Rebecca T. Horvat

Detection of Human Herpesvirus 8 DNA in Kaposi's Sarcoma Lesions and Peripheral Blood of Human Immunodeficiency Virus-Positive Patients and Correlation with Serologic Measurements

Polymerase chain reaction (PCR) was used to examine human herpesvirus 8 (HHV-8) DNA from Kaposis sarcoma (KS) lesions, normal skin, and peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV)-infected patients who did or did not have KS. Of 9 KS biopsies, 8 were positive for five HHV-8 open-reading frames and ranged from 1 viral genome per 2.5-12.7 cells. Two putative replicative gene RNAs were detected by reverse transcription-PCR at low levels in 1 KS lesion. HHV-8 DNA was detected in 4 of 8 PBMC samples from patients with KS and in 2 of 18 PBMC samples from patients without KS. Sera were tested for reactivity with BCBL-1 cells (HHV-8 positive): High immunofluorescence antibody titers against HHV-8 lytic and latent antigens were detected in samples from KS-positive patients, and >20 polypeptides from induced BCBL-1 cells were recognized. Sera from 6 of 18 patients without KS showed low levels of antibodies against HHV-8 lytic and latent antigens.

Francisella tularensis Induces Extensive Caspase-3 Activation and Apoptotic Cell Death in the Tissues of Infected Mice

ABSTRACT Although Francisella tularensis subsp. tularensis is known to cause extensive tissue necrosis, the pathogenesis of tissue injury has not been elucidated. To characterize cell death in tularemia, C57BL/6 mice were challenged by the intranasal route with type A F. tularensis, and the pathological changes in infected tissues were characterized over the next 4 days. At 3 days postinfection, well-organized inflammatory infiltrates developed in the spleen and liver following the spread of infection from the lungs. By the next day, extensive cell death, characterized by the presence of pyknotic cells containing double-strand DNA breaks, was apparent throughout these inflammatory foci. Cell death was not mediated by activated caspase-1, as has been reported for cells infected with other Francisella subspecies. Mouse macrophages and dendritic cells that had been stimulated with type A F. tularensis did not release interleukin-18 in vitro, a response that requires the activation of procaspase-1. Dying cells within type A F. tularensis-infected tissues expressed activated caspase-3 but very little activated caspase-1. When caspase-1-deficient mice were challenged with type A F. tularensis, pathological changes, including extensive cell death, were similar to those seen in infected wild-type mice. In contrast, type A F. tularensis-infected caspase-3-deficient mice showed much less death among their F4/80+ spleen cells than did infected wild-type mice, and they retained the ability to express tumor necrosis factor alpha and inducible NO synthase. These findings suggest that type A F. tularensis induces caspase-3-dependent macrophage apoptosis, resulting in the loss of potentially important innate immune responses to the pathogen.

Nationwide Antibiogram Analysis Using NCCLS M39-A Guidelines

ABSTRACT Lack of standardization in antibiogram (ABGM) preparation (the overall profile of antimicrobial susceptibility results of a microbial species to a battery of antimicrobial agents) has not been addressed until recently. The objective of this study was to analyze current antibiograms using the recently published NCCLS M39-A guidelines for preparation of antibiograms to identify areas for improvement in the reporting of antibiogram susceptibility data. Antibiograms from across the United States were obtained by various methods, including direct mailings, Internet searches, and professional contacts. Each ABGM collected was analyzed using prospectively defined elements from the M39-A guidelines. Additionally, seven quality indicators were also evaluated to look for the reporting of any atypical or inappropriate susceptibility data. The 209 antibiograms collected from 149 institutions showed at least 85% compliance to 5 of the 10 M39-A elements analyzed. Clinically relevant elements not met included annual analysis, duplicate isolate notation, and the exclusion of organisms with fewer than 10 isolates. As for the quality indicators evaluated, unexpected results included the 7% of antibiograms that reported <100% vancomycin susceptibility for Staphylococcus aureus, 24% that had inconsistent beta-lactam susceptibility for Staphylococcus aureus, 20% that reported <100% imipenem susceptibility for Escherichia coli, and 37% that reported >0% ampicillin susceptibility for Klebsiella pneumoniae. These findings suggest that antibiograms should be reviewed thoroughly by infectious disease specialists (physicians and pharmacists), clinical microbiologists, and infection control personnel for identification of abnormal findings prior to distribution.

Effect of Duplicate Isolates of Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus on Antibiogram Data

ABSTRACT Duplicate Staphylococcus aureus isolates were analyzed to determine the impact of multiple isolates from the same patient on annual antibiogram data. During a 6-year period (1996 to 2001), 3,227 patients with 4,844 S. aureus isolates were evaluated. A total of 39% of patients with methicillin-resistant S. aureus (MRSA) (n = 860) and 23% of patients with methicillin-susceptible S. aureus (MSSA) (n = 2,367) infections had duplicate isolates. Cumulative data show that 91% of the patients during this 6-year period with duplicate isolates (2 to 13 duplicates/year) did not switch between MSSA and MRSA but retained the original S. aureus strain whether it was MSSA or MRSA. Rates of MRSA were calculated for each year by using all isolates and then eliminating duplicates. The impact of duplicate MRSA and MSSA isolates was evaluated by using the ratio of isolates per patient such that ratios of >1.0 indicate >1 isolate per patient. The 6-year ratio for MRSA was 1.90 isolates/patient, and the ratio for MSSA was 1.35. A significant difference (P < 0.05) was noted in the MRSA rates in 4 of 6 years when duplicate isolates were removed. Common phenotypic antibiogram patterns were compared for all MRSA isolates during the 6-year period, and 64% were of a single antibiogram phenotype. Eighty-eight percent of patients with duplicate MRSA isolates had phenotypically identical multiple isolates. The rate of MRSA differs when duplicate isolates are removed from the antibiogram data.

