Biomarker models as surrogates for the disposition index in the Insulin Resistance Atherosclerosis Study.

AIMS: Insulin sensitivity and acute insulin response measure key components of Type 2 diabetes aetiology that contribute independently to risk in the Insulin Resistance Atherosclerosis Study. As insulin sensitivity and acute insulin response are not routinely measured in a clinical setting, we evaluated three fasting biomarker models, homeostasis model assessment of insulin sensitivity (HOMA-%S), ß-cell function (HOMA-%B) and a Diabetes Risk Score, as potential surrogates for risk associated with insulin sensitivity, acute insulin response and the interaction of these two measures, the disposition index. METHODS: Models were calculated from baseline plasma biomarker concentrations for 664 participants who underwent a frequently sampled intravenous glucose tolerance test. To assess relationships among biomarker models and test measures, we calculated improvement in risk estimation gained by combining each fasting measure with each frequently sampled intravenous glucose tolerance test measure using logistic regression. RESULTS: The strongest correlates of acute insulin response, insulin sensitivity and disposition index were HOMA-%B (r(s)(2) = 0.23), HOMA-%S (r(s)(2) = 0.48) and Diabetes Risk Score (r(s)(2) = 0.34), respectively. Individual areas under the curves for prediction of diabetes were 0.549 (HOMA-%B), 0.694 (HOMA-%S), 0.700 (insulin sensitivity), 0.714 (acute insulin response), 0.756 (Diabetes Risk Score) and 0.817 (disposition index). Models combining acute insulin response with Diabetes Risk Score (area under the curve 0.798) or HOMA-%S (area under the curve 0.805) nearly equalled disposition index, outperforming other individual measures (P < 0.05). Insulin sensitivity plus Diabetes Risk Score (area under the curve 0.760) was better than insulin sensitivity (P = 0.03), but not better than Diabetes Risk Score alone. HOMA-%S plus insulin sensitivity (area under the curve 0.704) was not significantly better than either alone. CONCLUSIONS: The Diabetes Risk Score and HOMA-%S were excellent surrogates for insulin sensitivity, capturing the predictive power of insulin sensitivity. Diabetes Risk Score captured some of the additional predictive power of acute insulin response, but the HOMA models did not. No fasting model was as predictive as disposition index, but the Diabetes Risk Score was the best surrogate.

The four mouse IgG isotypes differ extensively in bactericidal and opsonophagocytic activity when reacting with the P1.16 epitope on the outer membrane PorA protein of Neisseria meningitidis.

Mouse monoclonal antibodies (MoAbs) of the four IgG isotypes, all specific for the P1.16 epitope on the meningcoccal PorA protein, were tested for functional activities. The avidities of the antibodies, measured by NH4SCN elution in enzyme-linked immunosorbent assay, showed similar values for all the MoAbs. The serum bactericidal activity (SBA) defined as the lowest concentration of antibodies giving 50% reduction in the number of meningococcal colony-forming units using human serum as complement, showed a hierarchy of IgG3 >> IgG2b > IgG2a >> IgG1. For the opsonophagocytosis (OP), the hierarchy was IgG3 > IgG2b = IgG2a >> IgG1. OP was measured in flow cytometry using log-phase live meningococci as target cells, normal human peripheral blood polymorphonuclear cells (PMNs) as effector cells and human serum as a complement source. The mouse MoAbs were negative in OP when using human PMNs in the absence of complement. The results demonstrate the importance of choosing the right isotype of mouse MoAbs when using them to judge the potential vaccine importance of their corresponding antigen. If such MoAbs should be used for passive vaccination against infectious diseases, the isotype would presumably play an important role for their anticipated clinical effects.

Binding properties and anti-bacterial activities of V-region identical, human IgG and IgM antibodies, against group B Neisseria meningitidis.

