Performance of the new automated Abbott RealTime MTB assay for rapid detection of Mycobacterium tuberculosis complex in respiratory specimens.

The automated high-throughput Abbott RealTime MTB real-time PCR assay has been recently launched for Mycobacterium tuberculosis complex (MTBC) clinical diagnosis. This study would like to evaluate its performance. We first compared its diagnostic performance with the Roche Cobas TaqMan MTB assay on 214 clinical respiratory specimens. Prospective analysis of a total 520 specimens was then performed to further evaluate the Abbott assay. The Abbott assay showed a lower limit of detection at 22.5 AFB/ml, which was more sensitive than the Cobas assay (167.5 AFB/ml). The two assays demonstrated a significant difference in diagnostic performance (McNemar's test; P = 0.0034), in which the Abbott assay presented significantly higher area under curve (AUC) than the Cobas assay (1.000 vs 0.880; P = 0.0002). The Abbott assay demonstrated extremely low PCR inhibition on clinical respiratory specimens. The automated Abbott assay required only very short manual handling time (0.5 h), which could help to improve the laboratory management. In the prospective analysis, the overall estimates for sensitivity and specificity of the Abbott assay were both 100 % among smear-positive specimens, whereas the smear-negative specimens were 96.7 and 96.1 %, respectively. No cross-reactivity with non-tuberculosis mycobacterial species was observed. The superiority in sensitivity of the Abbott assay for detecting MTBC in smear-negative specimens could further minimize the risk in MTBC false-negative detection. The new Abbott RealTime MTB assay has good diagnostic performance which can be a useful diagnostic tool for rapid MTBC detection in clinical laboratories.

Stability of methylecgonidine and ecgonidine in sheep plasma in vitro.

BACKGROUND: Crack smokers are exposed to a pyrolysis product, methylecgonidine (MEG), which can be used as an analytical marker for crack smoking. Ecgonidine (EC), a hydrolytic product of MEG, has been identified in urine of crack smokers. MEG undergoes conversion to EC, complicating analysis and perhaps explaining a lack of forensic blood specimens containing MEG. METHODS: We developed gas chromatography-mass spectrometry (GC-MS) assays for MEG and EC. Plasma was collected from sheep blood containing 0, 0.06, or 0.24 mol/L (0%, 0.25%, or 1%) NaF. MEG was added to these plasmas, and they were incubated at -80, 1, 21, or 37 degrees C to determine whether there were temporal, temperature, or storage effects on MEG stability over 48 h. RESULTS: Decreased temperature and increased NaF concentrations limited MEG degradation and EC formation. MEG stored in plasma at -80 degrees C was stable up to 1 month, even in the absence of NaF. CONCLUSIONS: MEG is stable in sheep plasma collected in commercially available, evacuated blood-collection tubes containing NaF and stored at -80 degrees C. In vitro formation of EC can be minimized with appropriate sample handling, and its in vivo formation may provide a better marker of crack smoking than its parent pyrolysis product.

Detection of drug use during pregnancy.

Several methods of drug testing are efficacious in identifying and monitoring drug use during pregnancy. Urine screening remains the most commonly used method despite the limited period during which drugs can be detected. Hair has been recognized as a possible alternate test specimen, but wider acceptance of hair testing must await better understanding of drug disposition in hair, answers to the issues relating to interpretation, and the development of less demanding laboratory techniques. Regardless of the matrix used, proper interpretation of the results of drug testing requires familiarity with the sensitivity, specificity, and limitations of the laboratory methodologies employed. Moreover, unconfirmed positive results may actually be false-positives and must be interpreted with caution, particularly if they are the basis for major clinical decisions.

Drugs of abuse testing in a psychiatric outpatient service.

Drug testing of patients in a psychiatric outpatient service is an effective way to identify patients who relapse into renewed use of drugs of abuse and in monitoring the effectiveness of ongoing medical and psychological therapy. Most of this testing involves the analysis of urine specimens with immunoassays. Hair testing affords an alternative specimen matrix that is easy to obtain and not readily adulterated and offers the advantage of a wider surveillance window. Hair analysis is technically demanding, and the possibility of false-positives caused by environmental contamination renders it a controversial alternative. Sweat and saliva are potentially useful testing matrices, but their usefulness in clinical practice must await validation by additional clinical and laboratory experience. The correct interpretation of drug test results is predicated on knowing the performance characteristics of the analytical method, route of administration, and pharmacokinetics of the drug. All questionable positive results need confirmation testing to verify true positivity.

Detection of intrauterine illicit drug exposure by newborn drug testing. National Academy of Clinical Biochemistry.

