Detection and pharmacokinetics of a cytomegalovirus (CMV) DNA plasmid in human plasma during a clinical trial of an intramuscular CMV vaccine in hematopoietic stem cell transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) causes significant morbidity and mortality in solid organ and bone marrow transplant recipients. DNA vaccines can provide both humoral and cellular immunity without exposing immune-compromised persons to replication-competent CMV. METHODS: We studied the kinetics of CMV vaccine DNA in plasma. The samples were obtained from vaccine recipients who were enrolled in a double-blinded, placebo-controlled clinical trial of an intramuscular, plasmid-based, bivalent DNA vaccine for CMV in stem cell transplant recipients. Residual specimens on patients enrolled in the vaccine trial were saved until the trial was unblinded and published. Quantitative real-time polymerase chain reaction (PCR) was used to detect and quantify CMV glycoprotein B (gB) DNA in plasma from 4 recipients of the vaccine. The melting temperature of the vaccine gB amplicon was 62.4°C, compared to 68.8°C, which is seen with the wild-type virus. RESULTS: Sequence analysis revealed that there were 3 mismatches between the fluorescent resonance energy transfer probe and the vaccine DNA sequence. CONCLUSION: Because preemptive treatment of CMV disease in stem cell transplant patients is based on quantitative PCR analysis of viral sequences in plasma, it is important that vaccine sequences not be confused with those in wild-type virus. Confusion could lead to treatment with toxic medications, potentially compromising the transplant. Effects of PCR target choice and amplicon detection techniques on patient management and vaccine trials are discussed.

An international, prospective, multicenter evaluation of the combination of AdvanDx Staphylococcus QuickFISH BC with mecA XpressFISH for detection of methicillin-resistant Staphylococcus aureus isolates from positive blood cultures.

Sepsis caused by Staphylococcus aureus is a major health problem worldwide. Better outcomes are achieved when rapid diagnosis and determination of methicillin susceptibility enable early optimization of antimicrobial therapy. Eight large clinical laboratories, seven from the United States and one from Scotland, evaluated the combination of the Staphylococcus QuickFISH BC and the new mecA XpressFISH assay (both AdvanDx, Woburn, MA, USA) for the detection of methicillin-resistant S. aureus in positive blood cultures. Blood cultures flagged as positive by automated blood culture instruments and demonstrating only Gram-positive cocci in clusters on Gram stain were tested by QuickFISH, a 20-min assay. If only S. aureus was detected, mecA XpressFISH testing followed. The recovered S. aureus isolates were tested by cefoxitin disk diffusion as the reference method. The QuickFISH assay results were concordant with the routine phenotypic testing methods of the testing laboratories in 1,211/1,221 (99.1%) samples and detected 488/491 S. aureus organisms (sensitivity, 99.4%; specificity, 99.6%). Approximately 60% of the samples (730) contained coagulase-negative staphylococci or nonstaphylococci as assessed by the QuickFISH assay and were not tested further. The 458 compliant samples positive exclusively for S. aureus by the QuickFISH assay were tested by the mecA XpressFISH assay, which detected 209 of 211 methicillin-resistant S. aureus organisms (sensitivity, 99.1%; specificity, 99.6%). The mecA XpressFISH assay also showed high reproducibility, with 534/540 tests performed by 6 operators over 5 days achieving reproducible results (98.9% agreement). The combination of the Staphylococcus QuickFISH BC and mecA XpressFISH assays is sensitive, specific, and reproducible for the detection of methicillin-resistant S. aureus and yields complete results in 2 h after the blood culture turns positive.

Weissella confusa: an unexpected cause of vancomycin-resistant gram-positive bacteremia in immunocompromised hosts.

