Multi-center evaluation of the VITEK® MS system for mass spectrometric identification of non-Enterobacteriaceae Gram-negative bacilli.

Studies have demonstrated that matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid, accurate method for the identification of clinically relevant bacteria. The purpose of this study was to evaluate the performance of the VITEK MS v2.0 system (bioMérieux) for the identification of the non-Enterobacteriaceae Gram-negative bacilli (NEGNB). This multi-center study tested 558 unique NEGNB clinical isolates, representing 18 genera and 33 species. Results obtained with the VITEK MS v2.0 were compared with reference 16S rRNA gene sequencing and when indicated recA sequencing and phenotypic analysis. VITEK MS v2.0 provided an identification for 92.5 % of the NEGNB isolates (516 out of 558). VITEK MS v2.0 correctly identified 90.9 % of NEGNB (507 out of 558), 77.8 % to species level and 13.1 % to genus level with multiple species. There were four isolates (0.7 %) incorrectly identified to genus level and five isolates (0.9 %), with one incorrect identification to species level. The remaining 42 isolates (7.5 %) were either reported as no identification (5.0 %) or called "mixed genera" (2.5 %) since two or more different genera were identified as possible identifications for the test organism. These findings demonstrate that the VITEK MS v2.0 system provides accurate results for the identification of a challenging and diverse group of Gram-negative bacteria.

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.

Enteric fever in a 6-year-old traveler caused by Salmonella enterica serotypes Typhi and Paratyphi A: laboratory detection strategies and treatment options.

We report the first pediatric case of enteric fever caused by Salmonella enterica serotypes Typhi and Paratyphi A. Mixed infections are infrequently reported, potentially because detection of two different Salmonella serotypes in blood cultures is technically challenging. We suggest laboratory strategies to aid in the recognition of mixed infections.

Roseomonas mucosa isolated from bloodstream of pediatric patient.

We report a case of catheter-related bacteremia associated with Roseomonas mucosa isolated from an immunocompromised pediatric patient with a history of multiple episodes of urinary tract infection and bacteremia.

Molecular basis of antagonism between K70E and K65R tenofovir-associated mutations in HIV-1 reverse transcriptase.

The K70E mutation in HIV-1 reverse transcriptase was observed in 10% of virologic non-responders of the abacavir/lamivudine/tenofovir arm of ESS30009, alone, or in mixtures with K65R by population sequencing. Clonal analysis of six ESS30009 K70E isolates failed to identify double mutants carrying K65R+K70E. Site-directed K70E mutants had a replication capacity of 97+/-29%, but only 2.4+/-0.9% for K65R+K70E and 0.01% for K65R+K70E+M184V mutants. K65R+K70E phenotypic fold changes for abacavir, lamivudine and tenofovir were comparable to reported values for K65R alone. In molecular dynamic simulations, the epsilon-amino group of K65 was positioned 2.7+/-0.1A from the gamma-phosphate of the dTTP ligand and stabilized the triphosphate. In the R65 mutant, this distance increased to 4.2+/-0.4A and the interaction energy with the ligand was less favorable, but the K70 epsilon-amino group was repositioned closer to the gamma-phosphate and had a more favorable interaction energy. In the double mutant, E70 could not stabilize the gamma-phosphate, resulting in a more severe defect. The net effect of the atomic-level changes in the double mutant may be to destabilize the pyrophosphate leaving group of the ligand, more severely affecting the catalytic rate of the polymerization reaction than the R65 single mutation.

Increasing prevalence of HIV-1 protease inhibitor-associated mutations correlates with long-term non-suppressive protease inhibitor treatment.

Treatment of human immunodeficiency virus type 1 with protease inhibitors (PIs) is associated with the emergence of resistance-associated mutations. Treatment-characterized datasets have been used to identify novel treatment-associated protease mutations. In this study, we utilized two large reference laboratory databases (>115,000 viral sequences) to identify non-established resistance-associated protease mutations. We found 20 non-established protease mutations occurring in 82% of viruses with a PI resistance score of 4-7, 62% of viruses with a resistance score of 1-3, and 35% of viruses with no predicted PI resistance. We correlated mutational prevalence to treatment duration in a treatment-characterized dataset of 2161 patients undergoing non-suppressive PI therapy. In the non-suppressed dataset, 24 mutations became more prevalent and three mutations became less prevalent after more than 48 months of non-suppressive PI-therapy. Longer durations of non-suppressive treatment correlated with higher PI resistance scores. Mutations at eight non-established positions that were more common in viruses with the longest duration of non-suppressive therapy were also more common in viruses with the highest PI resistance score. Covariation analysis of 3036 protease amino acid substitutions identified 75 positive and nine negative correlations between resistance associated positions. Our findings support the utility of reference laboratory datasets for surveillance of mutation prevalence and covariation.

Ileal resection-induced gallstones: altered bilirubin or cholesterol metabolism?

