Simulation-based assessment of anesthesiology residents' competence: development and implementation of the Canadian National Anesthesiology Simulation Curriculum (CanNASC).

The specialty of anesthesiology will soon adopt the Competence By Design (CBD) approach to residency education developed by the Royal College of Physicians and Surgeons of Canada (RCPSC). A foundational component of CBD is frequent and contextualized assessment of trainees. In 2013, the RCPSC Anesthesiology Specialty Committee assembled a group of simulation educators, representing each of the 17 Canadian anesthesiology residency programs, to form the Canadian National Anesthesiology Simulation Curriculum (CanNASC) Task Force. The goals were to develop, implement, and evaluate a set of consensus-driven standardized mannequin-based simulation scenarios that every trainee must complete satisfactorily prior to completion of anesthesiology residency and certification. Curriculum development followed Kern's principles and was accomplished via monthly teleconferences and annual face-to-face meetings. The development and implementation processes included the following key elements: 1) Curriculum needs assessment: 368 of 958 invitees (38.4%) responded to a national survey resulting in 64 suggested scenario topics. Use of a modified Delphi technique resulted in seven important and technically feasible scenarios. 2) Scenario development: All scenarios have learning objectives from the National Curriculum for Canadian Anesthesiology Residency. Standardized scenario templates were created, and the content was refined and piloted. 3) Assessment: A validated Global Rating Scale (GRS) is the primary assessment tool, informed by using scenario-specific checklists (created via a modified Delphi technique) and the Anesthesia Non-Technical Skills GRS. 4) Implementation: Standardized implementation guidelines, pre-brief/debrief documents, and rater training videos, guide, and commentary were generated. National implementation of the scenarios and program evaluation is currently underway. It is highly feasible to achieve specialty-based consensus on the elements of a national simulation-based curriculum. Our process could be adapted by any specialty interested in implementing a simulation-based curriculum incorporating competency-based assessment on a national scale.

Assessing the clinical value of targeted massively parallel sequencing in a longitudinal, prospective population-based study of cancer patients.

INTRODUCTION: Recent discoveries in cancer research have revealed a plethora of clinically actionable mutations that provide therapeutic, prognostic and predictive benefit to patients. The feasibility of screening mutations as part of the routine clinical care of patients remains relatively unexplored as the demonstration of massively parallel sequencing (MPS) of tumours in the general population is required to assess its value towards the health-care system. METHODS: Cancer 2015 study is a large-scale, prospective, multisite cohort of newly diagnosed cancer patients from Victoria, Australia with 1094 patients recruited. MPS was performed using the Illumina TruSeq Amplicon Cancer Panel. RESULTS: Overall, 854 patients were successfully sequenced for 48 common cancer genes. Accurate determination of clinically relevant mutations was possible including in less characterised cancer types; however, technical limitations including formalin-induced sequencing artefacts were uncovered. Applying strict filtering criteria, clinically relevant mutations were identified in 63% of patients, with 26% of patients displaying a mutation with therapeutic implications. A subset of patients was validated for canonical mutations using the Agena Bioscience MassARRAY system with 100% concordance. Whereas the prevalence of mutations was consistent with other institutionally based series for some tumour streams (breast carcinoma and colorectal adenocarcinoma), others were different (lung adenocarcinoma and head and neck squamous cell carcinoma), which has significant implications for health economic modelling of particular targeted agents. Actionable mutations in tumours not usually thought to harbour such genetic changes were also identified. CONCLUSIONS: Reliable delivery of a diagnostic assay able to screen for a range of actionable mutations in this cohort was achieved, opening unexpected avenues for investigation and treatment of cancer patients.

Mutational profiling of familial male breast cancers reveals similarities with luminal A female breast cancer with rare TP53 mutations.