We report the first case of Weissella confusa bacteremia in an allogeneic hematopoietic stem cell transplant patient. After engraftment and discharge, the patient returned with fever and graft failure and was started on an empiric regimen of aztreonam and vancomycin. A blood culture grew an alpha-hemolytic, gram-positive coccus forming pairs and chains, originally thought to be a viridans Streptococcus and a skin contaminant. The isolation of the organism from multiple blood cultures, and the presence of vancomycin resistance prompted identification and additional susceptibility testing. The RapID(™) Str panel, which has W. confusa in its database, provided multiple incorrect identifications. The MicroScan WalkAway 96 SI, using PC-20 or -29 panels, also did not identify this bacterium, because it is not in their database. The organism was identified as W. confusa by 16S rDNA sequencing. Antibiotic susceptibility determination by Etest revealed vancomycin resistance and daptomycin susceptibility. Therapy was changed to daptomycin, and the infection resolved. Additionally, W. confusa sepsis, with multiple positive blood cultures, developed in a patient in the burn unit at our medical center. The patient's blood cultures remained positive until vancomycin was discontinued and daptomycin therapy initiated. Infections with vancomycin-resistant, gram-positive cocci are emerging among immuno compromised hosts. Under appropriate circumstances, clinicians need to request that the laboratory perform susceptibility testing and accurate identification, by nucleic acid sequencing if necessary. Sequencing of 16S rDNA is an important tool in the accurate identification of unusual pathogens.

Listeria grayi: vancomycin-resistant, gram-positive rod causing bacteremia in a stem cell transplant recipient.

We report the first case of Listeria grayi bacteremia in a stem cell transplant recipient. The patient developed bacteremia with a gram-positive rod that was initially thought to be Corynebacterium species and a skin contaminant. The organism grew in multiple blood cultures and therapy with vancomycin was initiated. The API Coryne (version 3.0) identified the organism as L. grayi. Susceptibility testing by Etest suggested that the organism was resistant to vancomycin, but susceptible to ampicillin. After therapeutic change from vancomycin to ampicillin, the bacteremia cleared. Empiric therapy with vancomycin for all gram-positive bacterial infections is not appropriate. Accurate identification and antibiotic susceptibility is important, particularly in those with persistent bacteremia.

Methicillin-resistant Staphylococcus aureus (MRSA) in hospitalized children: correlation of molecular analysis with clinical presentation and antibiotic susceptibility testing (ABST) results.

The molecular analysis of methicillin-resistant Staphylococcus aureus (MRSA) from 98 children admitted to the Children's Hospital of Michigan, Detroit, MI, with serious MRSA infections during 2006-2007 was correlated with risk factors, clinical features, and antibiotic susceptibility testing (ABST) results. Isolates were characterized by staphylococcal cassette chromosome (SCC) mec type, the presence of Panton-Valentine leukocidin (PVL) genes, repetitive sequence (rep) polymerase chain reaction (PCR) and pulsed-field gel electrophoresis (PFGE), requirement for surgical intervention, antibiograms, and response to therapy. rep-PCR was more rapid than PFGE typing and correlated well. SCCmec type IV-containing isolates caused 92.8% of all infections, but the demographics and diseases associated with subtypes IVa and IVd differed. Subtype IVa (all PFGE type USA300 and PVL-positive) was identified in 81/93 (87.1%) of patients with community-onset (CO) MRSA, including 21/35 of those with risk factors for health care-associated (HA) infection. All other clones were PVL-negative. Subtype IVd (10 isolates; 9 USA800 and 1 eMRSA15) caused mainly HA-MRSA and no skin and soft tissue infections (SSTI). Seven classic HA-MRSA strains (SCCmec types II [6; 3 USA100 and 3 USA600] and III [1; USA200]) caused HA and hospital-onset (HO) infections. Surgical intervention was required in 68/81 patients infected with USA300 and 8/17 of the others. Most USA300 were susceptible (S) to clindamycin (CD) and patients were treated with CD alone or in combination. The other isolates were generally treated with vancomycin (VA) alone or in combination.

Measurement of free fatty acids in cerebrospinal fluid from patients with hemorrhagic and ischemic stroke.

Free fatty acid (FFA) concentrations in cerebrospinal fluid (CSF) from patients with ischemic and hemorrhagic stroke (n=25) and in contemporary controls (n=73) were examined using HPLC. Concentrations of CSF FFAs from ischemic and hemorrhagic stroke patients obtained within 48 h of the insult were significantly greater than in control patients. Higher concentrations of polyunsaturated fatty acids (PUFAs) in CSF obtained within 48 h of insult were associated with significantly lower (P<0.05) admission Glasgow Coma Scale scores and worse outcome at the time of hospital discharge, using the Glasgow Outcome Scale (P<0.01).

Trichosporon asahii, a non-Candida yeast that caused fatal septic shock in a patient without cancer or neutropenia.

