John A. Branda

An essential E box in the promoter of the gene encoding the mRNA cap-binding protein (eukaryotic initiation factor 4E) is a target for activation by c-myc.

The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF4E]) binds the m7 GpppN cap on mRNA, thereby initiating translation. eIF4E is essential and rate limiting for protein synthesis. Overexpression of eIF4E transforms cells, and mutations in eIF4E arrest cells in G, in cdc33 mutants. In this work, we identified the promoter region of the gene encoding eIF4E, because we previously identified eIF4E as a potential myc-regulated gene. In support of our previous data, a minimal, functional, 403-nucleotide promoter region of eIF4E was found to contain CACGTG E box repeats, and this core eIF4E promoter was myc responsive in cotransfections with c-myc. A direct role for myc in activating the eIF4E promoter was demonstrated by cotransfections with two dominant negative mutants of c-myc (MycdeltaTAD and MycdeltaBR) which equally suppressed promoter function. Furthermore, electrophoretic mobility shift assays demonstrated quantitative binding to the E box motifs that correlated with myc levels in the electrophoretic mobility shift assay extracts; supershift assays demonstrated max and USF binding to the same motif. cis mutations in the core or flank of the eIF4E E box simultaneously altered myc-max and USF binding and inactivated the promoter. Indeed, mutations of this E box inactivated the promoter in all cells tested, suggesting it is essential for expression of eIF4E. Furthermore, the GGCCACGTG(A/T)C(C/G) sequence is shared with other in vivo targets for c-myc, but unlike other targets, it is located in the immediate promoter region. Its critical function in the eIF4E promoter coupled with the known functional significance of eIF4E in growth regulation makes it a particularly interesting target for c-myc regulation.

Multicenter Evaluation of the Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System for Identification of Gram-Positive Aerobic Bacteria

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) is gaining momentum as a tool for bacterial identification in the clinical microbiology laboratory. Compared with conventional methods, this technology can more readily and conveniently identify a wide range of organisms. Here, we report the findings from a multicenter study to evaluate the Vitek MS v2.0 system (bioMérieux, Inc.) for the identification of aerobic Gram-positive bacteria. A total of 1,146 unique isolates, representing 13 genera and 42 species, were analyzed, and results were compared to those obtained by nucleic acid sequence-based identification as the reference method. For 1,063 of 1,146 isolates (92.8%), the Vitek MS provided a single identification that was accurate to the species level. For an additional 31 isolates (2.7%), multiple possible identifications were provided, all correct at the genus level. Mixed-genus or single-choice incorrect identifications were provided for 18 isolates (1.6%). Although no identification was obtained for 33 isolates (2.9%), there was no specific bacterial species for which the Vitek MS consistently failed to provide identification. In a subset of 463 isolates representing commonly encountered important pathogens, 95% were accurately identified to the species level and there were no misidentifications. Also, in all but one instance, the Vitek MS correctly differentiated Streptococcus pneumoniae from other viridans group streptococci. The findings demonstrate that the Vitek MS system is highly accurate for the identification of Gram-positive aerobic bacteria in the clinical laboratory setting.

Two-Tiered Antibody Testing for Lyme Disease With Use of 2 Enzyme Immunoassays, a Whole-Cell Sonicate Enzyme Immunoassay Followed by a VlsE C6 Peptide Enzyme Immunoassay

BACKGROUND Lyme disease (LD) antibody testing currently involves a 2-tiered algorithm with a whole-cell sonicate (WCS) enzyme immunoassay (EIA), followed by IgM/IgG Western immunoblots. A single EIA using the C6 peptide of the Borrelia burgdorferi variable-major protein-like sequence expressed lipoprotein provides similar or better sensitivity but less specificity, compared with standard 2-tiered testing. Here, we investigated an alternative 2-tiered strategy, in which the first step remained a WCS EIA, but immunoblotting was replaced by a C6 EIA. METHODS We determined the sensitivity of the 3 testing strategies with use of 91 serum samples from research study patients with LD and 78 serum samples from patients with LD whose samples were submitted to our hospitals clinical laboratory. Specificity was measured using 54 patients with other illnesses and 1246 healthy subjects from areas where the infection is endemic and nonendemic. RESULTS The 2-EIA algorithm in early LD had similar sensitivity as C6 testing alone, and both strategies had better sensitivity than did standard 2-tiered testing (61% and 64%, respectively, vs 48%; P = .03 and P = .008). For late disease, all 3 strategies had 100% sensitivity. The specificity of the 2-EIA algorithm was equal to that of standard 2-tiered testing, and both 2-tiered strategies were more specific than C6 testing alone (for both, 99.5% vs 98.4%; P = .01). The positive predictive value of the 2-EIA algorithm was 70%, compared with 66% for standard 2-tiered testing and 43% for the C6 EIA alone. CONCLUSIONS The 2-EIA strategy matched the individual strengths of the C6 EIA and Western blotting, without the drawbacks. The 2 EIAs provided sensitivity comparable to that of the C6 EIA but maintained the specificity of standard 2-tiered testing.