BACKGROUND: Male breast cancer (MBC) is still poorly understood with a large proportion arising in families with a history of breast cancer. Genomic studies have focused on germline determinants of MBC risk, with minimal knowledge of somatic changes in these cancers. METHODS: Using a TruSeq amplicon cancer panel, this study evaluated 48 familial MBCs (3 BRCA1 germline mutant, 17 BRCA2 germline mutant and 28 BRCAX) for hotspot somatic mutations and copy number changes in 48 common cancer genes. RESULTS: Twelve missense mutations included nine PIK3CA mutations (seven in BRCAX patients), two TP53 mutations (both in BRCA2 patients) and one PTEN mutation. Common gains were seen in GNAS (34.1%) and losses were seen in GNAQ (36.4%), ABL1 (47.7%) and ATM (34.1%). Gains of HRAS (37.5% vs 3%, P=0.006), STK11 (25.0% vs 0%, P=0.01) and SMARCB1 (18.8% vs 0%, P=0.04) and the loss of RB1 (43.8% vs 13%, P=0.03) were specific to BRCA2 tumours. CONCLUSIONS: This study is the first to perform high-throughput somatic sequencing on familial MBCs. Overall, PIK3CA mutations are most commonly seen, with fewer TP53 and PTEN mutations, similar to the profile seen in luminal A female breast cancers. Differences in mutation profiles and patterns of gene gains/losses are seen between BRCA2 (associated with TP53/PTEN mutations, loss of RB1 and gain of HRAS, STK11 and SMARCB1) and BRCAX (associated with PIK3CA mutations) tumours, suggesting that BRCA2 and BRCAX MBCs may be distinct and arise from different tumour pathways. This has implications on potential therapies, depending on the BRCA status of MBC patients.

Complications of endotracheal intubation in the critically ill.

OBJECTIVE: Assess the risk of complications during endotracheal intubation (ETI) and their association with the skill level of the intubating physician. DESIGN: Prospective cohort study of 136 patients intubated by the intensive care team during a 5-month period. Standardized data forms were used to collect detailed information on the intubating physicians, supervisors, techniques, medications and complications. SETTING: Canadian academic intensive care unit. MEASUREMENTS AND RESULTS: All intubations were successful and there were no deaths during intubation. Non-experts were supervised in 92% of procedures. Expert operators were successful within two attempts in 94%, compared to only 82% of non-experts (P = 0.03), with 13.2% of all intubations requiring > or =3 attempts. Furthermore, 10.3% of intubations required 10 or more minutes. Difficult intubation (3 or more attempts by an expert) occurred in 6.6%. Overall risk of complications was 39%, including: severe hypoxemia (19.1%), severe hypotension (9.6%), esophageal intubation (7.4%) and frank aspiration (5.9%). ICU and hospital mortality were 15.4 and 29.4%, respectively. Compared with non-expert intubating physicians, propensity score-adjusted odds ratios (95% confidence interval) for expert physicians were 0.92 (95% CI: 0.28, 3.05, P = 0.89) for any complication, 0.45 (95% CI: 0.09, 2.20, P = 0.32) for ICU mortality and 0.47 (95% CI: 0.13, 1.70, P = 0.25) for hospital mortality. Two or more attempts at ETI was independently associated with an increased risk of severe complications (OR 3.31, 95% CI: 1.30, 8.40, P = 0.01). CONCLUSIONS: These prospective data show a high risk of serious complications, and difficult intubations, that are associated with ETI of the critically ill. DESCRIPTOR: Artificial airways and complications.

An infusion program resource nurse consult service: our experience in a major Canadian teaching hospital.

Infusion resource teams are comprised of nurses specially trained and experienced in infusion therapy. Our multidisciplinary team provides clinical, educational, and research support to a 1000-bed Canadian tertiary hospital. To characterize the infusion resource nurse service, 789 recorded consults for 250 patients during a 12-month period study were reviewed. Noncritical medicine and surgical wards accounted for a similar number of consults, with the highest volume (31% of total consults) being generated by the general and vascular surgery wards. Vein status was visible and either "fair" or "good" in approximately half of all consults, but 39% of consults were visible and "poor." Most consults (81% of total) resulted in the initiation of peripheral intravenous catheters into an area of nonflexion in an upper extremity and successful peripheral catheter initiations were accomplished in 96% of all cases. Our multidisciplinary infusion program approach to vascular access support appears to be a well-utilized and an effective resource for this hospital.

Characteristic hydrogen concentrations for various redox processes in batch study.

