Gail L. Woods

NOSOCOMIAL INVASIVE ASPERGILLOSIS IN LYMPHOMA PATIENTS TREATED WITH BONE MARROW OR PERIPHERAL STEM CELL TRANSPLANTS

OBJECTIVESnTo determine the prevalence of aspergillosis in lymphoma patients housed in a protective environment while undergoing a bone marrow transplant or peripheral stem cell transplant and its relation to lymphoma type, type of transplant, period of neutropenia, method of diagnosis, species of Aspergillus, and the use of empiric amphotericin B.nnnDESIGNnClinical, autopsy, and microbiology records were reviewed retrospectively to determine the presence or absence of invasive aspergillosis. All positive specimens underwent further review to determine parameters outlined above.nnnSETTINGnThe review took place at the University of Nebraska Medical Center with lymphoma patients housed in the oncology/hematology special care unit, which consists of 30 single-patient rooms under positive pressure with high-efficiency particulate air filtration.nnnPATIENTSn417 lymphoma patients admitted to the oncology/hematology special care unit who underwent 427 courses of high-dose chemotherapy with or without total body irradiation followed by a stem cell rescue.nnnRESULTSnTwenty-two cases (5.2%) of nosocomial invasive aspergillosis (14 caused by Aspergillus flavus, 2 by Aspergillus terreus, 2 by Aspergillus fumigatus, and 4 by characteristic histology) were diagnosed. The prevalence of disease according to transplant was 8.7% for allogeneic bone marrow transplant (2/23 treatments), 5.6% for autologous peripheral stem cell transplant (9/161), and 4.5% for autologous bone marrow transplant (11/243). Fifteen patients were presumptively diagnosed prior to death (68.2%) most commonly by histologic examination of skin biopsies. All 22 patients received amphotericin B therapy, 17 prior to aspergillosis diagnosis, and 7 (31.8%) survived. No patient with disseminated disease survived.nnnCONCLUSIONSnEven when housing lymphoma patients undergoing myeloablative therapy in a protective environment containing high-efficiency particulate air filtration, there was a risk of developing aspergillosis. These data also showed that antemortem diagnosis with aggressive amphotericin B therapy was most effective in the management of infected lymphoma patients when engraftment occurred and the disease did not become disseminated.

Cytomegalovirus infection and disease after liver transplantation. An overview.

Cytomegalovirus is the single most important pathogen in clinical transplantation. Although much progress has been made in our understanding of the molecular biology and epidemiology of CMV infection and in our ability to diagnosis and treat CMV disease, it remains a major cause of morbidity but is no longer a major cause of mortality after liver transplantation. Risk factors for CMV disease after liver transplantation include donor and recipient serologic status, the use of antilymphocyte therapy, and retransplantation. CMV disease occurs early after transplantation, and the most frequent site of disease is the hepatic allograft. We have treated 79 patients with intravenous ganciclovir, with ultimate control of disease achieved in 69 patients (87.3%). Preliminary results using intravenous immunoglobulin and oral acyclovir for CMV prophylaxis in high-risk patients have been encouraging. In addition to producing clinical syndromes, CMV may have direct immunologic effects and is a marker of the net state of immunosuppression.

Detection of cytomegalovirus by 24-well plate centrifugation assay using a monoclonal antibody to an early nuclear antigen and by conventional cell culture

During a 12-month period, two methods for detection of cytomegalovirus (CMV) in 1624 clinical specimens were compared: (1) centrifugal inoculation of MRC-5 cells on coverslips in 24-well plates and staining with a monoclonal antibody to CMV early nuclear antigen after incubation for 40 h (EA assay), and (2) conventional tube cell culture. CMV was identified in 183 (11.3%) specimens from 113 different patients. The EA assay was positive for CMV in 144/183 specimens (79%), and CMV was detected by recognition of specific cytopathic effect (CPE) in conventional cell culture in 143/183 (78%). Both methods yielded CMV in 56% of the specimens (104/183). CMV was detected by EA assay alone in 22% (40/183) and only by CPE in 21% (39/183) of the positive specimens. When all specimen types were considered, there was no significant difference in the detection of CMV between the two methods. However, bronchoalveolar lavage (BAL) fluids yielded CMV more frequently by EA assay than by CPE (58 compared to 48 of 574, p = 0.0178), and CMV was detected in blood specimens more often by CPE than by EA assay (20 compared to one of 149, p less than 0.0001). In addition to CMV, other viruses were recovered by conventional tube cell culture, including herpes simplex virus (HSV) type 1 from 17 BAL fluids (two of which were positive for CMV by EA assay) and one liver biopsy and adenovirus serotype 4 from four separate urine specimens and three gastrointestinal tract biopsies from one patient.(ABSTRACT TRUNCATED AT 250 WORDS)

Resistance in Enterobacteriaceae: results of a multicenter surveillance study, 1995-2000.

