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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. [ABSTRACT FROM AUTHOR]

Background: Stenotrophomonas maltophilia is a significant nosocomial pathogen, responsible for an increasing number of infections, particularly in immunocompromised patients., Aim: This study describes the clinical and microbiological characteristics of patients with Stenotrophomonas maltophilia bacteraemia., Methods: We reviewed the charts of 102 patients with Stenotrophomonas maltophilia bacteraemia over a seven-year period from 2001 to 2007 in two tertiary care centres in New York, USA., Findings: There were 79 episodes (77.5%) related to nosocomial acquisition, 21 (20.6%) were healthcare-associated and two episodes (2%) were community-acquired. The most common source of bacteraemia was an infected central catheter in 44 patients (43.1%); 17 (16.6%) were related to neutropenic sepsis; nine (8.8%) were from an abdominal source; six (5.9%) were from a respiratory source, and the source of the bacteraemia was unclear in 26 cases (25.5%). The majority (94.1%) of the patients had central venous access devices. Intensive care unit stay, intubation, septic shock, neutropenia at the time of bacteraemia or carbapenem antibiotic use within 30 days of the episode were associated with mortality according to univariate analysis. By multivariate analysis, hypotensive shock and carbapenem use within 30 days of the episode were factors significantly correlated with mortality. The 102 isolates were mostly susceptible in vitro to trimethoprim-sulfamethoxazole (97.1%), levofloxacin (92.9%), ceftazidime (53.0%) and ticarcillin-clavulanic acid (49.2%)., Conclusion: Our data describe the characteristics of patients with Stenotrophomonas maltophilia bacteraemia and emphasize the importance of careful evaluation of vascular access devices in those patients., (Copyright © 2012 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Resistance to HIV-1 infection and delayed disease progression have been associated with a 32-bp deletion (Delta32) in the gene encoding the CCR5 chemokine receptor. In the present study we describe the modification of a nucleic acid sequence-based amplification (NASBA)-based CCR5 genotyping assay for a NucliSens Basic Kit (Organon Teknika, Durham, N.C.) format using a new target-specific sandwich oligonucleotide detection methodology. The new method permitted the use of generic electrochemiluminescent probes supplied in the NucliSens Basic Kit, whereas the original NASBA method required expensive target-specific ruthenium detection probes. The Basic Kit CCR5 Delta32 genotypic analysis was in 100% concordance with both the original NASBA assay and DNA PCR results. This study also evaluated the use of multiple specimen types, including peripheral blood mononuclear cells (PBMC), whole blood, dried blood spots, buccal scrapings, and plasma, for CCR5 genotype analysis. The sensitivities of the three assays were comparable when PBMC or whole blood was the specimen source. In contrast, when dried blood spots, buccal scrapings, or plasma was used as the sample source, the sensitivity of DNA PCR was 80.95, 42.8, or 0%, respectively, compared to 100% sensitivity obtained with the original NASBA and Basic Kit NASBA assays. Our study indicates that the NucliSens Basic Kit NASBA assay is very sensitive and specific for CCR5 Delta32 genotyping using multiple sample types.

A nucleic acid sequence-based amplification (NASBA) assay for qualitative detection of human cytomegalovirus (CMV) pp67 mRNA was evaluated in a multicenter study. Negative results were obtained for all specimens from 50 CMV-seronegative and 50 CMV-seropositive low-risk whole-blood donors. No interference with CMV mRNA amplification was observed in the testing of 288 specimens containing various potential interfering substances, nonspecifically reacting substances (including mRNA from other herpesviruses), and three anticoagulants. A total of 95% (50 of 51) of CMV-positive (cell culture- and antigenemia immunofluorescence [AG-IFA]-positive) clinical specimens were positive by the NASBA assay. Results from different operators over multiple testing days were consistent for each of four panel members containing different concentrations of CMV mRNA, indicating the reproducibility of the assay. The estimated 95% reliable upper detection limit of the assay was 600 mRNA copies; the lower limit of detection was less than 25 mRNA copies. The clinical utility of the assay was evaluated with longitudinally collected specimens from solid-organ transplant patients (n = 21). A total of 98% (81 of 83) of the specimens from CMV-negative patients were negative by the NASBA assay, while 90% (10 of 11) of patient specimens that were positive by cell culture or AG-IFA were positive by the NASBA assay. Positive NASBA assay results were obtained earlier than AG-IFA or cell culture results for 55% of the patients and at the same time for the remainder of the patients (45%). The overall agreement between the NASBA assay and current reference tests was 86% when active CMV infection was present. These studies indicate that the CMV pp67 mRNA NASBA assay has reproducible and sensitive performance characteristics that should enable more rapid diagnosis of CMV infection.