The dissolved hydrogen concentrations under various redox processes were investigated based on batch experiments. Chloroethenes including tetrachloroethene (PCE), cis-dichloroethene (cis-DCE) and vinylchloride (VC) were respectively used as culture substrates. For each chloroethene, a series of bottles were prepared with the additions of different electron acceptors or donors such as nitrate, manganese oxide, ferrous iron, sulfate, carbondioxide and volatile fatty acids. Hydrogen concentrations as well as redox species were measured over time to ensure the achievements of characteristic hydrogen levels in various enrichment batches. The results showed that redox processes with nitrate, manganese oxide and ferric iron as the electron acceptors exhibited hydrogen threshold values close to PCE/TCE dechlorination, whereas cis-DCE and VC dechlorinations exhibited hydrogen threshold values in the range of sulfate reduction and methanogenesis, respectively. Characteristic hydrogen concentrations for various redox processes were as follows (nM): denitrification, 0.1-0.4; manganese reduction, 0.1-2.0; iron reduction, 0.1-0.4; sulfate reduction, 1.5-4.5; methanogenesis, 2.5-24; PCE/TCE dechlorination, 0.6-0.9; eis-DCE dechlorination, 0.1-2.5; and VC dechlorination, 2-24.

Halohydrin dehalogenases are structurally and mechanistically related to short-chain dehydrogenases/reductases.

Halohydrin dehalogenases, also known as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases, catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl function in halohydrins to yield epoxides. Three novel bacterial genes encoding halohydrin dehalogenases were cloned and expressed in Escherichia coli, and the enzymes were shown to display remarkable differences in substrate specificity. The halohydrin dehalogenase of Agrobacterium radiobacter strain AD1, designated HheC, was purified to homogeneity. The k(cat) and K(m) values of this 28-kDa protein with 1,3-dichloro-2-propanol were 37 s(-1) and 0.010 mM, respectively. A sequence homology search as well as secondary and tertiary structure predictions indicated that the halohydrin dehalogenases are structurally similar to proteins belonging to the family of short-chain dehydrogenases/reductases (SDRs). Moreover, catalytically important serine and tyrosine residues that are highly conserved in the SDR family are also present in HheC and other halohydrin dehalogenases. The third essential catalytic residue in the SDR family, a lysine, is replaced by an arginine in halohydrin dehalogenases. A site-directed mutagenesis study, with HheC as a model enzyme, supports a mechanism for halohydrin dehalogenases in which the conserved Tyr145 acts as a catalytic base and Ser132 is involved in substrate binding. The primary role of Arg149 may be lowering of the pK(a) of Tyr145, which abstracts a proton from the substrate hydroxyl group to increase its nucleophilicity for displacement of the neighboring halide. The proposed mechanism is fundamentally different from that of the well-studied hydrolytic dehalogenases, since it does not involve a covalent enzyme-substrate intermediate.

Complex dynamics of adaptation in a nonaxenic microcystis culture. 2. Computer simulation of dinotrophenol effects.

A hypothesis was modeled to account for complex 20-day dynamics in a culture of blue-green algae Microcystis and heterotrophic bacteria exposed to 2,4-dinitrophenol (DNP). In trials with little or no added DNP, a limiting factor (light or CO(2)) may cause algal density to fluctuate after 14 days of increase. Such factors may be unimportant at levels of DNP that restrict photosynthesis. Bacterial growth may be limited by organic substrate, and bacteria may be more resistant to DNP than blue-green algae. Hence, at intermediate levels of DNP, substrate provided by increased algal death stimulates bacterial growth more than DNP retards it, causing a bacterial peak. Sorption of DNP to cells may cause the DNP decline. Greater growth and slower DNP decline in experiments with preexposed organisms indicate lower DNP sorption affinity in preexposed cells. Bacterial assimilation of DNP-containing substrate may cause the reappearance of DNP. The model reproduced the fluctuation in algal density after growth was limited and better growth and lower DNP decline with preexposed organisms. Reappearance of DNP occurred, but was not obvious. Bacterial dynamics were least well reproduced. Changes in bacterial constants most affected output. Despite model inadequacies, probable aspects of toxicant action in nature have been revealed. Ecological relationships among populations of different species and genetic differences among individuals may have led to lower than expected toxicity, adaptation, and even growth stimulation. Responses of single species tested in isolation may be inadequate to predict toxicant impact.

Haloalkane-utilizing Rhodococcus strains isolated from geographically distinct locations possess a highly conserved gene cluster encoding haloalkane catabolism.