Lymphoepithelioma-like carcinoma of the colon: report of a case with histologic, immunohistochemical, and molecular studies for Epstein-Barr virus.

We report a case of lymphoepithelioma-like carcinoma of the colon in a 62-year-old Hispanic male with multiple other tumors, including thyroid, breast, pharyngeal, and prostate carcinomas; a neurofibrosarcoma; and a meningioma. The association with Epstein-Barr virus infection and colonic lymphoepithelioma-like carcinoma was studied using immunohistochemical and polymerase chain reaction techniques.

Ribosomal RNA Sequence Analysis of Brucella Infection Misidentified as Ochrobactrum anthropi Infection

ABSTRACT A Brucella isolate was identified from purulent material collected during a hip surgery. Two previous blood cultures from the same patient yielded Ochrobactrum anthropi. After rRNA sequencing, all the isolates were identified as Brucella species and subsequently serotyped as Brucella suis. Misidentification of Brucella species remains a problem with bacterial identification systems.

Coactivating Signals for the Hepatic Lymphocyte Gamma Interferon Response to Francisella tularensis

ABSTRACT The facultative intracellular bacterium Francisella tularensis is capable of causing systemic infections in various hosts, including mice and humans. The liver is a major secondary site of F. tularensis infection, but hepatic immune responses to the pathogen remain poorly defined. Immune protection against the pathogen is thought to depend on the cytokine gamma interferon (IFN-γ), but the cellular basis for this response has not been characterized. Here we report that natural killer cells from the livers of naïve uninfected mice produced IFN-γ when challenged with live bacteria in vitro and that the responses were greatly increased by coactivation of the cells with either recombinant interleukin-12 (IL-12) or IL-18. Moreover, the two cytokines had strong synergistic effects on IFN-γ induction. Neutralizing antibodies to either IL-12 or IL-18 inhibited IFN-γ production in vitro, and mice deficient in the p35 subunit of IL-12 failed to show IFN-γ responses to bacterial challenge either in vitro or in vivo. Clinical isolates of highly virulent type A Francisella tularensis subsp. tularensis organisms were comparable to the live attenuated vaccine strain of Francisella tularensis subsp. holarctica in their ability to induce IL-12 and IFN-γ expression. These findings demonstrate that cells capable of mounting IFN-γ responses to F. tularensis are resident within the livers of uninfected mice and depend on coactivation by IL-12 and IL-18 for optimum responses.

Identification of donor melanoma in a renal transplant recipient.

A patient with chronic renal failure received a closely matched cadaveric kidney. Approximately 3 months after transplantation, the patient developed a metastatic malignant melanoma. A large retroperitoneal mass consisting of large pleomorphic polygonal neoplastic cells was found close to the donated kidney. This tumor was diagnosed as a malignant melanoma. DNA analysis of this tumor, the donated kidney, and the recipient indicated that the melanoma originated from the donor. Although this is not the first report of a donated melanoma, it is the first report of definitive DNA analysis of the origin of the malignant cells.

Evaluation of three analyte-specific reagents for detection and typing of herpes simplex virus in cerebrospinal fluid.

The performance of three analyte-specific reagents (ASRs); Cepheid herpes simplex virus (HSV) Typing Primer Probe set (CD), Eragen MultiCode-Rtx HSV-1/2 kit primer mix (ER), and Roche LightCycler HSV-1/2 Primer/Hybridization Probes (RD), was evaluated for detection and typing of herpes simplex virus (HSV-1 and HSV-2) in cerebrospinal fluid (CSF) specimens. Of 68 CSF specimens, HSV-1 was detected in 8 specimens and HSV-2 was detected in 17 specimens. ER detected all 25 HSV-positive specimens, whereas CD and RD detected 24 and 23 HSV-positive specimens, respectively. The results of HSV typing with the 3 ASRs were in complete agreement. The analytical sensitivity of all ASRs was determined to be about 10(1) copies/reaction. Our results demonstrate that the performances of all 3 ASRs are comparable and reliable for routine clinical testing in detection and typing of HSV DNA in CSF.

Antibiograms: New NCCLS Guidelines, Development, and Clinical Application

Antibiograms are an important resource for health care providers. All clinicians involved in antibiotic selection and monitoring should become familiar with the NCCLS M39-A document “Analysis and Presentation of Cumulative Antimicrobial Susceptibility Test Data: Approved Guideline.” Providers who interpret and apply antibiogram data in clinical practice should know about susceptibility testing methods, the susceptibility breakpoint determination process, and problems associated with antibiogram data analysis. The M39-A guidelines contain more than 40 recommendations, including the following: antibiogram data should be analyzed at least annually, an attempt should be made to remove duplicate isolates, and only organisms with 10 isolates or more should be presented. Accurate antibiograms facilitate improved empiric antibiotic selection and more precise monitoring of bacterial resistance in the hospital. This article reviews common antimicrobial susceptibility testing methods and relevant issues, highlights the major NCCLS M39-A recommendations, discusses the antibiogram preparation process and challenges in data interpretation, and provides a general overview of how antibiogram data may be applied to clinical practice.