Trichosporon asahii (formerly Trichosporon beigelii) is an emerging fungal pathogen seen particularly in immunologically compromised patients. There are now approximately 100 reported cases of hematogenously disseminated infections with this life-threatening yeast, and no effective antifungal therapy is available. The present case is unusual because the patient did not have neutropenia or evidence of a malignancy.

Quantification of free fatty acids in human cerebrospinal fluid.

Free fatty acids (FFA) in cerebrospinal fluid (CSF) are well-recognized markers of brain damage in animal studies. Information is limited regarding human CSF in both normal and pathological conditions. Samples of CSF from 73 patients, who had undergone lumbar puncture for medically indicated reasons, came from a core laboratory upon completion of ordered tests. Using high performance liquid chromatography, mean FFA concentrations (microg/L +/- SEM) were: arachidonic 26.14 +/- 3.44; docosahexaenoic 60.74 +/- 5.70; linoleic 105.07 +/- 10.98; myristic 160.38 +/- 16.17; oleic 127.91 +/- 10.13; and palmitic 638.34 +/- 37.27. No differences in FFA concentrations were seen with gender, race, age, and/or indication for lumbar puncture. This is the first study to document normal human CSF FFA concentrations in a large series. Further characterization of FFA in pathological conditions may provide markers for evaluating clinical treatments and assisting in prognostication of neurological disease.

Pap smear follow-up of possible role of mucopurulent exudate as a prognosticator of a negative pregnancy outcome.

Our objective was to study a cohort of women by various means to detect sexually transmitted diseases (STD) and to correlate the presence of mucopurulent exudate (PEX) on Papanicolaou (Pap) smears with pregnancy outcome. Bacteriologic cultures taken from swabs for chlamydia and gonorrhea were correlated with Gen-Probe results. Smears were examined for trichomonas, yeast, HPV, obscuring mucopurulent exudate, and squamous intraepithelial abnormalities. There was no positive correlation between STD and negative pregnancy outcome. Mucopurulent exudate on Pap smears was very high in this population. Continuing study of this population of women is needed to see if larger cohorts will demonstrate any positive correlations between PEX and pregnancy outcome. Placing women with obscuring mucopurulent exudate in a "high-risk" category for complications may prevent adverse side effects to the birth product. The Pap smear works as well as the Gram stain for detection of bacterial vaginosis and cervicitis. Eliminating the Gram stain from routine pelvic exam results in cost savings.

Antibacterial activity of antibiotic coated silicone grafts.

PURPOSE: Postoperative infection remains one of the most serious complications of implantation of penile prostheses. Attempts to reduce the rate of infection by spraying the prosthesis with an antibiotic solution prior to implantation, along with perioperative antibiotics, have failed to eradicate infection. No published studies have evaluated the effect of antibiotic coating of penile prostheses. In this study, we evaluate the antibacterial effect of antibiotic-coated silicone strips as a surrogate for the penile prosthesis. MATERIALS AND METHODS: Strips coated with several different antibiotics were dipped in bacterial solutions containing Staphylococcus epidermidis or S. aureus and implanted subcutaneously in adult Sprague-Dawley rats. After a week, the strips were removed, and the number of bacteria on the strips and in the surrounding tissue were determined. The in vitro antibiotic activity of the antibiotic-coated strips against the same organisms was also determined. RESULTS: In the group of rats that received silicone strips contaminated in vitro with S. epidermidis, six of nine control rats yielded strips and tissues containing heavy bacterial growth. None of six strips coated with rifampin/minocycline yielded bacterial growth, nor did any of the seven strips coated with vancomycin. One of seven rats that received amikacin-coated strips had infection of the strip. The tissue results were similar to the strip results. In the group using S. aureus as the contaminating bacterium, the strips and tissues from eight of nine control rats yielded bacteria. None of the six rifampin/minocycline-coated strips yielded bacteria, while two of seven vancomycin-treated strips and two of six amikacin-coated strips were infected with S. aureus. The difference in bacterial growth between controls and antibiotic-coated strips reached a level of statistical significance for the rifampin/minocycline and vancomycin groups and was highly significant for the rifampin/minocycline groups. CONCLUSION: The experimental results presented here suggest that coating silicone graft material with antibiotics, particularly rifampin/minocycline, can reduce the incidence of graft colonization in contaminated wounds in rats, even in the absence of systemic antibiotics. These graft materials may prove useful in preventing the infection of penile prostheses.

