe that topiramate may cause a false positive in an overnight 1-mg dexamethasone suppression test (DST) for hypercortisolism screening.MethodsWe present a case in which topiramate induced dexamethasone metabolism, leading to a false positive on the DST.ResultsA 44-year-old female with an incidentally found adenoma in the right adrenal gland underwent a DST for hypercortisolism screening. The patient was taking topiramate prescribed by a psychiatrist for an affective disorder, and insufficient cortisol suppression (11.9 mcg/ dL) was observed. Her free cortisol in 24-hour urine was normal, and insufficient suppression was established in a second determination (9.3 mcg/dL). Finally, her psychiatrist switched her treatment from topiramate to bupropion, and the measurements were repeated. When she was not taking topiramate, correct suppression with 1 mg of dexamethasone was obtained (1.7 mcg/dL), and her free cortisol in 24-hour urine was again normal, thereby excluding the presence of hypercortisolism. On reviewing the literature, topiramate was not found to have been previously described as a cause of a false positive on DST, but it was proposed as a cause of hypoadrenalism in a patient taking oral corticosteroid replacement due to its capacity to induce dexamethasone metabolism.ConclusionTopiramate treatment may well be a cause of false positives in DSTs, and its presence should be taken into consideration when screening for hypercortisolism. (Endocr Pract. 2014;20:e116-e118)     

Quantification of the D2-Glycoprotein in Amniotic Fluid and Serum from Pregnancies with Fetal Neural Tube Defects

Abstract: D2 is a glycoprotein existing in both membrane-bound and soluble forms. Employing a specific rabbit antibody against purified human brain D2, we developed an enzyme-linked immunosorbent assay (ELISA) for the quantification of D2 and applied it to amniotic fluids from 87 normal and 36 pathological pregnancies. With a cut-off point of 150 ng D2/ml, no false positive D2 values were obtained in any of the amniotic fluids from normal fetuses, although the alpha-fetoprotein concentrations were slightly increased in 13 cases. No false negative D2 values were found in any of the 18 investigated amniotic fluids from fetuses with anencephaly. Of 8 amniotic fluids from fetuses with spina bifida, 2 false negative D2 values were found. No false negative alphafetoprotein values were found in any of the cases with neural tube defects in this study. In 10 amniotic fluids from fetuses with other malformations, 5 samples showed raised D2 concentrations. The D2 level in sera from 10 women carrying normal fetuses and 16 women carrying malformed fetuses was also determined, but no statistically significant difference in D2 level was found in the pathological sera when compared with normal sera. It was concluded that the determination of D2 concentrations in amniotic fluid by means of the D2-ELISA may be used as an additional test in the screening of fetal malformations in early pregnancy.     

Comparing disease screening tests when true disease status is ascertained only for screen positives

Disease screening is a fundamental part of health care. To evaluate the accuracy of a new screening modality, ideally the results of the screening test are compared with those of a definitive diagnostic test in a set of study subjects. However, definitive diagnostic tests are often invasive and cannot be applied to subjects whose screening tests are negative for disease. For example, in cancer screening, the assessment of true disease status requires a biopsy sample, which for ethical reasons can only be obtained if a subject's screening test indicates presence of cancer. Although the absolute accuracy of screening tests cannot be evaluated in such circumstances, it is possible to compare the accuracies of screening tests. Specifically, using relative true positive rate (the ratio of the true positive rate of one test to another) and relative false positive rate (the ratio of the false positive rates of two tests) as measures of relative accuracy, we show that inference about relative accuracy can be made from such studies. Analogies with case-control studies can be drawn where inference about absolute risk cannot be made, but inference about relative risk can. In this paper, we develop a marginal regression analysis framework for making inference about relative accuracy when only screen positives are followed for true disease. In this context factors influencing the relative accuracies of tests can be evaluated. It is important to determine such factors in order to understand circumstances in which one test is preferable to another. The methods are applied to two cancer screening studies, one concerning the effect of race on screening for prostate cancer and the other concerning the effect of tumour grade on the detection of cervical cancer with cytology versus cervicography screening.     

The toxin binding inhibition test as a reliable in vitro alternative to the toxin neutralization test in mice for the estimation of tetanus antitoxin in human sera

A method for the screening of human sera for tetanus antibodies has been developed and evaluated. The toxin binding inhibition test (ToBI-test) is based on inhibition of the binding of tetanus toxin to an antitoxin-coated immunoassay microtitre plate by tetanus antibodies. Serum samples from 191 healthy adults with different vaccination histories have been titrated for tetanus antibodies by the toxin neutralization (TN) test in mice, by toxoid-ELISA and by the ToBI-test. In every respect, the ToBI-test proved to be the best in vitro alternative to the TN-test in mice. Comparisons showed a higher degree of correlation between the ToBI-test and the TN-test than between the toxoid-ELISA and the TN-test. Furthermore, no overestimation of antibody content was seen in titrating low titre sera by the ToBI-test. In contrast, several false positive results were seen when using the toxoid-ELISA. It is concluded that the ToBI-test is a reliable and precise alternative to the TN-test and can be performed under simple laboratory conditions in a short time.     