Multicenter Study Evaluating the Vitek MS System for Identification of Medically Important Yeasts

ABSTRACT The optimal management of fungal infections is correlated with timely organism identification. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) is revolutionizing the identification of yeasts isolated from clinical specimens. We present a multicenter study assessing the performance of the Vitek MS system (bioMérieux) in identifying medically important yeasts. A collection of 852 isolates was tested, including 20 Candida species (626 isolates, including 58 C. albicans, 62 C. glabrata, and 53 C. krusei isolates), 35 Cryptococcus neoformans isolates, and 191 other clinically relevant yeast isolates; in total, 31 different species were evaluated. Isolates were directly applied to a target plate, followed by a formic acid overlay. Mass spectra were acquired using the Vitek MS system and were analyzed using the Vitek MS v2.0 database. The gold standard for identification was sequence analysis of the D2 region of the 26S rRNA gene. In total, 823 isolates (96.6%) were identified to the genus level and 819 isolates (96.1%) were identified to the species level. Twenty-four isolates (2.8%) were not identified, and five isolates (0.6%) were misidentified. Misidentified isolates included one isolate of C. albicans (n = 58) identified as Candida dubliniensis, one isolate of Candida parapsilosis (n = 73) identified as Candida pelliculosa, and three isolates of Geotrichum klebahnii (n = 6) identified as Geotrichum candidum. The identification of clinically relevant yeasts using MS is superior to the phenotypic identification systems currently employed in clinical microbiology laboratories.

2-Tiered Antibody Testing for Early and Late Lyme Disease Using Only an Immunoglobulin G Blot with the Addition of a VlsE Band as the Second-Tier Test

BACKGROUND Standard 2-tiered immunoglobulin G (IgG) testing has performed well in late Lyme disease (LD), but IgM testing early in the illness has been problematic. IgG VlsE antibody testing, by itself, improves early sensitivity, but may lower specificity. We studied whether elements of the 2 approaches could be combined to produce a second-tier IgG blot that performs well throughout the infection. METHODS Separate serum sets from LD patients and control subjects were tested independently at 2 medical centers using whole-cell enzyme immunoassays and IgM and IgG immunoblots, with recombinant VlsE added to the IgG blots. The results from both centers were combined, and a new second-tier IgG algorithm was developed. RESULTS With standard 2-tiered IgM and IgG testing, 31% of patients with active erythema migrans (stage 1), 63% of those with acute neuroborreliosis or carditis (stage 2), and 100% of those with arthritis or late neurologic involvement (stage 3) had positive results. Using new IgG criteria, in which only the VlsE band was scored as a second-tier test among patients with early LD (stage 1 or 2) and 5 of 11 IgG bands were required in those with stage 3 LD, 34% of patients with stage 1, 96% of those with stage 2, and 100% of those with stage 3 infection had positive responses. Both new and standard testing achieved 100% specificity. CONCLUSIONS Compared with standard IgM and IgG testing, the new IgG algorithm (with VlsE band) eliminates the need for IgM testing; it provides comparable or better sensitivity, and it maintains high specificity.

Identification of Enterobacteriaceae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using the VITEK MS system.

This multicenter study evaluated the accuracy of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry identifications from the VITEK MS system (bioMérieux, Marcy l’Etoile, France) for Enterobacteriaceae typically encountered in the clinical laboratory. Enterobacteriaceae isolates (n = 965) representing 17 genera and 40 species were analyzed on the VITEK MS system (database v2.0), in accordance with the manufacturer’s instructions. Colony growth (≤72 h) was applied directly to the target slide. Matrix solution (α-cyano-4-hydroxycinnamic acid) was added and allowed to dry before mass spectrometry analysis. On the basis of the confidence level, the VITEK MS system provided a species, genus only, or no identification for each isolate. The accuracy of the mass spectrometric identification was compared to 16S rRNA gene sequencing performed at MIDI Labs (Newark, DE). Supplemental phenotypic testing was performed at bioMérieux when necessary. The VITEK MS result agreed with the reference method identification for 96.7 % of the 965 isolates tested, with 83.8 % correct to the species level and 12.8 % limited to a genus-level identification. There was no identification for 1.7 % of the isolates. The VITEK MS system misidentified 7 isolates (0.7 %) as different genera. Three Pantoea agglomerans isolates were misidentified as Enterobacter spp. and single isolates of Enterobacter cancerogenus, Escherichia hermannii, Hafnia alvei, and Raoultella ornithinolytica were misidentified as Klebsiella oxytoca, Citrobacter koseri, Obesumbacterium proteus, and Enterobacter aerogenes, respectively. Eight isolates (0.8 %) were misidentified as a different species in the correct genus. The VITEK MS system provides reliable mass spectrometric identifications for Enterobacteriaceae.