We have constructed chimaeric (ch) mouse/human antibodies with identical binding regions isolated from the V-genes of two mouse parent hybridoma cell lines, with specificity against the P1.7 and P1.16 epitopes on the outer-membrane protein PorA on meningococci. The chimaeric antibodies can be used to analyse relationships between specificity, binding activity (avidity and kinetics), isotype (antibody class and antibody subclass) and in vitro anti-bacterial activity of meningococcal antibodies. The antibody sets represented the human isotypes IgG1, IgG3 and IgM, which dominate during immune response against protein antigens. The binding activities were quite similar for all these isotypes, surprisingly also for the pentameric IgM. Interestingly, monomeric IgM, prepared from pentameric IgM by partially reduction and alkylation, had similar binding activities as the original pentameric IgM. Regarding in vitro anti-bacterial activity, chIgG1 was superior in SBA (serum bactericidal activity) compared with chIgG3, while chIgG3 was more efficient in OP (opsonophagocytosis; measured by flow cytometry) than chIgG1. ChIgM showed slightly higher SBA than chIgG1 on molar basis, and much higher OP than chIgG3 and chIgG1. A lower concentration of antibodies was needed against the P1.16 than against the P1.7 epitope to induce SBA, but this was not the case for OP.

Epitope analyses of pneumococcal surface protein A: a combination of two monoclonal antibodies detects 94% of clinical isolates.

Immunisation of BALB/c mice with seven heat-treated Norwegian clinical isolates of Streptococcus pneumoniae of different serotypes elicited mainly monoclonal antibodies (mAbs) to pneumococcal surface protein A (PspA). It was remarkable that the fusions resulted only in a few mAbs directed against other protein antigens. Dot blot analysis with 16 mAbs using clinical isolates representing 23 different capsular types and the uncapsulated reference strain R36A showed that some of the mAbs bound to PspA epitopes expressed by a low number of strains whereas others bound to broadly distributed epitopes. On the basis of their reactivities, seven of these mAbs could be divided into two groups recognising different subsets of pneumococci. The three mAbs in the narrow reacting group bound to epitopes found in 21-25% of the strains whereas the four mAbs in the broad reacting group detected more than 57% of the analysed strains. The epitopes for these seven antibodies were surface exposed on live exponential phase grown pneumococci as shown by flow cytometry. The finding that a combination of mAb 180,C-1 (IgG2a) from the first group and mAb 170,E-11 (IgG2a) from the second group detected 94% of the examined strains is interesting because PspA has been reported by others to be a serological highly variable protein.

Analysis of the expression of the putatively virulence-associated neisserial protein RmpM (class 4) in commensal Neisseria and Moraxella catarrhalis strains.

The RmpM protein has been reported to be present only in pathogenic Neisseria species. In the present study we demonstrate that this protein is also present at least in N. lactamica and N. sicca strains. The N. lactamica protein reacts with a RmpM-specific monoclonal antibody (185,H-8), having a molecular mass ( approximately 31 kDa) slightly lower than that of the meningococcal RmpM, and mouse antibodies from sera against outer membrane vesicles from both N. lactamica and N. sicca strains cross-react with the meningococcal RmpM. PCR and hybridization experiments with a complete rmpM probe agree with the immunodetection experiments. Our results strongly suggest that the meningococcal RmpM should not be considered a virulence marker, and the presence of this protein in the commensal species agrees with its role as a structural protein, proposed for the RmpM, which should be considerably conserved in the Neisseria species.

Single-copy gene detection using branched DNA (bDNA) in situ hybridization.

We have developed a branched DNA in situ hybridization (bDNA ISH) method for detection of human papillomavirus (HPV) DNA in whole cells. Using human cervical cancer cell lines with known copies of HPV DNA, we show that the bDNA ISH method is highly sensitive, detecting as few as one or two copies of HPV DNA per cell. By modifying sample pretreatment, viral mRNA or DNA sequences can be detected using the same set of oligonucleotide probes. In experiments performed on mixed populations of cells, the bDNA ISH method is highly specific and can distinguish cells with HPV-16 from cells with HPV-18 DNA. Furthermore, we demonstrate that the bDNA ISH method provides precise localization, yielding positive signals retained within the subcellular compartments in which the target nucleic acid sequences are localized. As an effective and convenient means for nucleic acid detection, the bDNA ISH method is applicable to the detection of cancers and infectious agents. (J Histochem Cytochem 49:603-611, 2001)

PorB3 outer membrane protein on Neisseria meningitidis is poorly accessible for antibody binding on live bacteria.