Identification of intrauterine drug-exposed newborns with toxicological screening may have benefits including close follow-up of the infant by both medical and social services. Applying specific written guidelines to select newborns for drug testing decreases bias and protects the physicians and hospitals involved. All drugs reported as positive should be confirmed by an appropriate second test. Urine and meconium testing are the best current options for identifying drug-exposed neonates. Urine testing sensitivity is low because of problems encountered in urine collections and the high thresholds used in current urine assays. The disadvantage to meconium testing is the increased labor and time required to work with this material. Testing of newborn hair is unlikely to be widely used until technically less demanding assays become available. Testing of amniotic fluid or gastric lavage is still in the developmental stages. Adopting lower urine assay thresholds for newborn samples would increase sensitivity and would be an appropriate modification of current methodologies.

Inhibition of apolipoprotein B net synthesis and secretion from cultured rat hepatocytes by the calcium-channel blocker diltiazem.

1. The effect of the Ca2+-channel blocker diltiazem on hepatic apolipoprotein B (apo B) synthesis and secretion was studied in 12-18 h cultures of collagenase-dispersed rat hepatocytes. 2. The presence of diltiazem in the medium decreased apo B secretion by hepatocytes in a concentration-dependent manner. At 25 microM, diltiazem inhibited apo B secretion by approx. 36%, but there was no evidence of intracellular accumulation of apo B. 3. The inhibition of apo B secretion by hepatocytes was significantly correlated with cell-associated diltiazem (r = 0.72, P less than 0.01). 4. The rate of apo B secretion remained linear over 16 h even in the presence of 50 microM-diltiazem. 5. At diltiazem concentrations in the medium which were inhibitory for apo B secretion, [14C]acetate incorporation into cellular lipids and [35S]methionine incorporation into protein were enhanced. 6. Diltiazem inhibited the secretion of the apo B variants with a preferential inhibition of the higher-molecular-mass form of apo B (apo BH) over the lower-molecular-mass form (apo BL) at diltiazem concentrations in the medium greater than 25 microM. 7. Together, these results suggest that Ca2+ may play an important role in the synthesis and secretion of apo B-containing lipoproteins.

Monitoring of anti-inflammatory drugs.

Advances in analytic technology have allowed clinical laboratories to accurately measure plasma levels of NSAIDs. With the exception of salicylate, however, there is no relationship, or only a weak one, between plasma NSAID levels and clinical responses. Therefore, therapeutic drug monitoring of NSAIDs other than salicylate is of little value to clinicians in their management of patients with rheumatoid arthritis. On occasion, however, it may be helpful to monitor specific patients suspected of noncompliance by measuring plasma drug levels. Routine therapeutic drug monitoring of these NSAIDs must await further work on the refinement of quantitating clinical response and the possible relationship between free drug level and drug effects.

Salicylate measurement: clinical usefulness and methodology.

The salicylates are the most commonly used analgesic, antipyretic, and anti-inflammatory drugs. They are available in hundreds of preparations, many of which are over-the-counter medications. The easy access to large quantities of the drug and the widespread perception that the drug is harmless have contributed to salicylate intoxication becoming a serious and common problem, particularly among the pediatric and geriatric populations. Salicylate is still the major drug for the treatment of rheumatic diseases. The use of salicylate in high doses for the management of these patients requires close monitoring of serum salicylate levels because of the large interindividual variation in dose-serum level relationships and the narrowness of the therapeutic range. Thus, both for the management of patients intoxicated with salicylate and patients who are on high-dose salicylate therapy, the measurement of serum salicylate levels is an important clinical laboratory service. Recent research on the inhibitory effect of aspirin on platelet aggregation has led to the prophylactic use of aspirin in low doses as an antithrombotic drug. This new therapeutic use of aspirin can be aided by monitoring low serum levels of salicylate and perhaps aspirin itself. This article reviews the current state of the knowledge of the pharmacokinetics and clinical toxicology of salicylate, the clinical usefulness of salicylate measurement by the clinical laboratory, and recent development in the analytical technology for salicylate analysis.

Lipoprotein and protein binding of the calcium channel blocker diltiazem.

The plasma protein binding properties of the calcium channel blocker diltiazem were studied using a three-chamber equilibrium dialysis system. Diltiazem is 81% bound to human sera with significant inter-individual variation. The relative binding of diltiazem by lipoproteins and alpha 1-acid glycoprotein was higher than by albumin. The binding to low density lipoprotein was strong and appeared not to be associated with the surface apoprotein.

Activities of some enzymes in serum after therapy with intracoronary streptokinase in acute myocardial infarction.

Enzyme kinetics for creatine kinase (CK), CK-MB, aspartate aminotransferase (AST), and lactate dehydrogenase (LD) in serum were followed in 14 patients who had suffered acute myocardial infarction and who were given intracoronary streptokinase shortly (mean 4.9 h, SD 2.6 h) after onset of symptoms. In the 10 patients for whom thrombolysis was successful, CK activity peaked earlier (12.8 vs 21.6 h) and at higher values (3548 vs 2436 U/L) than in the four patients for whom the treatment was unsuccessful. The mean maximum rate of increase in CK was threefold greater in the former group (574 vs 169 U/L per hour), but the total amount of CK released into the circulation and the fractional disappearance rates were similar for both groups. The profiles for AST and CK-MB for successfully treated patients closely resembled those for CK. LD, however, peaked significantly later than CK (25.7 vs 12.8 h). Early peaking of CK or CK-MB after nonsurgical reperfusion can be potentially useful as a noninvasive in vitro index to the success of therapy of myocardial infarction with thrombolytic agents.