OBJECTIVESnTo assess changes over time in susceptibility of Enterobacteriaceae from patients in ICUs, compare susceptibility rates of isolates from patients in ICUs with those from inpatients outside ICUs, and explore phenotypic patterns of cross-resistance and co-resistance.nnnDESIGNnFrom 1995 to 2000, centers participating in the ICU Surveillance Study tested 100 to 200 consecutive nosocomial gram-negative bacilli by broth microdilution.nnnSETTINGnEach year, 42 to 97 U.S. hospitals tested isolates.nnnRESULTSnIn all years, imipenem was the most potent agent tested, followed by amikacin and ertapenem. Extended-spectrum beta-lactam and monobactam agents had good activity against Escherichia coli and Klebsiella species, but limited activity against Enterobacter species. Susceptibility to imipenem and amikacin did not fluctuate during the analysis period, whereas susceptibility to ceftazidime, ceftriaxone, and ciprofloxacin decreased 2% to 5%. The decline was most pronounced for susceptibility of Escherichia coli to ciprofloxacin: 98.7% of ICU isolates were susceptible in 1995 versus 93.2% in 2000. Susceptibility of ICU isolates was lower than that of non-ICU isolates, except for ciprofloxacin, for which the reverse was true. Cross-resistance was common among extended-spectrum cephalosporins and penicillins, but uncommon between imipenem and ertapenem. Only imipenem and ertapenem remained highly active against Enterobacteriaceae with a phenotype suggesting possible production of an extended-spectrum beta-lactamase and those with a phenotype suggesting possible Amp C hyperproduction.nnnCONCLUSIONSnImipenem was the most active agent against nosocomial Enterobacteriaceae. Susceptibility to ciprofloxacin decreased from 1995 to 2000, particularly in Escherichia coli, and, in contrast to other agents, was lower among non-ICU isolates.

Rapid 24-well plate centrifugation assay for detection of influenza A virus in clinical specimens

Two methods for detection of influenza virus in 234 clinical respiratory specimens were compared: (i) a 24-well plate-centrifugation assay using Madin Darby canine kidney (MDCK) cells and staining with monoclonal antibody pools to influenza A and B (Centers for Disease Control, Atlanta, GA) after incubation for 16 h and 40 h, and (ii) conventional tube cell culture using MDCK cells and primary rhesus monkey kidney cells. Influenza A was identified in 23 specimens (10%). No influenza B was recovered. The rapid centrifugation and tissue culture methods were positive for influenza A in 21 (91%) and 16 (70%) of the 23 specimens, respectively. Fourteen specimens were positive by both methods, 2 were positive by tissue culture alone, and 7 were positive by rapid centrifugation only. Of the 21 specimens positive by rapid centrifugation, 16 (76%) were detected after overnight incubation, and 5 (24%) were positive only after incubation for 40 h. Cytopathic effect was observed in 13 (81%) of the 16 isolates identified by tissue culture after an average of 6 days, and 3 (19%) were identified only by hemadsorption and staining with monoclonal antibodies at day 10. Compared with conventional tissue culture, the 24-well plate centrifugation assay is a more rapid and more sensitive method for detecting influenza virus in clinical specimens.

The effect of dexamethasone on the detection of cytomegalovirus in tissue culture and by immunofluorescence

With the development of both monoclonal antibodies to the immediate early nuclear antigen (EA) of cytomegalovirus (CMV) and different methods used for its detection, the time required for diagnosis of infection has become significantly shorter. However, discrepancies between tissue culture (TC) and the EA assay methods have raised questions concerning which method is more sensitive and whether a positive test result truly represents infection or latency. We have found that dexamethasone (Dex), when incorporated into the growth medium at a concentration of 10(-5) M, decreases the variability between the two techniques and increases the sensitivities of both TC and EA assay. However, for Dex to be effective, it was necessary to prepare, seed and grow the indicator cells in the presence of 10(-5) M Dex. Additionally, the cells had to be in contact with Dex for approximately 24 h before any effect was observed. Except for this step, all other procedures were the same as have been described elsewhere. In this study, four methods were compared: TC, TC with Dex (TC-D), EA, and EA with Dex (EA-D). Of 251 clinical specimens (200 microliter/well), 46 (18%) were positive for CMV. Of the 46, 30 (65%) were positive by TC, 39 (85%) by TC-D, 39 (85%) by EA, and 42 (91%) by EA-D. Without Dex the combination of TC plus EA detected only 41 of 46 (84%) positive samples. In contrast, the presence of Dex allowed detection of all 46 positive samples by TC-D and EA-D. Also, more fluorescent forming units (FFU) were detected by EA-D than EA (mean 14.8 FFU), and cytopathic effect was detected sooner (mean 9 days) by TC-D than by TC. Dex significantly increased the detection of CMV by TC (P less than 0.05). In addition, the use of any one of these methods alone did not effectively detect all positive samples. We suggest the combination of TC-D and EA-D for detection of all samples positive for CMV and to detect other viruses that may be missed by the EA method.