Lovastatin effect on ergosterol production and growth of Tolypocladium inflatum 106]

The effect of lovastatin on Tolypocladium inflatum 106 was studied. The strain was shown to be highly sensitive to lovastatin when its MIC was determined by the agar diffusion method and under submerged conditions that was considered possible to use the strain as a test culture in screening of new natural compounds with hypolipidemic action and to study its specificity. It was demonstrated that the effect of lovastatin on ergosterol synthesis in T. inflatum 106 was of specific dose-dependent polymodal nature.     

Investigation of arcobacters in meat and faecal samples of clinically healthy cattle in Turkey

AIMS: To investigate the presence of Arcobacter spp. in minced beef meat (n = 97) and rectal faecal samples (n = 200) collected from cattle immediately after slaughter at a local abattoir in Turkey. METHODS AND RESULTS: Meat samples were examined using three different isolation procedures (CAT-supplemented media, de Boer arcobacter isolation method and membrane filtration method), but only one method (CAT-supplemented media) was employed for faecal samples. The isolated Arcobacter strains were identified by genus- and species-(multiplex) specific PCR assays. Arcobacter spp. were isolated from 5 and 9.5% of meat and faecal samples respectively. Although the only Arcobacter sp. found in meat samples was Arcobacter butzleri, all three pathogenic species--A. butzleri, A. cryaerophilus and A. skirrowii--were detected in the rectal swabs. No Arcobacter was isolated when the de Boer method was used for minced meat samples but the same five meat samples were found positive for arcobacters when CAT-supplemented media and membrane filtration method were used. CONCLUSIONS: The membrane filtration method was found to be superior to the CAT-supplemented media, because it led to a reduction in competing microflora. However, the necessity for one filter and medium for each sample makes this method somewhat expensive. The multiplex-PCR (m-PCR) assay shortened significantly the time required for the identification of Arcobacter spp. and also removed the possibility of false positive results due to other campylobacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports the isolation of Arcobacter spp. in cattle for the first time in Turkey. The m-PCR assay enables the identification and differentiation of all arcobacters simultaneously in one-step PCR.     

Overnight Dexamethasone Suppression Test: A Reliable Screen for Cushing's Syndrome in the Obese

Objective: Reevaluation of the validity of the 1-mg overnight dexamethasone suppression test (ODST) as a screening test for Cushing's syndrome in obese patients. Research Methods and Procedures: Eighty-six obese patients (body mass index, 30 to 53 kg/m²) that were referred to a general endocrine outpatient clinic for evaluation of simple obesity, diabetes mellitus, hypertension, polycystic ovary disease, or pituitary tumor. One milligram dexamethasone was administered orally at 11:00 pm , and serum cortisol levels were measured the following morning between 8:00 am and 9:00 am . Suppression of serum cortisol to <80 nM (3 μg/dL) was chosen as the cut-off point for normal suppression. Patients with serum cortisol levels ≥80 nM were evaluated for Cushing's syndrome. Results: Suppression of morning cortisol levels to <80 nM occurred in 79 of the 86 obese patients. Seven patients had serum cortisol levels higher than 80 nM; five were eventually diagnosed with Cushing's syndrome and two were considered false positive results in view of normal 24-hour free urinary cortisol and normal suppression on a low dose dexamethasone suppression test (0.5 mg of dexamethasone every 6 hours for 2 days). We found a false positive rate of 2.3% for the ODST using a cut-off serum cortisol of 80 nM. Discussion: The ODST is a valid screening test for Cushing's syndrome in the obese population. The false positive rate was 2.3%, even when using a strict cut-off serum cortisol of 80 nM. Abnormal cortisol suppression in obese patients should be investigated and not be considered false positive results.     