It is reported here that the PorB3 porin proteins of serotype 4 and 15 are poorly accessible for antibody binding on live Neisseria meningitidis bacteria, whereas the allelic PorB2 and the PorA outer membrane protein appear to be highly accessible. This was revealed by flow cytometry analysis using several mouse monoclonal antibodies (mAbs) as well as PorB3 specific antibodies isolated from post vaccination and patient sera. However, strong antibody binding to the PorB3 protein was observed after killing the bacteria with ethanol. The reason for the lack of epitope exposure could be a shielding effect of the carbohydrate chains of lipopolysaccharides (LPS) possibly combined with short extra-cellular loops in the PorB3 protein. The findings indicate that the PorB3 protein is not an optimal target for protective antibodies induced by vaccination.

Streptococcus pneumoniae heat shock protein 70 does not induce human antibody responses during infection.

Mouse monoclonal antibodies (mAbs) were developed against Streptococcus pneumoniae in search for potential common pneumococcal proteins as vaccine antigens. mAb 230,B-9 (IgG1) reacted by immunoblotting with a 70-kDa protein which was isolated by immunoaffinity chromatography and subsequent preparative electrophoresis. N-terminal amino acid sequencing showed homology to that of heat shock protein 70 (hsp70). The hsp70 epitope reactive with mAb 230,B-9 was found in all the pneumococci examined as well as in other streptococci and enterococci. The epitope was not expressed in several other examined Gram-positive or -negative bacteria. Pneumococcal hsp70 has by other investigators been proposed to be a vaccine candidate. Binding experiments using flow cytometry showed that the epitope was not surface-exposed on live exponential phase grown S. pneumoniae. Human patient sera did not react with affinity-purified pneumococcal hsp70. Therefore the pneumococcal hsp70 does not seem to be of special interest in a vaccine formulation. The human sera contained antibodies to high molecular proteins co-purified with hsp70. Some of these proteins could be the pneumococcal surface protein A.

Prevalence of primary resistance to zidovudine and lamivudine in drug-naive human immunodeficiency virus type-1 infected patients: high proportion of reverse transcriptase codon 215 mutant in circulating lymphocytes and free virus.

The presence of primary zidovudine (AZT)-resistance (mutation T215Y/F) or lamivudine (3TC)-resistance (mutation M184V) was evaluated in 90 drug-naive patients infected with human immunodeficiency virus type-1 (HIV-1) between 1987 and 1997. The proportion of mutant strains in proviral samples or plasma viral samples was determined using a differential hybridization assay. Mutation T215Y/F was found in five (5.6%) patients infected between 1994 and 1997, whereas none of these patients harbored the mutation M184V. The T215Y/F mutation was present in the virus and/or provirus and persisted for at least two years. In one patient, the mutant provirus was associated with only wild-type free virus. Four of these patients were followed, and two were treated subsequently to a regimen containing AZT but with low response. The persistence of primary resistance mutations might depend on the proportion of these mutations at the time of infection, although mutant provirus might not give rise to replicating variants.

Direct mecA detection from blood culture bottles by branched-DNA signal amplification.

A branched-DNA (bDNA) signal amplification method was used to detect the mecA gene directly from blood culture broth growing staphylococci. BACTEC blood culture bottles with positive growth indices and containing staphylococcus-like organisms as shown by Gram stain were tested for the presence of the mecA gene. Comparison of test results was done among 225 patients (one blood culture from each patient). Compared with PCR, the sensitivity and specificity of the bDNA method are 100 and 99%, respectively. The bDNA test is carried out in a 96-well format and requires approximately 6 h to perform. Our preliminary results suggest that direct detection of the mecA gene by bDNA signal amplification is (i) sensitive enough to detect mecA directly from blood culture bottles without the requirement for subculture and (ii) as sensitive and specific as the PCR-based method.

Hepatitis G virus and human immunodeficiency virus coinfection: response to interferon-alpha therapy.

The prevalence and consequences of hepatitis G virus (HGV) infection were determined in 180 patients with human immunodeficiency virus (HIV) infection (predominantly male homosexuals) who participated in a trial that compared treatment with zidovudine versus interferon (IFN)-alpha versus the combination. HGV RNA levels were measured by branched DNA signal amplification assay. Initially, 66 (37%) had HGV RNA. Sexual transmission was the sole risk factor for infection in all but 4 subjects. Pretreatment clinical features were similar between HGV RNA-positive and -negative patients. After 6 months, only 5% treated with zidovudine became HGV RNA negative, compared with 95% who received IFN-alpha alone and 66% on combination therapy with low-dose IFN-alpha. After therapy, HGV RNA levels returned to baseline in most subjects. Thus, HGV infection is common among HIV-infected homosexual males but does not appear to influence clinical features in early HIV infection. HGV RNA levels are suppressed by IFN but not by zidovudine.