Self-poisoning with enteric-coated aspirin.

A case of self-poisoning by enteric-coated aspirin tablets is described. Absorption of salicylate was delayed, resulting in serum kinetics different from that obtained with regular aspirin. When the ingested aspirin is enteric coated, the use of Done's nomogram may be inappropriate.

Clinical evaluation of a radiometric assay specific for creatine kinase isoenzyme MB.

A new radiometric assay specific for creatine kinase isoenzyme MB was evaluated with respect to its precision and agreement with a conventional electrophoretic CK-MB assay for the diagnosis of myocardial infarction. The reference interval we find for serum CK-MB in healthy subjects is 0--30 micrograms/L. The coefficients of variation at 197 and 40 micrograms of CK-MB per liter, were 5.2 and 11.5%, respectively. In a clinical study of 52 consecutive patients admitted into a Coronary Care Unit with a diagnosis of suspected myocardial infarction, there was overall agreement in CK-MB results by the two assays for 51 of 52 patients. A more sensitive and quantitative assay that is specific for CK-MB can be helpful in cases where diagnosis of myocardial infarction is equivocal.

Wheat embryo ribonucleates. IX. Generation of N2-dimethylguanylate when bulk wheat embryo tRNA is used as substrate for wheat embryo S-adenosylmethionine-tRNA methyltransferases, in vitro.

Although the cellular ribonucleates in normally growing cells are virtually saturated with respect to their customary complement of methyl substituents, it has often been reported that 'marginal' levels of (homologous) methylation can be detected when ribonucleates and enzymes from the same source material are incubated, together with S-adenosylmethionine, in vitro. Experiments were designed to acquire new insights that might be useful for circumscribing the number of possible interpretations that could be advanced to account for the introduction of 'supernumerary' methyl groups during (homologous) methylation of wheat RNA by wheat enzymes, in vitro. For a large fraction of the supernumerary methyl groups that can be introduced into wheat RNA, in vitro, it was not possible to adduce convincing evidence in support of the view that any appreciable quantity of methyl groups is ever introduced at these same sites, in vivo. The possibility that these supernumerary methyl groups might have transient existence, in vivo, and the potential physiological significance of any such occurrence are dealt with as part of a more general discussion of the experimental findings.

Wheat embryo ribonucleates. V. Generation of N2--dimethylguanylate when 'fully sequenced' homogeneous species of transfer RNA are used as substrates for wheat embryo methyltransferases.

When S-adenosly[methyl-14-C]methionine and various species of transfer RNA are used as substrates for wheat embryo methyltransferases, the principal site of guanylate-N-2 methylation can be shown to be a G-residue between the stems of the dihydrouridine and anticodon loops. This common site of guanylate-N-2 methylation is referred to as the interstem target site. 2. When the interstem target site is the non-terminal G-residue in a G-C-G-C sequence, as in the cases of Escherichia coli tRNA1-Leu, tRNA-Ile, and tRNA3-Ser, there is preponderant dimethylation to yield N-2-dimethylguanylate. 3. When the interstem target site is part of a U-C-G-U sequence, as in the case of E. coli tRNAf-Met, there is diminished dimethylation and correspondingly increased monomethylation to yield N-2-monomethylguanylate. 4. When the interstem target site is the non-terminal G-residue in an A-U-G-G sequence, as in the case of yeast tRNA-Asp, there is negligible dimethylation and almost exclusive monomethylation to yield N-2-monomethylguanylate. 5. The concerted way in which the primary, secondary, and tertiary structures of tRNA molecules might influence the efficacy of these methylations is the subject of a brief discussion. Attention is also focused on the evolutionary and molecular basis for the generally non-random distributions of methylated oligonucleotide sequences in ribosomal and transfer ribonucleates.

Isolation and partial characterization of three Escherichia coli mutants with altered transfer ribonucleic acid methylases.

Seven transfer ribonucleic acid (tRNA) methylase mutants were isolated from Escherichia coli K-12 by examining the ability of RNA prepared from clones of unselected mutagenized cells to accept methyl groups from S-adenosylmethionine catalyzed by crude enzymes from wild-type cells. Five of the mutants had an altered uracil-tRNA methylase; consequently their tRNA's lacked ribothymidine. One mutant had tRNA deficient in 7-methylguanosine, and one mutant contained tRNA lacking 2-thio-5-methylaminomethyluridine. The genetic loci of the three tRNA methylase mutants were distributed over the E. coli genome. The mutant strain deficient in 7-methylguanosine biosynthesis showed a reduced efficiency in the suppression of amber mutations carried by T4 or lambda phages.