Multi-centre evaluation of mass spectrometric identification of anaerobic bacteria using the VITEK® MS system

Accurate and timely identification of anaerobic bacteria is critical to successful treatment. Classic phenotypic methods for identification require long turnaround times and can exhibit poor species level identification. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an identification method that can provide rapid identification of anaerobes. We present a multi-centre study assessing the clinical performance of the VITEK(®) MS in the identification of anaerobic bacteria. Five different test sites analysed a collection of 651 unique anaerobic isolates comprising 11 different genera. Multiple species were included for several of the genera. Briefly, anaerobic isolates were applied directly to a well of a target plate. Matrix solution (α-cyano-4-hydroxycinnamic acid) was added and allowed to dry. Mass spectra results were generated with the VITEK(®) MS, and the comparative spectral analysis and organism identification were determined using the VITEK(®) MS database 2.0. Results were confirmed by 16S rRNA gene sequencing. Of the 651 isolates analysed, 91.2% (594/651) exhibited the correct species identification. An additional eight isolates were correctly identified to genus level, raising the rate of identification to 92.5%. Genus-level identification consisted of Actinomyces, Bacteroides and Prevotella species. Fusobacterium nucleatum, Actinomyces neuii and Bacteroides uniformis were notable for an increased percentage of no-identification results compared with the other anaerobes tested. VITEK(®) MS identification of clinically relevant anaerobes is highly accurate and represents a dramatic improvement over other phenotypic methods in accuracy and turnaround time.

Performance of United States Serologic Assays in the Diagnosis of Lyme Borreliosis Acquired in Europe

BACKGROUND Physicians in the United States sometimes need to evaluate a patient for suspected Lyme borreliosis (LB) who may have acquired the infection in Europe. Using serum samples from European LB patients, we compared the performance of European and US serodiagnostic tests, including newer-generation assays containing Vmp-like sequence, expressed or its C6 peptide. METHODS The sensitivity of each assay was determined using 64 serum samples from LB patients with early or late disease manifestations who acquired the infection in Europe. Specificity was measured using 100 sera from healthy subjects from a nonendemic area. RESULTS For the detection of European-acquired infection, conventional 2-tiered testing (enzyme-linked immunosorbent assay [ELISA] followed by immunoblotting) using US assays had an overall sensitivity and specificity of 52% and 100%, compared with 81% (P = .0007) and 99% (P = 1.00) using analogous European tests. The sensitivity of a US C6 ELISA used as a stand-alone test (88% overall) was statistically comparable to that of conventional 2-tiered testing using European tests (P = .47) and was 100% specific. Similarly, an alternative 2-tiered algorithm using a standard US ELISA followed by a C6 ELISA was comparably sensitive (84% overall) compared with conventional 2-tiered testing using European assays (P = .82), and specificity remained 100%. CONCLUSIONS European assays outperformed analogous US assays in a conventional 2-tiered testing algorithm. However, a C6 ELISA used as a stand-alone test or in the second tier of a 2-tiered algorithm performed comparably to conventional 2-tiered testing using European assays, and can be used for evaluation of any patient, regardless of travel history.

A Rational Approach to the Stool Ova and Parasite Examination

BACKGROUND Examination of multiple stool specimens per patient to rule out parasitic infection continues to be recommended in the literature. Attractive alternatives have been proposed, such as examination of a single specimen, but data to support their use have been inconclusive. METHODS We reviewed the results of comprehensive stool ova and parasite examinations performed during a 1-year period to determine the incremental value of examining >1 specimen. Next, we implemented rejection criteria, allowing analysis of only a single specimen in most cases, and studied the impact of the change by reviewing data from a subsequent year. RESULTS Prior to implementation of rejection criteria, 91% of parasites were detected in the first specimen submitted, although many clinical evaluations (72%) involved the submission of only 1 stool specimen. When at least 3 specimens were submitted, the sensitivity of examining the first in the series was 72%. Even the latter sensitivity provides negative predictive values of approximately 98%, approximately 97%, approximately 95%, or approximately 93% when the prevalence of parasites among those tested is 5%, 10%, 15%, or 20%, respectively. Examination of additional specimens after examination of the first specimen that yielded a positive finding revealed previously undetected parasites in only 10% of cases. After the application of rejection criteria, the parasite detection rate did not change significantly. CONCLUSIONS Comprehensive examination of a single stool specimen is sufficient for most patients, when the prevalence of infection among the tested population is up to 20%. Rational use of the stool ova and parasite examination relies on communication between clinician and laboratory, and retention of deferred specimens in case examination of additional specimens is clinically warranted.

Performance of the Vitek MS v2.0 system in distinguishing Streptococcus pneumoniae from nonpneumococcal species of the Streptococcus mitis group.

ABSTRACT The Vitek MS v2.0 matrix-assisted laser desorption ionization–time of flight mass spectrometry system accurately distinguished Streptococcus pneumoniae from nonpneumococcal S. mitis group species. Only 1 of 116 nonpneumococcal isolates (<1%) was misidentified as S. pneumoniae. None of 95 pneumococcal isolates was misidentified. This method provides a rapid, simple means of discriminating among these challenging organisms.