The sequences of the 16S rRNA and haloalkane dehalogenase (dhaA) genes of five gram-positive haloalkane-utilizing bacteria isolated from contaminated sites in Europe, Japan, and the United States and of the archetypal haloalkane-degrading bacterium Rhodococcus sp. strain NCIMB13064 were compared. The 16S rRNA gene sequences showed less than 1% sequence divergence, and all haloalkane degraders clearly belonged to the genus Rhodococcus. All strains shared a completely conserved dhaA gene, suggesting that the dhaA genes were recently derived from a common ancestor. The genetic organization of the dhaA gene region in each of the haloalkane degraders was examined by hybridization analysis and DNA sequencing. Three different groups could be defined on the basis of the extent of the conserved dhaA segment. The minimal structure present in all strains consisted of a conserved region of 12.5 kb, which included the haloalkane-degradative gene cluster that was previously found in strain NCIMB13064. Plasmids of different sizes were found in all strains. Southern hybridization analysis with a dhaA gene probe suggested that all haloalkane degraders carry the dhaA gene region both on the chromosome and on a plasmid (70 to 100 kb). This suggests that an ancestral plasmid was transferred between these Rhodococcus strains and subsequently has undergone insertions or deletions. In addition, transposition events and/or plasmid integration may be responsible for positioning the dhaA gene region on the chromosome. The data suggest that the haloalkane dehalogenase gene regions of these gram-positive haloalkane-utilizing bacteria are composed of a single catabolic gene cluster that was recently distributed worldwide.

Utilization of trihalogenated propanes by Agrobacterium radiobacter AD1 through heterologous expression of the haloalkane dehalogenase from Rhodococcus sp. strain M15-3.

Trihalogenated propanes are toxic and recalcitrant organic compounds. Attempts to obtain pure bacterial cultures able to use these compounds as sole carbon and energy sources were unsuccessful. Both the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA) and that from Rhodococcus sp. strain m15-3 (DhaA) were found to dehalogenate trihalopropanes to 2,3-dihalogenated propanols, but the kinetic properties of the latter enzyme are much better. Broad-host-range dehalogenase expression plasmids, based on RSF1010 derivatives, were constructed with the haloalkane dehalogenase from Rhodococcus sp. strain m15-3 under the control of the heterologous promoters P(lac), P(dhlA), and P(trc). The resulting plasmids yielded functional expression in several gram-negative bacteria. A catabolic pathway for trihalopropanes was designed by introducing these broad-host-range dehalogenase expression plasmids into Agrobacterium radiobacter AD1, which has the ability to utilize dihalogenated propanols for growth. The recombinant strain AD1(pTB3), expressing the haloalkane dehalogenase gene under the control of the dhlA promoter, was able to utilize both 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane as sole carbon sources. Moreover, increased expression of the haloalkane dehalogenase resulted in elevated resistance to trihalopropanes.

Rubella virus and chronic joint disease: is there an association?

Synovial fluid samples and/or biopsies from 79 patients with various chronic inflammatory joint diseases or traumatic joint injury were tested for rubella virus (RV) in order to confirm or refute results from other studies that suggested RV as a cause of chronic inflammatory joint disease. Sixty-eight of the 72 patients tested had RV antibodies. RV RNA was detected by reverse transcription-PCR in the synovial fluid cells from two patients. RV was also isolated by cell culture from the synovial fluid of one of these two patients. This patient was a 42-year-old female with common variable immune deficiency and Mycoplasma hominis arthritis, while the other was a 68-year-old female with rheumatoid arthritis. While these results fail to confirm that RV is associated with chronic inflammatory joint disease, they suggest that RV may persist within a joint and be reactivated when cell-mediated immunity is suppressed.

Molecular analysis of rubella virus epidemiology across three continents, North America, Europe, and Asia, 1961-1997.

E1 gene nucleotide sequences of 63 rubella virus isolates from North America, Europe, and Asia isolated between 1961 and 1997 were compared phylogenetically. Two genotypes were evident: Genotype I contained 60 viruses from North America, Europe, and Japan, and genotype II contained 3 viruses from China and India. The genotype I isolates prior to 1970 grouped into a single diffuse clade, indicating intercontinental circulation, while most post-1975 viruses segregated into geographic clades from each continent, indicating evolution in response to vaccination programs. The E1 amino acid sequences differed by no more than 3%; thus, no major antigenic variation was apparent. Among 4 viruses from congenital rubella syndrome that occurred following reinfection, only one amino acid substitution occurred in several important epitopes, indicating that antigenic drift is not important in this phenomenon. However, 2 viruses isolated from chronic arthritis exhibited changes in these epitopes. Isolates of the RA 27/3 vaccine strain were readily identifiable by nucleotide sequence.