Ileal resection has been shown to increase the risk of cholelithiasis. Earlier studies in humans suggested that ileal resection increases the cholesterol saturation index. Recent data from patients on long-term parenteral nutrition and from animals, however, have suggested that ileal resection predisposes to pigment gallstone formation. We therefore tested the hypothesis that ileal resection alters bile calcium and bilirubin metabolism without affecting the cholesterol saturation index. Adult male prairie dogs underwent either sham laparotomy (eight prairie dogs) or ileal resection (16 prairie dogs). All animals were fed a trace cholesterol (nonlithogenic) diet before and for 4 weeks after operation. Pigment gallstones were present in 44% of the ileal-resected animals and in none of the sham animals (p less than 0.05). Calcium bilirubinate crystals were present in 94% of the ileal-resected animals and in none of the sham animals (p less than 0.01). Gallbladder bile calcium (25.6 +/- 2.4 versus 17.2 +/- 1.1 mg/dl; p less than 0.05) and total bilirubin (29.3 +/- 4.0 versus 9.4 +/- 1.8 mg/dl; p less than 0.01) concentrations were significantly greater in ileal-resected animals. The cholesterol saturation index of gallbladder bile, however, was no different in ileal-resected (0.53 +/- 0.04) and in sham-operated animals (0.50 +/- 0.04). Although initial studies suggested that the cholesterol saturation index of hepatic bile was increased after ileal resection, a second set of experiments demonstrated that this phenomenon resulted from washout of bile salts that were already in extremely low concentrations in hepatic bile. We conclude that alterations in bilirubin, but not cholesterol, metabolism result in pigment gallstone formation after ileal resection.

Sensitivity of multiplex real-time PCR reactions, using the LightCycler and the ABI PRISM 7700 Sequence Detection System, is dependent on the concentration of the DNA polymerase.

The introduction of multiplex PCR techniques to clinical laboratories has provided a means to streamline assays and to produce multiple results with minimal effort. While this methodology is very beneficial, care must be taken to ensure that reactions are properly optimized to allow for maximum sensitivity. This study was conducted to determine whether the sensitivity of multiplex-real-time PCR assays could be improved by increasing the concentration of DNA polymerase within a reaction. Multiplex reactions were designed to simultaneously detect the human HLA-DQ gene and a sequence from the UL83 region of the CMV genome. Two real-time PCR systems, one utilizing AmpliTaq Gold DNA polymerase and the ABI 7700 Sequence Detection System, and one utilizing FastStart Taq DNA polymerase and the Roche LightCycler were tested. The results indicated that increasing the AmpliTaq Gold concentration from 0.050 to 0.10 U/microl and the FastStart Taq concentration from 0.1875 to 0.375 U/microl increased detection sensitivity from 5,000 to 50 CMV copies per PCR reaction. In separate experiments, commercially prepared mastermixes were utilized for both real-time PCR platforms as per the manufacturer's suggestions or with the addition of supplemental DNA polymerase. In assays designed to detect 4 CMV genome copies per reaction, the addition of 2.5 U of AmpliTaq Gold to TaqMan Universal Mastermix increased the detection rate from 21 to 67%, and the addition of 5 U of FastStart Taq to FastStart DNA Master Hybridization Probes mastermix increased the detection rate from 17 to 56%. These results indicate that increasing the DNA polymerase concentration in multiplex real-time PCR reactions may be a simple way to optimize assay sensitivity.

The cholecysto-sphincter of Oddi reflex.

Multiple factors including hormonal and neural influences as well as intravascular volume, body temperature, and pharmacologic agents have been shown to influence sphincter of Oddi function. Recent studies have also suggested that mechanical or electrical stimulation of the gallbladder and the degree of gallbladder filling may affect the frequency and amplitude of sphincter of Oddi phasic contractions. However, the effect of gallbladder filling on sphincter of Oddi phasic wave direction has not previously been studied. In acute terminal experiments, eight adult male prairie dogs underwent cannulation of the gallbladder with a pressure-monitored perfusion catheter. The common bile duct was cannulated proximally with a drainage catheter and distally with a triple-lumen, side-hole, closed-tipped catheter. The distal port of this catheter was positioned in the sphincter of Oddi (SO) while the proximal port was in the distal common bile duct (CBD). Distal CBD and SO phasic wave activity was recorded first with the gallbladder perfused with lactated Ringer's solution at 0.1 ml/min (mean intragallbladder pressure 8.1 +/- 0.3 mm Hg) and then with the gallbladder (GB) empty. Sphincter of Oddi phasic wave frequency was 6.7 +/- 0.9/min with the GB full and 5.4 +/- 0.6/min with the GB empty (P less than 0.02). Phasic wave amplitude was also greater with the GB full versus empty in both the distal CBD (6.4 +/- 0.6 vs 4.3 +/- 0.5 mm Hg, P less than 0.05) and the SO (9.5 +/- 1.5 vs 6.4 +/- 0.8, P = 0.075). Baseline pressures and the direction of phasic waves were unaffected by the degree of gallbladder filling.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation of immunity in experimental syphilis with serum-mediated aggregation of Treponema pallidum rare outer membrane proteins.