This study examined the clinical correlation between the presence of human cytomegalovirus (HCMV) pp67 mRNA in cerebrospinal fluid (CSF) and active HCMV central nervous system (CNS) disease in patients with human immunodeficiency virus type 1 (HIV-1). In total, 76 CSF specimens collected from 65 HIV-1-positive patients diagnosed with HCMV CNS disease, other non-HCMV-related CNS diseases, or no CNS disease were tested for the presence of HCMV pp67 mRNA using the NucliSens cytomegalovirus (CMV) pp67 assay (Organon Teknika, Durham, N.C.). The results were compared to those of a nested PCR for the detection of HCMV glycoprotein B DNA and to those obtained by viral culture (54 samples). CSF specimens collected from patients without HCMV CNS disease yielded the following results: pp67 assay negative, 62 of 62 specimens; culture negative, 41 of 41 specimens; and PCR negative, 56 of 62 specimens (6 specimens were positive). CSF specimens collected from patients with HCMV CNS disease yielded the following results: pp67 assay positive, 9 of 13 specimens; PCR positive, 13 of 13 specimens; and culture positive, 2 of 13 specimens. After resolution of the discordant results, the following positive and negative predictive values (PPV and NPV, respectively) for the diagnosis of HCMV CNS disease were determined. The PPV for PCR, pp67 assay, and culture were 68.4, 100, and 100%, respectively, and the NPV for PCR, pp67 assay, and culture were 100, 97.0, and 82. 7%, respectively. The sensitivities for DNA PCR, pp67 assay, and culture for the detection of HCMV were 100, 84.6, and 18%, respectively, and the clinical specificities were 90.5, 100, and 100%, respectively. This study indicates that the detection of HCMV pp67 mRNA in CSF has good correlation with active HCMV CNS disease, whereas CSF culture is insensitive and qualitative DNA PCR may detect latent nonreplicating virus in CSF from patients without HCMV CNS disease.

The relationship between specimen input volume and the frequency of reported human immunodeficiency virus type 1 (HIV-1) RNA copy numbers by nucleic acid amplification technology (the NASBA HIV-1 RNA QT system) was investigated. Results obtained with both clinical specimens and dilution panels indicated that both the absolute number of reported results and the reported HIV-1 RNA copy number were directly proportional to the specimen input volumes evaluated (0.1, 0.5, and 1.0 ml). Conversion of the reported HIV-1 RNA copy numbers to a constant 1.0-ml volume indicated that the numerical relationship among the specimen input volumes and the HIV-1 RNA copy numbers was multiplicative. The HIV-1 RNA copy numbers reported for the 0.5-ml input volume were approximately 5-fold increased over those reported for the 0.1-ml input volume, and those reported for the 1.0-ml input volume were 10-fold increased over those reported for the 0.1-ml input volume. For the specimen input volumes investigated, a common linear range of 264 to 5,400,000 HIV-1 RNA copies was observed. The use of increased specimen input volumes did not result in a loss of assay specificity, as the results reported for specimens from 50 seronegative blood donors were negative at all three specimen input volumes. In conclusion, an increase in the input volume of specimens analyzed by nucleic acid amplification technology can be useful for the enhanced detection of HIV-1 RNA.