The clinical significance of simultaneous infection with hepatitis G virus in patients with chronic hepatitis C.

OBJECTIVES: Hepatitis G virus (HGV) is a recently discovered member of the flavivirus family that has been associated with acute and chronic hepatitis. HGV infection has been reported to coexist in 10-20% of patients with chronic hepatitis C. The significance of simultaneous infection with HGV and hepatitis C virus (HCV) remains to be clarified, as do the effects on HGV of therapeutic interventions such as interferon treatment or liver transplantation. THE AIMS OF OUR STUDY WERE: 1) to examine the frequency of HGV infection in the settings of liver transplantation and interferon therapy for hepatitis C; and 2) to compare HGV RNA levels before and after liver transplantation or interferon treatment. METHODS: Pre-treatment sera were available in 65 patients with chronic hepatitis C treated with interferon; pretransplant sera were available in 49 patients transplanted for end stage liver disease associated with chronic hepatitis C. Information collected included age, sex, risk factors for hepatitis, concurrent liver disease, patient and allograft survival, biochemical response to interferon, histological activity index, and degree of fibrosis/cirrhosis. HCV genotyping was performed by sequencing the NS-5 region. HGV quantitation was performed using a research-based branched DNA (bDNA) assay with a set of probes directed at the 5' untranslated region. RESULTS: HGV was detected in 10 of 49 patients (20%) before transplant and in 13 of 65 patients (20%) treated with interferon. There was a female predominance among HGV-positive compared with HGV-negative transplant patients (80% vs 20%; p < 0.01), but such a difference was not observed in the interferon-treated group. Hepatic iron concentration was lower in hepatic explants from patients who were HGV-positive than in those who were HGV-negative (318 +/- 145 microg/g dry weight vs 1497 +/- 2202 microg/g dry weight; p = 0.02). HCV exposure after 1980 was more common in the HGV-positive patients than in those who were HGV-negative for the entire study population (10 of 20 [50%] vs 16 of 66 [24%]; p = 0.03), as well as for the nontransplant subgroup (8 of 12 [67%] vs 12 of 39 [31%]; p = 0.03). HGV RNA levels declined at 1 yr after transplant in seven of eight patients. Among nine patients tested during or after interferon treatment, HGV RNA levels declined from pretreatment levels in all and disappeared in three. CONCLUSIONS: Among patients with chronic hepatitis C treated with either interferon or liver transplantation, the frequency of coinfection with HGV is about 20%. HGV may be a more recent virus in the US than HCV. Coinfection with HGV does not appear to affect the likelihood of response to interferon in patients with hepatitis C. Finally, HGV RNA levels appear to decline after both liver transplantation and interferon therapy, suggesting possible suppression by increased HCV replication in the former case, and a possible drug treatment effect in the latter.

Functional activities and epitope specificity of human and murine antibodies against the class 4 outer membrane protein (Rmp) of Neisseria meningitidis.

Antibodies against the class 4 outer membrane protein (OMP) from Neisseria meningitidis have been purified from sera from vaccinees immunized with the Norwegian meningococcal group B outer membrane vesicle vaccine. The human sera and purified antibodies reacted strongly with the class 4 OMP in immunoblots, whereas experiments with whole bacteria showed only weak reactions, indicating that the antibodies mainly reacted with parts of the class 4 molecule that were not exposed. The purified human anti-class 4 OMP antibodies and the monoclonal antibodies (MAbs) were neither bactericidal nor opsonic against live meningococci. Three new MAbs against the class 4 OMP were generated and compared with other, previously described MAbs. Three linear epitopes in different regions of the class 4 OMP were identified by the reaction of MAbs with synthetic peptides. The MAbs showed no blocking effect on bactericidal activity of MAbs against other OMPs. However, one of the eight purified human anti-class 4 OMP antibody preparations, selected from immunoblot reactions among sera from 27 vaccinees, inhibited at high concentrations the bactericidal effect of a MAb against the class 1 OMP. However, these antibodies were not vaccine induced, as they were present also before vaccination. Therefore, this study gave no evidence that vaccination with a meningococcal outer membrane vesicle vaccine containing the class 4 OMP induces blocking antibodies. Our data indicated that the structure of class 4 OMP does not correspond to standard beta-barrel structures of integral OMPs and that no substantial portion of the OmpA-like C-terminal region of this protein is located at the surface of the outer membrane.