Screening of yeast strains for phytase activity

A screening method was developed to elucidate the ability of different yeast strains to utilize phytic acid as sole phosphorus source. The growth test in liquid culture in a microtiter plate with phytic acid as sole phosphorus source was shown to be a reliable, fast and easy-to-use screening method. We tested 122 strains from 61 species with our method and observed growth differences among species and strains that were not detectable on solid medium. Specific phytase activities were measured for 10 yeasts strains, selected due to their strong growth in the liquid medium. Strains of Arxula adeninivorans and Pichia anomala reached the highest volumetric phytase activities. Arxula adeninivorans also displayed the highest intra- and extracellular specific activities. There were large differences in both extra- and intracellular phytase activities among species. Strain-specific extracellular phytase activities were detected in P. anomala . The presence of free phosphate in the media completely suppressed the extracellular phytase activity and also reduced intracellular phytase activity for all tested yeast strains.     

Clinical evaluation of non-invasive prenatal screening in 32,394 pregnancies from Changzhi maternal and child health care hospital of Shanxi China

BackgroundNon-invasive prenatal screening (NIPS) is a highly sensitive and specific screening test to detect fetal chromosomal abnormalities. The primary objective of this study was to evaluate the NIPS as an effective method for prenatal detection of aneuploidies in both high-risk and low-risk pregnancies.MethodsIn current study, we performed NIPS in 32,394 pregnancies, out of which results were available in 32,361 (99.9%) of them. Illumina sequencing was performed for NIPS screening. Hypothesis Z test was used to classify fetal autosomal aneuploidy of T21, T18, and T13. Karyotyping was performed to determine the true negative and true positive NIPS results.ResultsAmong the 32,361 confirmed samples, 164 cases had positive results and 32197 cases had negative results. Of these positive cases, 116 cases were trisomy 21, 34 cases were trisomy 18 and 14 cases were trisomy 13. No false negative results were found in this cohort. The overall sensitivity and specificity were 100% and 99.91%, respectively. There was no significant difference in test performance between the 7,316 high-risk and 25,045 low-risk pregnancies, (sensitivity, 100% vs 100% (P>0.05); specificity, 99.96% vs 99.95% (P > 0.05)). Factors contributing to false-positive results included fetal copy number variants (CNVs), fetal mosaicism and typically producing Z scores between 3 and 4. Moreover, we analyzed NIPS wholegenome sequencing to investigate the Single Nucleotide Polymorphisms (SNPs) associations with drug response or risk of disease. As compare to the 1000g East Asian genome data, the results revealed a significant difference in 7,285,418 SNPs variants of Shanxi pregnant women including 19,293 clinvar recorded variants and 7,266,125 non-clinvar recorded.ConclusionsOur findings showed that NIPS was an effective assay that may be applied as routine screening for fetal trisomies in the prenatal setting. In addition, this study also provides an accurate assessment of significant differences in 7,285,418 SNPs variants in Shanxi pregnant women that were previously unavailable to clinicians in Shanxi population.     

Screening for possible human carcinogens and mutagens. False positives, false negatives: statistical implications

A screening method aimed at identifying potential human carcinogens using either animal cancer bioassays or short-term genotoxic assays has 4 possible results: true positive, true negative, false positive and false negative. Such a categorisation is superficially similar to the results of hypothesis testing in a statistical analysis. In this latter case the false positive rate is determined by the significance level of the test and the false negative rate by the statistical power of the test. Although the two types of categorisation appear somewhat similar, different statistical issues are involved in their interpretation. Statistical methods appropriate for the analysis of the results of a series of assays include the use of Bayes' theorem and multivariate methods such as clustering techniques for the selection of batteries of short-term test capable of a better prediction of potential carcinogens. The conclusions drawn from such studies are dependent upon the estimates of values of sensitivity and specificity used, the choice of statistical method and the nature of the data set. The statistical issues resulting from the analysis of specific genotoxicity experiments involve the choice of suitable experimental designs and appropriate analyses together with the relationship of statistical significance to biological importance. The purpose of statistical analysis should increasingly be to estimate and explore effects rather than for formal hypothesis testing.     

A Comparative Study of the Triphenyltetrazolium Chloride (Uroscreen) Test and Conventional Methods for the Detection of Bacteriuria

Acute urinary tract infection may be preceded by and active pyelonephritis may be associated with asymptomatic bacteriuria. Treatment of asymptomatic bacteriuria may prevent or arrest active, chronic pyelonephritis and its sequelae. Consequently, there is a need for a reliable and simple screening procedure to detect asymptomatic bacteriuria in large segments of the population.The reliability and practicability of tests advocated for the detection of bacteriuria, including the new chemical triphenyltetrazolium chloride (T.T.C.) (Uroscreen) test, were evaluated. Reliability was assessed by correlating results of these tests with bacterial counts of tested urines. Significant bacteriuria is defined as the presence of 100,000 or more organisms per ml. of urine.The T.T.C. (Uroscreen) test was positive in 92.5% of cases of bacteriuria; there were 7.5% false-negative and 2.8% false-positive results. Bacteria on Gram-stained smear were found in 95.5% of the cases of bacteriuria and in 14.6% of those with non-infected urine; pyuria (more than three leukocytes per high-power field), in 60% of those with bacteriuria and in 15.9% of those with presumably non-infected urine. Bacteria were conspicuous in the urinary sediment in 91.1% of cases of bacteriuria and in 3.7% of presumably non-infected urines.The T.T.C. (Uroscreen) test fulfilled the criteria for a reliable and simple screening procedure. It should be used concomitantly with other screening tests when the urine is examined routinely.     