Measurement of minimum substrate concentration (Smin) in a recycling fermentor and its prediction from the kinetic parameters of Pseudomonas strain B13 from batch and chemostat cultures.

The minimum substrate concentration required for growth, Smin, was measured for Pseudomonas sp. strain B13 with 3-chlorobenzoate (3CB) and acetate in a recycling fermentor. The substrates were provided alone or in a mixture. Smin values predicted with kinetic parameters from resting-cell batches and chemostat cultures differed clearly from the values measured in the recycling fermentor. When 3CB and acetate were fed as single substrates, the measured Smin values were higher than the individual Smin values in the mixture. The Smin in the mixture reflected the relative energy contributions of the two substrates in the fermentor feed. The energy-based maintenance coefficients during zero growth in the recycling fermentor were comparable for all influent compositions (mean +/- standard deviation, 0.34 +/- 0.07 J mg [dry weight]-1 h-1). Maintenance coefficient values for acetate were significantly higher in chemostat experiments than in recycling-fermentor experiments. 3CB maintenance coefficients were comparable in both experimental systems. The parameters for 3CB consumption kinetics varied remarkably with the experimental growth conditions in batch, chemostat, and recycling-fermentor environments. The results demonstrate that the determination of kinetic parameters in the laboratory for prediction of microbial activity in complex natural systems should be done under conditions which best mimic the system under consideration.

Nucleotide sequence analysis of a major antigenic domain of the E1 glycoprotein of 22 rubella virus isolates.

We have determined the nucleotide sequence of the region of the rubella virus genome which encodes amino acids 195-296 of the E1 glycoprotein (E1-195-296) from a panel of 22 rubella viruses obtained from Europe, USA and Asia between 1963-1995. E1-195-296 contains neutralizing and haemagglutinating determinants, and may represent a major antigenic domain. The nucleotide sequence divergence of the 22 rubella viruses compared to the Therien strain sequence ranged from 0.65-7.14%. The greatest sequence divergence occurred in two rubella viruses of Indian origin, and was more than twofold greater than that previously reported for rubella virus. The majority of nucleotide changes occurring in the 22 viruses did not effect the deduced amino acid sequence of E1-195-296. Two rubella viruses isolated from cases of reinfection in pregnancy did not exhibit nucleotide sequence variation resulting in changes in the deduced amino acid sequence of E1-195-296, suggesting that antigenic change within this region of E1 is not associated with rubella reinfection. A rubella virus isolated from a synovial fluid sample exhibited a nucleotide substitution in a putative neutralization domain contained within E1-195-296. Phylogenetic analysis was performed to study the relationship between E1-195-296 coding sequences of the 22 viruses in this report and corresponding sequences of other rubella viruses in the databank.

Detection of the 5' region of the rubella virus genome in clinical samples by polymerase chain reaction.

BACKGROUND: Maternal rubella infection in early pregnancy has a high probability of causing congenital rubella infection. In some cases it may be difficult to establish the risk of congenital infection and polymerase chain reaction (PCR) based techniques are therefore being applied to prenatal diagnosis. OBJECTIVES: To investigate whether the non-structural region of the rubella virus (RV) genome can be detected in clinical samples using PCR, thereby providing a prenatal assay independent of those currently used to detect the structural protein coding region. STUDY DESIGN: Oligonucleotide primers coding for RV nucleotides 1-17 and 541-558 from the non-structural protein coding region of the RV genome were used in a reverse transcription polymerase chain reaction (NS RT-PCR) to amplify 558 nucleotides of RV cDNA. Amplification of RV specific sequences was confirmed by Southern hybridization. RESULTS: The specificity of the assay was confirmed by the detection of RV RNA from both wild-type and vaccine strains of RV, pharyngeal swabs from two adult males with acute rubella and products of conception from three women with serologically confirmed primary rubella in pregnancy. RV RNA was not detected in uninfected HEL and Vero cells or peripheral blood mononuclear cells. The results were concordant with those of an RT-PCR directed to the E1 protein coding region and with virus isolation. CONCLUSIONS: Detection of the non-structural coding region of the RV genome in clinical samples suggests that NS RT-PCR could be used as a confirmatory assay for prenatal diagnosis of congenital rubella, and that it will be of value for the identification of nucleotide changes in the 5' region of the RV genome.