We have previously shown by freeze-fracture electron microscopy that serum from infection-immune syphilitic rabbits aggregates the low-density membrane-spanning Treponema pallidum rare outer membrane proteins (TROMPs). The purpose of this study was to determine if a relationship could be demonstrated between acquired immunity in experimental rabbit syphilis, serum complement-dependent treponemicidal antibody, and antibody directed against TROMPs as measured by the aggregation of TROMP particles. Three groups of T. pallidum-infected rabbits were treated curatively with penicillin at 9 days, 30 days, and 6 months postinfection to generate various degrees of immunity to challenge reinfection. Sera from rabbits completely susceptible to localized and disseminated reinfection possessed a low titer of treponemicidal antibody (/=50% of a treponemal suspension) and showed a correspondingly low level of TROMP aggregation (16.5% of the total number of outer membrane particles counted) similar to normal serum controls (13. 4%); the number of particles within these aggregates never exceeded three. Sera from partially immune rabbits, which were susceptible to local reinfection but had no evidence of dissemination, showed an increase in the titer of treponemicidal antibody (1:16) compared to the completely susceptible group (

Immunization with Treponema pallidum outer membrane vesicles induces high-titer complement-dependent treponemicidal activity and aggregation of T. pallidum rare outer membrane proteins (TROMPs).

The purpose of this study was to determine whether immunization with purified outer membrane vesicles (OMV) from Treponema pallidum (T.p. ) could elicit Abs capable of killing this organism. It is well established that the immunization of rabbits or mice with killed T.p. or with recombinant T.p. Ags has failed to generate serum killing activity comparable with that of infection-derived immunity. Because of the small amount of T.p. OMV obtainable, a single mouse was immunized with purified OMV. The mouse anti-OMV serum and infection-derived immune rabbit serum (IRS) were compared by reactivities on two-dimensional T.p. immunoblots and by the T.p. immobilization test, a complement-dependent killing assay. Whereas IRS detected >40 Ags, the anti-OMV serum identified only 6 Ags corresponding to proteins identified previously in the outer membrane. T.p. immobilization testing showed that IRS had a 100% killing titer of 1:44 and a 50% killing titer of 1:662. By comparison, the mouse anti-OMV serum had a significantly greater 100% killing titer of 1:1,408 and a 50% killing titer of 1:16,896. Absorption of the anti-OMV serum to remove Ab against outer membrane-associated lipoproteins did not change the 100% killing titer. Freeze-fracture analysis of T.p. incubated in IRS or anti-OMV serum showed that T.p. rare membrane-spanning outer membrane proteins were aggregated. This is the first demonstration of high-titer killing Abs resulting from immunization with defined T.p. molecules; our study indicates that the targets for these Abs are T. p. rare outer membrane proteins.

Treponemicidal antibody measured by the "washed-killing" assay correlates with immunity in experimental rabbit syphilis.

BACKGROUND AND OBJECTIVES: The authors have previously shown that complement-dependent treponemicidal antibody measured by the "washed-killing" assay is directed exclusively against surface-exposed targets on Treponema pallidum, presumably the Treponema pallidum rare outer membrane proteins detected by freeze-fracture electron microscopy. GOAL OF THIS STUDY: Because immune mechanisms against Treponema pallidum rare outer membrane proteins are likely to be central to a protective host response, it was examined whether a relationship could be established between treponemicidal levels as measured by the "washed-killing" assay and host immunity in experimental syphilis. STUDY DESIGN: Three groups of Treponema pallidum-infected rabbits were treated curatively with penicillin at 9 days, 30 days, and 6 months post-infection to generate animals with varying degrees of immunity to challenge re-infection. The level of complement-dependent treponemicidal activity in sera obtained before infection (basal) and before intradermal challenge was determined by the "washed-killing" assay and compared with that detected using conventional in vitro immobilization. RESULTS: Using the "washed-killing" assay, a close quantitative correlation as measured by a treponemal immobilizing endpoint titer was demonstrable between prechallenge treponemicidal antibody and the status of immunity to re-infection. Sera from rabbits completely susceptible to symptomatic and disseminated asymptomatic re-infection lacked treponemicidal antibody. Sera from challenged rabbits with a relatively low degree of immunity to symptomatic disease showed endpoints of < or = 4. Rabbits with a relatively high degree of immunity to symptomatic reinfection and resistant to disseminated disease had endpoints that ranged from 6 to 96. Rabbits completely resistant to challenge exhibited endpoints ranging from 96 to 128. CONCLUSION: Treponemicidal antibody measured by the "washed-killing" assay correlated closely with the status of immunity in experimental rabbit syphilis. Thus, antibody measured by this assay may be directed against key protective Treponema pallidum surface immunogens.