Counter Current Line Absorption Immunoelectrophoresis is an Alternative Diagnostic Screening Test to Counter Current Immunoelectrophoresis in Aleutian Disease (AD) Eradication Programs

Counter current immunoelectrophoresis (CCIE) is the diagnostic method used in the ongoing Aleutian disease virus eradication program on Danish mink farms. There has been an increasing demand for an alternative diagnostic test especially to evaluate suspected false positive CCIE reactions. We compared test results of a number of negative and positive mink sera in indirect counter current immunoelectrophoresis (ICCIE), counter current line absorption immunoelectrophoresis (CCLAIE) and radio immuno assay (RIA) with test results from counter current immunoelectrophoresis and found that counter current line absorption immunoelectrophoresis is the best alternative diagnostic screening test to counter current immunoelectrophoresis for Aleutian disease eradication programs. Not only proved the CCLAIE test to be useful for evaluation of doubtfully positive CCIE reactions, but it was found to have a higher sensitivity than the CCIE test.     

Neonatal screening for cystic fibrosis: result of a pilot study using both immunoreactive trypsinogen and cystic fibrosis gene mutation analyses

We have evaluated a two-tier neonatal cystic fibrosis (CF) screening of immunoreactive trypsinogen (IRT) followed by CFTR gene mutation analysis using a systematic scanning of exons 7, 10, and 11, and, if necessary, by direct DNA sequencing. Over an 18-month period we screened 32,300 neonates born in the western part of Britanny. The first tier, involving IRT screening at 3 days of age, utilizes a low elevation of the trypsinogen level (600 ng/ml), which is highly sensitive. The second tier, which corresponds to the exhaustive screening for mutations in three exons of the gene, is highly specific for this population (Britanny). The false positive rate is very low, and no false negatives have been reported to date. This strategy has allowed the identification of five novel alleles (V322A, V317A, 1806 del A, R553G, G544S). Moreover the test can be adapted to other countries in which the distribution of mutations is established.     

The Laboratory Diagnosis of Whooping Cough by Fluorescent Antibody and by Culture Methods

Attempts were made to hasten the identification of Bordetella pertussis by using the fluorescent antibody (FA) technique and to improve the isolation rate by culture methods. First, a comparison was made between the number of identifications of the organism by growth on Bordet-Gengou medium containing penicillin G and by staining smears of pharyngeal exudate with FA. Of 100 specimens, 29 were positive on culture and 46 were positive by the FA method. Five of the 46 were false positives. Second, 170 specimens were inoculated in quadruplicate on Bordet-Gengou medium containing either penicillin G or methicillin, and on Lacey medium containing either penicillin G or methicillin. In order there were 39, 41, 56 and 57 isolations. The isolation of Bord. pertussis on methicillin-containing media was easier than on penicillin-containing media. When culture and FA methods were compared, the most reliable was found to be the inoculation of specimens on Lacey medium.     

Bacteriological Examination of Unbottled Soft Drinks

A study of 300 samples, representing 14 different unbottled drinks, indicated that there are three vitally important criteria pertaining to their bacteriological examination. First, the total viable counts may be better accomplished by the pour-plate method, using enriched media, with incubation at either 30 or 37 C. Second, a comparative study of the coliaerogenes group and the enterococci as indices of pollution unquestionably favors the latter as the reliable indicator, owing to false interpretations of the presumptive test and to lack of accurate definition of fecal and nonfecal coliforms recovered from positive cases. The use of enterococci, however, did not provide as reliable an indicator as the pour-plate method. Third, the results with enterococci, in defining the probable source of pollution, are more precise. Experiments judiciously selected and simultaneously conducted revealed that the heat and heat-tellurite resistance tests, and the tetrazolium-reduction test, matched in relating 98.9% of available enterococci to an animal source. Negligible but vital discrepancies were obtained with the two odd strains which qualified as human-derived according to the heat and heat-tellurite resistance tests. The differential criterion of Skadhauge and Barnes, based on the failure of animal-derived enterococci to grow in the presence of a low concentration of potassium tellurite, did not apply to the other two methods, since 99.5% of the recovered strains were found tolerant to the specified tellurite concentration.     