Preparation of genotype-specific HCV RNA transcripts for assessing HCV detection and quantification assays.

Hepatitis C virus (HCV), the etiological agent responsible for the majority of cases of parenterally acquired liver disease, is found throughout the world. HCV is an enveloped virus with a small, single-stranded RNA genome. Because it uses an error-prone, RNA-dependent RNA polymerase, HCV has a high spontaneous mutation rate, and isolates of HCV display significant genetic heterogeneity. Isolates of HCV have been classified into at least six major genotypes and multiple subtypes based on sequencing and phylogenetic analysis (1). These genetic variants of HCV show a diverse geographical distribution. HCV types 1a, 1b, 2b, and 3a are the most prevalent in the US and western Europe (2,3), although all six major genotypes have been noted.

Quantitation of estrogen receptor mRNA in breast carcinoma by branched DNA assay.

A quantitative nucleic acid hybridization assay for determination of estrogen receptor (ER) mRNA in breast carcinoma is described. The assay, which is based on the branched DNA (bDNA) technology, requires 20 mg of tissue, is simple, highly specific, and reproducible, and correlates reasonably well with an established methodology (r = 0.87). The assay has a dynamic range of 3 x 10(3)-6 x 10(7) copies of ER mRNA per well. ER message as high as 2.5 x 10(6) copies per well could be detected in normal breast tissues. Thus a sensitivity of 3 x 10(3) ER copies per well was sufficient to analyze clinical specimens. In the present studies, accurate measurement of tissue weight enabled direct reporting of the ER mRNA values as the end point results. The bDNA assay provides a useful tool for the detection and quantitation of ER mRNA in research and routine clinical laboratories.

Quantitation of progesterone receptor mRNA in breast carcinoma by branched DNA assay.

Expression of progesterone receptor (PR) mRNA is indicative of a normal gene regulation mechanism mediated by functional estrogen receptor (ER). A simple assay which can reliably detect and quantitate PR mRNA levels in a small amount of tissue will be of value for studying functional status of ER. We have developed a quantitative nucleic acid hybridization assay for PR mRNA in breast carcinoma. The assay, which is based on the branched DNA (bDNA) technology, is simple, highly specific, and reproducible, requires 20 mg of tissue, and correlates reasonably well (r = 0.86) with an established methodology. The assay has a dynamic range of 3 x 10(3)-6 x 10(7) copies of PR mRNA per well. PR message as high as 3.9 x 10(5) copies per well could be detected in normal breast tissues. Thus a sensitivity of 3 x 10(3) PR copies per well was sufficient for testing clinical samples. In the present studies, accurate measurement of tissue weight enabled direct reporting of the PR mRNA values as the end point results. The bDNA assay provides a useful tool for the detection and quantitation of PR mRNA in research and routine clinical laboratories.

Hepatitis after liver transplantation: the role of the known and unknown viruses.

This study was designed to determine the cause of posttransplantation hepatitis in patients undergoing transplantation for liver disease of nonviral cause; the role of acquired hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis G virus (HGV) in posttransplantation hepatitis; and the course of posttransplantation hepatitis of unknown cause. Two hundred forty-three patients underwent transplantation for nonviral liver diseases (mean age, 48 years; 103 men, 140 women). Serological and virological assays for HBV and HCV were performed pretransplantation to exclude preexisting infection and posttransplantation to investigate the cause of posttransplantation hepatitis. Histology was graded on all available biopsy specimens; posttransplantation hepatitis was assessable in 150 patients. Posttransplantation hepatitis was present in 29% (44 of 150) of the patients after a median follow-up of 47 months (range, 1 to 101 months). Actuarial survival was significantly lower in patients with posttransplantation hepatitis compared with patients without (71% v 89% at 5-year follow-up; P = .03). HCV and HBV were identified posttransplantation in 14% and 9% of patients with hepatitis, respectively. After the exclusion of HCV and HBV infection, 22% (33 of 150) of the patients had posttransplantation hepatitis of unknown cause. HGV was present in 58% of these patients, but HGV was equally prevalent in patients without posttransplantation hepatitis. When patients with HBV and HCV were excluded, there was no difference in survival between patients with posttransplantation hepatitis compared with patients without (P = .08, log-rank test). Posttransplantation hepatitis was present in approximately 30% of the patients undergoing transplantation for nonviral diseases, with a median follow-up of 47 months. Known hepatitis viruses (HBV, HCV) were present in one fourth of the patients with posttransplantation hepatitis; 22% (33 of 150) of the patients had hepatitis of unknown cause, suggesting that other, as yet undiscovered, hepatitis viruses may exist.