The human CCR5 chemokine receptor functions as a coreceptor with CD4 for infection by macrophage-tropic isolates of human immunodeficiency virus type 1 (HIV-1). A mutated CCR5 allele which encodes a protein that does not function as a coreceptor for HIV-1 has been identified. Thus, expression of the wild-type and/or mutation allele is relevant to determining the infectability of patient peripheral blood mononuclear cells (PBMC) and affects disease progression in vivo. We developed a qualitative CCR5 genotyping assay using NASBA, an isothermal nucleic acid amplification technology. The method involves three enzymes and two oligonucleotides and targets the CCR5 mRNA, which is expressed in PBMC at a copy number higher than 2, the number of copies of DNA present encoding the gene. The single oligonucleotide set amplifies both alleles, and genotyping is achieved by separate hybridizations of wild-type- and mutation-specific probes directly to the single-stranded RNA amplification product. Assay sensitivity and specificity were demonstrated with RNAs produced in vitro from plasmid clones bearing the DNA encoding each allele. No detectable cross-reactivity between wild-type and mutation probes was found, and 50 copies of each allele were readily detectable. Analysis of patient samples found that 20% were heterozygous and 1% were homozygous for the CCR5 mutation. Thus, NASBA is a sensitive and specific means of rapidly determining CCR5 genotype and provides several technical advantages over alternative assay systems.

Together with CD4(+)-cell counts as an indicator of immune function, the use of human immunodeficiency virus type 1 (HIV-1) RNA levels as a direct marker of viral load has gained widespread attention for evaluation of patient clinical status. Results obtained with other HIV-1 markers for this purpose are often inconsistent. This study examined the relationship between various HIV-1 markers by using clinical specimens (plasma) from HIV-1-infected individuals at different stages of disease progression and supernatant fluid from four human T-lymphocyte cell lines chronically infected with HIV-1. Cell culture specimens were collected periodically over 7 days and were tested for HIV-1 RNA levels with a nucleic acid amplification assay, for p24 with an enzyme-linked immunosorbent assay, and for reverse transcriptase activity by isotope uptake. An increase in the level of each marker was observed over the 7-day period with each of the four HIV-1 strains tested (LAV1, HTLV-IIIB, MN, and ARV2); with these specimens, the frequency of detection for each marker was 100%. In the clinical specimens, HIV-1 RNA was detected more often (143 of 183 specimens [78%]) than was p24 (87 of 183 [48%]); little correlation between the levels of the two markers was seen. In these clinical specimens evaluated, CD4(+)-cell counts were better correlated with the frequency and levels of HIV-1 RNA than with p24. In specimens (n = 38) collected serially from six HIV-1-infected subjects, HIV-1 RNA was detected more often (33 of 38 [85%]) than p24 (23 of 38 [59%]). When reported by the assays used, the levels of both HIV-1 markers fluctuated over time for each of the subjects. Although the markers correlated in the in vitro systems studied, the observed differences in the correlation of levels and frequencies of HIV-1 markers in vivo indicate that p24 has less clinical utility than does viral load testing when used in conjunction with CD4(+)-cell counts as a measure of immune system functioning.

The Salmonella typhimurium InvA protein is a component of a sec-independent secretion apparatus necessary for full virulence of the bacteria. We generated a monoclonal antibody to the C-terminal portion of the InvA protein that recognized proteins in S. typhimurium and weakly in Y. enterocolitica, but not in several other species of bacteria, including S. flexneri. S. typhimurium grown without agitation produced relatively constant amounts of membrane InvA throughout the growth cycle, whereas bacteria grown with agitation had a sharp increase in the amount of membrane InvA at late exponential phase. Levels of InvA present in Salmonella membranes under some growth conditions do not appear to correlate with levels of invasion under the same conditions.

The enteric bacteria Salmonella typhimurium has the ability to invade (enter) nonphagocytic cells. The internalization process occurs as a result of an intimate interaction between the bacteria and the host cell, in which S. typhimurium triggers a cascade of host cell-signaling events leading to the formation of host cell membrane ruffles and bacterial uptake. Using high resolution scanning electron microscopy, we have observed that contact with cultured epithelial cells results in the formation of appendages on the surface of S. typhimurium. The formation of such appendages did not require de novo protein synthesis, and it was transient, since these surface structures were no longer present on bacteria that had initiated the internalization event. Salmonella mutants defective in the transient formation of these surface organelles were unable to enter into cultured epithelial cells, indicating that such structures are required for bacterial internalization.