Evaluation of new culture media for rapid detection and isolation of salmonellae in foods.

Conventional methods for Salmonella detection in foods can require up to 6 and at least 4 days. We have observed that the total analysis time can be reduced to 48 h by using Salmosyst broth as a liquid medium for both preenrichment and selective enrichment and Rambach agar (RA), a new selective plate medium. In samples of artificially contaminated ground beef Salmonella enteritidis was detected at a concentration of 0.4 CFU/g (10 CFU/25 g) by both a conventional method and the new method. Of 519 samples of foods for sale, 38 were Salmonella positive by both methods while 471 were negative. Nine samples which were negative by the conventional method were positive by the Salmosyst-RA method, while one sample positive by the first method was negative by the last. Therefore, the Salmosyst-RA method showed 97.9% sensitivity compared with the 81.2% sensitivity of the conventional method. The new method was also highly specific (98% specificity) in presumptive identification of Salmonella colonies. Furthermore, a 6-h preenrichment in Salmosyst broth has been proved sufficient for the repair of heat-injured Salmonella cells and for subsequent recovery by selective enrichment. In conclusion, the Salmosyst-RA method shows several advantages over both conventional and rapid noncultural methods: (i) only two media are required instead of the five media for conventional methods; (ii) in real time it is comparable to other rapid noncultural methods, which require 30 to 31 h; (iii) it is highly sensitive and specific; and (iv) it allows the isolation of Salmonella strains which can be characterized by appropriate phenotypic and genotypic typing methods for epidemiological investigations.     

Evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular level

Detecting positive Darwinian selection at the DNA sequence level has been a subject of considerable interest. However, positive selection is difficult to detect because it often operates episodically on a few amino acid sites, and the signal may be masked by negative selection. Several methods have been developed to test positive selection that acts on given branches (branch methods) or on a subset of sites (site methods). Recently, Yang, Z., and R. Nielsen (2002. Codon-substitution models for detecting molecular adaptation at individual sites along specific lineages. Mol. Biol. Evol. 19:908-917) developed likelihood ratio tests (LRTs) based on branch-site models to detect positive selection that affects a small number of sites along prespecified lineages. However, computer simulations suggested that the tests were sensitive to the model assumptions and were unable to distinguish between relaxation of selective constraint and positive selection (Zhang, J. 2004. Frequent false detection of positive selection by the likelihood method with branch-site models. Mol. Biol. Evol. 21:1332-1339). Here, we describe a modified branch-site model and use it to construct two LRTs, called branch-site tests 1 and 2. We applied the new tests to reanalyze several real data sets and used computer simulation to examine the performance of the two tests by examining their false-positive rate, power, and robustness. We found that test 1 was unable to distinguish relaxed constraint from positive selection affecting the lineages of interest, while test 2 had acceptable false-positive rates and appeared robust against violations of model assumptions. As test 2 is a direct test of positive selection on the lineages of interest, it is referred to as the branch-site test of positive selection and is recommended for use in real data analysis. The test appeared conservative overall, but exhibited better power in detecting positive selection than the branch-based test. Bayes empirical Bayes identification of amino acid sites under positive selection along the foreground branches was found to be reliable, but lacked power.     

A model‐based solution for observational errors in laboratory studies

Molecular techniques for detecting microorganisms, macroorganisms and infectious agents are susceptible to false‐negative and false‐positive errors. If left unaddressed, these observational errors may yield misleading inference concerning occurrence, prevalence, sensitivity, specificity and covariate relationships. Occupancy models are widely used to account for false‐negative errors and more recently have even been used to address false‐positive errors, too. Current modelling options assume false‐positive errors only occur in truly negative samples, an assumption that yields biased inference concerning detection because a positive sample could be classified as such not because the target agent was successfully detected, but rather due to a false‐positive test result. We present an extension to the occupancy modelling framework that allows false‐positive errors in both negative and positive samples, thereby providing unbiased inference concerning occurrence and detection, as well as reliable conclusions about the efficacy of sampling designs, handling protocols and diagnostic tests. We apply the model to simulated data, showing that it recovers known parameters and outperforms other approaches that are commonly used when confronted with observation errors. We then apply the model to an experimental data set on Batrachochytrium dendrobatidis, a pathogenic fungus that is implicated in the global decline or extinction of hundreds of amphibian species. The model‐based approach we present is not only useful for obtaining reliable inference when data are contaminated with observational errors, but also eliminates the need for establishing arbitrary thresholds or decision rules that have hidden and unintended consequences.