Sequence variation in 5' termini of rubella virus genomes: changes affecting structure of the 5' proximal stem-loop.

Variation within a 523 nucleotide region proximal to the 5' terminus of seven rubella virus strains has been analysed. Compared to the Therien strain twenty sites of nucleotide variation have been identified, three of which are in the 5' untranslated region. Individual strains have between three and nine nucleotide differences, only three of which result in amino acid substitutions. TO-336 has a serine for threonine at amino acid (aa) 42 and CM arginine for histidine at aa 159. RA27/3 has arginine for lysine at aa 3 and serine for threonine at aa 42. Nucleotide differences which affect a stem-loop structure reported to be important for binding of host cell proteins have been identified.

Use of PCR for prenatal and postnatal diagnosis of congenital rubella.

A reverse transcription-nested PCR assay (RT-PCR) was evaluated for diagnosis of congenitally acquired rubella in utero and during infancy. RT-PCR was compared with virus isolation for retrospective detection of rubella virus in placental and fetal tissues obtained after termination of pregnancy following primary rubella or rubella virus reinfection. Concordant results were obtained for 85% of samples; rubella virus RNA was detected by RT-PCR alone in four samples, and rubella virus was detected by isolation alone in two samples. Samples were also obtained for prenatal diagnosis of congenital infection; rubella virus RNA was detected in three of seven chorionic villus samples and one of three amniotic fluid samples by RT-PCR, while rubella virus was isolated in only one chorionic villus sample. To demonstrate that the RNA extracted from chorionic villus samples contained amplifiable RNA, a nested RT-PCR was used to detect keratin mRNA. Rubella virus was detected in placenta in two cases in which the fetus was uninfected, and there was no evidence of rubella virus in the placenta from one case in which the fetus was infected. Thus, detection of rubella virus in chorionic villus samples by RT-PCR may not always correctly predict fetal rubella virus infection. RT-PCR was successfully used for the diagnosis of congenitally acquired rubella in infancy. Rubella virus RNA was detected in cyropreserved or formalin-fixed lens aspirates obtained from infants in India with serologically confirmed congenital rubella but not in samples from controls with inherited cataract.

Thermostability of respiratory terminal oxidases in the lipid environment.

The effect of the lipid environment on the thermostability of three respiratory terminal oxidases was determined. Cytochrome-c oxidase from beef heart and Bacillus stearothermophilus were used as representative proteins from mesophilic and thermophilic origin, respectively. Quinol oxidase from the archaeon Sulfolobus acidocaldarius represented the model for a extreme thermoacidophilic enzyme. All three integral membrane proteins were tested for their thermal inactivation in detergent and after reconstitution in liposomes composed of phospholipids of Escherichia coli or tetraether lipids from S. acidocaldarius. When preincubated at 0 degrees C, all three enzymes exhibited biphasic thermal inactivation curves. Data could be analysed according to a two-state model that defines two conformations of the enzyme, differing in their thermostability. Monophasic inactivation curves were observed when the enzymes were preincubated at higher temperatures prior to thermal inactivation. Lipids rendered the beef-heart cytochrome-c oxidase and S. acidocaldarius quinol oxidase more thermostable as compared to detergent solution. In contrast, the B. stearothermophilus oxidase, an intrinsically thermostable enzyme, was as thermostable in detergent as in the reconstituted state. These data suggest that the lipid environment can be an important factor in the thermostability of membrane proteins.

PCR for detection of rubella virus RNA in clinical samples.

A reverse transcription nested PCR (RT-PCR) assay for the detection of rubella virus RNA using primers from the E1 open reading frame was established. This assay was found to be sensitive (detecting approximately two synthetic RNA copies and RNA extracted from 0.1 50% tissue culture infective dose of rubella virus) and specific; five wild-type rubella strains and four vaccine strains were detected, and no nonspecific amplification of 16 other RNA viruses or RNAs from seven cell types occurred. Rubella virus RNA was detected in 12 pharyngeal swabs from patients with serologically confirmed rubella; these RT-PCR results were in complete agreement with virus isolation. Analysis of products of conception obtained after confirmed primary maternal rubella infection by RT-PCR gave 92% agreement (12 of 13 samples) with virus isolation. No false-positive results were obtained. The potential use of this assay for prenatal diagnosis of congenital rubella infection and for investigating aspects of the pathogenesis of chronic disease is discussed.