Quantification of cytokine mRNA in peripheral blood mononuclear cells using branched DNA (bDNA) technology.

Changes in the patterns of cytokine expression are thought to be of central importance in human infectious and inflammatory diseases. As such, there is a need for precise, reproducible assays for quantification of cytokine mRNA that are amenable to routine use in a clinical setting. In this report, we describe the design and performance of a branched DNA (bDNA) assay for the direct quantification of multiple cytokine mRNA levels in peripheral blood mononuclear cells (PBMCs). Oligonucleotide target probe sets were designed for several human cytokines, including TNFalpha, IL-2, IL-4, IL-6, IL-10, and IFNgamma. The bDNA assay yielded highly reproducible quantification of cytokine mRNAs, exhibited a broad linear dynamic range of over 3-log10, and showed a sensitivity sufficient to measure at least 3000 molecules. The potential clinical utility of the bDNA assay was explored by measuring cytokine mRNA levels in PBMCs from healthy and immunocompromised individuals. Cytokine expression levels in PBMCs from healthy blood donors were found to remain relatively stable over a one-month period of time. Elevated levels of IFNgamma mRNA were detected in PBMCs from HIV-1 seropositive individuals, but no differences in mean levels of TNFalpha or IL-6 mRNA were detected between seropositive and seronegative individuals. By providing a reproducible method for quantification of low abundance transcripts in clinical specimens, the bDNA assay may be useful for studies addressing the role of cytokine expression in disease.

Direct quantitation of HIV by flow cytometry using branched DNA signal amplification.

Adaptation of the branched DNA signal amplification technology to flow cytometry has resulted in a quantitative nuclei-acid assay with significant advantages over the microwell-based format. In this assay, microbeads, rather than microwell plates, are derivatized with nucleic-acid capture probes and the derivatized beads are used to capture single nucleic-acid targets, which then capture fluorescent reporter probes via branched DNA. The assay detects DNA or RNA targets, has a current lower sensitivity limit of 500 human immunodeficiency virus (HIV) RNA molecules and responds linearly to target level from 500 to at least 50,000 molecules. Since microbeads can easily interrogate large volumes, viral lysis and genomic RNA capture can proceed in one step from comparatively large volumes, and sample preparation is greatly simplified compared to the microwell-format bDNA assay.

Branched-DNA assay for detection of the mecA gene in oxacillin-resistant and oxacillin-sensitive staphylococci.

The identification of methicillin-resistant staphylococcus isolates in the clinical laboratory has typically been performed by using methods that detect phenotypic expression of resistance determinants. However, these methods may be difficult to interpret and some isolates do not express resistance until selective pressure is administered. Assays that detect genetic determinants are not subject to these limitations and have been effective in distinguishing isolates that are capable of expressing the resistance phenotype. In this study, a novel branched-DNA (bDNA) hybridization assay was used to test for the mecA gene in 416 clinical staphylococcal isolates. The results were compared with those obtained by a PCR-based assay and oxacillin disk diffusion. For 155 Staphylococcus aureus and 261 coagulase-negative Staphylococcus isolates, the bDNA assay and PCR results were 100% concordant. Among the S. aureus isolates, 20 were MecA+ and 135 were MecA-. For the coagulase-negative staphylococci, 150 were MecA+ and 111 were MecA-. The results from the genotypic detection methods were compared with those obtained by oxacillin disk diffusion. No discrepancies were detected among the S. aureus isolates; however, 10 coagulase-negative isolates were MecA+ but oxacillin sensitive and 1 isolate was MecA- but oxacillin resistant. Oxacillin resistance was induced in 6 of the 10 MecA+ isolates previously classified as oxacillin sensitive. These results suggest that the bDNA method described here is a sensitive and efficient method for detection of methicillin resistance in staphylococci and that genetic detection methods may be useful for detection of potential methicillin resistance in the clinical laboratory.