Failure of the Sterile Air-Flow Component of a Protected Environment Detected by Demonstration of Chaetomium Species Colonization of Four Consecutive Immunosuppressed Occupants

Four bone marrow transplant recipients consecutively occupying the same room on our Oncology-Hematology Special Care Unit (OHSCU) became colonized with Chaetomium species between January and April, 1987. These patients, aged 27 to 43 years, were immunocompromised as a result of intensive chemotherapy, and were consequently at increased risk for development of invasive fungal infection. At the time of Chaetomium colonization, all patients were febrile, two had transient new infiltrates on chest x-ray, and three were receiving amphotericin B therapy. Subsequent environmental cultures revealed Chaetomium contamination of the OHSCU air-handling system, including the HEPA (high-efficiency particulate air) filters in seven of the nine rooms comprising the unit. Because fungal colonization of HEPA filters used to create a protective environment for immunocompromised patients can occur and can serve as a source for patient infections, guidelines concerning proper surveillance of these HEPA filters should be established. We suggest that before a new patient enters a protected room, the clean side of the HEPA filter should be cultured. If fungi are recovered from that culture, we would recommend changing the filter.

Detection of adenovirus by rapid 24-well plate centrifugation and conventional cell culture with dexamethasone

Two methods for rapid detection of adenovirus were tested: (i) 24-well plate centrifugation followed by staining with a monoclonal antibody after incubation for 24 h and 48 h, and (ii) pretreatment of A549 cells used in conventional cell culture and 24-well plate centrifugation with 10(-5)M dexamethasone. Twenty-seven clinical isolates of adenovirus and 12 specimens from which adenovirus had been recovered were included in the analysis. Both isolates and specimens had been frozen at -70 degrees C for up to 6 months. By 24-well plate centrifugation both with and without dexamethasone, 21 (78%) and 27 (100%) isolates were positive for adenovirus at 24 h and 48 h, respectively. Of the specimens, 6 (50%) and 8 (67%) were positive by 24-well plate centrifugation without dexamethasone at 24 h and 48 h, respectively, whereas with dexamethasone 3 (25%) were positive at 24 h and 7 (58%) were positive at 48 h. Overall, combining isolates and specimens, the sensitivity of 24-well plate centrifugation for detection of adenovirus at 24 h was 69% without dexamethasone and 62% with dexamethasone, and at 48 h the sensitivity was 90% without dexamethasone and 87% with dexamethasone. The specificity under all conditions tested was 100%. In conventional tissue culture dexamethasone inhibited recovery of adenovirus. Without dexamethasone, adenovirus was recovered from all 39 samples within 7 days after inoculation; however with dexamethasone pretreatment, the virus was detected in only 31 (79%) of the samples tested in the same period of time.

Detection of herpes simplex virus in clinical specimens using a DNA probe after centrifugal inoculation of A549 cells

Two methods for detection of herpes simplex virus (HSV) in 216 clinical specimens were compared: (a) 24-well plate centrifugation using A-549 cells followed by nucleic acid hybridization (Ortho Diagnostic Systems, Inc., Raritan, NJ) after incubation for 16 to 18 h, and (b) conventional tube cell culture using A-549 cells. HSV was identified by conventional tube cell culture in 44 of 216 specimens (20%) and in 36 specimens (17%) by the centrifugation-hybridization method (P less than 0.01). HSV was recovered by tissue culture from all specimens positive by centrifugation-hybridization. The sensitivity, specificity, and positive and negative predictive values of the centrifugation-hybridization technique for detection of HSV in clinical specimens were 82, 100, 100, and 96%, respectively. Centrifugal inoculation of A549 cells in 24-well plates followed by nucleic acid hybridization after overnight incubation should not replace conventional tube cell culture for detection of HSV in clinical specimens.