Cutaneous infections due to Acanthamoeba in patients with acquired immunodeficiency syndrome.

We report 2 cases of cutaneous Acanthamoeba infection in patients with acquired immunodeficiency syndrome. The disease, which manifests as subcutaneous nodules, mimics other more commonly encountered clinical entities. A high index of suspicion, familiarity with the clinical and histologic appearance of skin lesions, and communication between clinicians and pathologists are crucial for early diagnosis and treatment of this potentially fatal infection.

Phialophora verrucosa infection in a BMT patient.

Phialophora is a dematiaceous fungus isolated from soil and wood. Human infections including chromoblastomycosis, mycotic keratitis, cutaneous infections, and prosthetic valve endocarditis have been reported. We report a case of fatal hemorrhage due to Phialophora verrucosa in a patient with prolonged neutropenia undergoing autologous bone marrow transplant (BMT) for acute myelogenous leukemia (AML). Bacterial infections complicated induction and consolidation chemotherapies. Liposomal amphotericin B (LAMB) was given from day +33 to day +72 for febrile neutropenia. Death occurred on day +74 due to tracheal hemorrhage. Autopsy revealed granulation tissue on the posterior wall of the trachea with fungal hyphae on histopathology; the tissue grew Phialophora verrucosa. In vitro susceptibility studies revealed a minimum inhibitory concentration to AmB of 0.1 microg/ml. This represents the first reported case of invasive P. verrucosa in a BMT patient leading to fatal hemorrhage, despite large cumulative doses of LAMB to which the organism remained susceptible.

Evaluation of the Gen-Probe amplified Mycobacterium tuberculosis direct detection test.

Mycobacterium tuberculosis direct (MTD) testing was performed on 423 specimens (from 293 patients) submitted for mycobacterial culture, including 121 from 47 patients with current or recent cultures positive for tuberculosis. Resolved sensitivity and specificity were 91.4% and 99.3%, respectively. The assay detected 12 of 14 patients with one specimen and 31 of 32 with > or = 2 specimens. The 10 missed specimens included 2 resembling blood clots and 6 smear negative or 1+ specimens; however, a 2+ and a 4+ specimen were missed, which implies inhibitors. Some smear positive/culture negative and smear negative/culture negative specimens from treated patients gave positive results. The number of smear negative, culture positive specimens from untreated patients was too low to evaluate rigorously. Consideration of these observations and of costs suggests that the assay is appropriate only for untreated patients with smear-positive, nonbloody specimens. They should not be reported as negative for tuberculosis until three smear positive specimens have given negative MTD results. Factors involved in the generation of false positive results, including amplicon contamination, are evaluated.

Adrenal insufficiency, recurrent bacteremia, and disseminated abscesses caused by Nocardia asteroides in a patient with acquired immunodeficiency syndrome.

Nocardia asteroides is an opportunistic pathogen of increasing incidence in human immunodeficiency virus (HIV)-infected persons. The lungs are the most common site of infection, followed by the brain; involvement of other extrapulmonary sites is less common. We describe a patient with acquired immunodeficiency syndrome who presented with a number of unique manifestations of nocardial infection: the first reported case of bilateral adrenal abscesses with adrenal insufficiency, the first case of a renal abscess due to N. asteroides alone, and the first case of recurrent, symptomatic bacteremia. A review of the literature on nocardial infections in HIV-positive individuals is presented.

Human herpesvirus 6 DNA in blood cells of human immunodeficiency virus-infected men: correlation of high levels with high CD4 cell counts.

The relationship between human herpesvirus 6 (HHV-6) and the progression of human immunodeficiency virus type 1 (HIV-1) infection to AIDS was evaluated using the polymerase chain reaction to quantitate HHV-6 genome equivalents in peripheral blood mononuclear cells (PBMC) and saliva of 32 HIV-1-seropositive men. Unlike other herpesviruses, HHV-6 was found with higher frequency and in higher copy numbers of HIV-infected men with high rather than low CD4 cell counts. Among subjects with > 400 CD4 cells/mL, 100% had detectable HHV-6 genomes in PBMC compared with 57.9% of subjects with < 400 CD4 cells/mL (P < .05). There was no significant correlation between HHV-6 antibody titer or number of amplifiable copies of HHV-6 DNA in saliva and stage of HIV disease or CD4 cell number.