Patricia Charache

Epstein-Barr virus in AIDS-related primary central nervous system lymphoma.

Primary central nervous system lymphoma occurs more often in patients with AIDS. Epstein-Barr virus (EBV) has been detected in these tumours, but the degree of association has not been defined because of both the highly restricted expression of EBV in malignant tissue and the lack of a technique that is reliable in formalin-fixed paraffin-embedded specimens. EBV-transformed lymphocytes contain short non-protein coding EBV transcripts (EBERs), which are expressed in much higher quantity than other EBV-latency transcripts. We describe a new strategy for detection of latent EBV with these transcripts as targets for in-situ hybridisation. 18 cases of AIDS-related primary CNS lymphoma from a consecutive necropsy series together with specimens from 3 further cases were studied. In each case, a strong positive signal over tumour cells indicated abundant expression of the EBV-EBER1 transcript. This 100% association suggests that the pathogenesis of these AIDS-associated lymphomas may differ from the systemic disease in which only 30-50% of tumours are associated with EBV. A pathogenetic role for EBV was further supported by showing expression of a viral protein (the latent membrane protein) that is implicated as an effector for EBV-associated lymphomagenesis. EBV might have a role as a tumour marker in the diagnosis and management of AIDS-related primary CNS lymphoma.

Association of BK virus with failure of prophylaxis against hemorrhagic cystitis following bone marrow transplantation.

PURPOSE Hemorrhagic cystitis (HC) after bone marrow transplantation (BMT) has been ascribed to cyclophosphamide metabolites. HC has also been associated with excretion of the BK type of polyomavirus. The relative contributions of cyclophosphamide metabolites and BK virus in the development of HC following BMT are unknown. PATIENTS AND METHODS We conducted a randomized trial to compare mesna with forced diuresis for prophylaxis against HC in 147 BMT recipients. We studied the association of BK virus with HC in 95 consecutive BMT recipients by prospectively monitoring urinary excretion of BK virus using polymerase chain reaction amplification of viral gene sequences. RESULTS HC occurred in 37 of 147 (25.2%) transplant recipients. The incidence of HC was similar in patients given mesna (26.8%, 19 of 71) or forced diuresis (23.7%, 18 of 76), and in recipients of allogeneic (27.2%, 18 of 64) or autologous marrow (22.9%, 19 of 83). The incidence of HC was unrelated to primary disease, preparative regimen, or occurrence of graft-versus-host disease (GVHD). Excretion of BK virus was demonstrated in 50 of 95 patients (52.6%); 38 patients (40%) had persistent BK viruria (> or = two consecutive positive samples). HC occurred in 19 of 38 patients (50%) with persistent BK viruria, in one of 12 (8.3%) with only a single urine sample positive for BK virus, and in none of 45 who did not excrete BK virus (P < .0001). Shedding of BK virus also had a strong temporal correlation with onset of HC (r = .95). CONCLUSION Mesna and forced diuresis are equally effective in abrogating the urothelial toxicity of preparative regimens for BMT. Since HC after BMT is virtually always associated with persistent BK viruria, strategies aimed at the prevention or elimination of viruria in BK seropositive recipients are warranted.

Prediction of severe disseminated adenovirus infection by serum PCR

Adenoviruses are increasingly recognised as viral pathogens that can cause fatal infections in immunocompromised patients, particularly recipients of haematopoietic stem-cell grafts. Adenovirus infections are not easily diagnosed and the development of a severe infection cannot be predicted by standard culture techniques. In a pilot study, we investigated the value of adenovirus DNA detection in serum as a marker of disseminated disease in 14 patients with defined patterns of adenovirus infections. The results show that the appearance of adenoviral DNA in serum preceded the development of a severe or fatal adenovirus infection. Because proper management is dependent on early diagnosis and differentiation from other conditions, this test may be a valuable tool in the management of adenovirus infection.

Empiric use of vancomycin during prolonged treatment-induced granulocytopenia. Randomized, double-blind, placebo-controlled clinical trial in patients with acute leukemia☆

Because gram-positive infections cause morbidity following intensive antileukemic chemotherapy, the effects of vancomycin versus placebo were evaluated in a randomized, double-blind, placebo-controlled trial in 60 adult patients with acute leukemia and first infectious fever during prolonged (mean of 32 days) granulocytopenia. Gram-positive sepsis was associated with first fever in 17 (28 percent) of the 60 patients. None of 31 patients randomly assigned to receive vancomycin demonstrated gram-positive infection, whereas 16 of 22 patients randomly assigned to receive placebo subsequently had gram-positive infection (seven had sepsis, and nine had local infections; p less than 0.005). All patients with gram-positive infection were then given vancomycin, and all showed prompt clinical responses. The predominant gram-positive organism causing infection was beta-lactam-resistant Staphylococcus epidermis (19 of 44 isolates). Patients randomly assigned to receive vancomycin had more rapid resolution of first infectious fever and fewer total febrile days during the granulocytopenic course than did patients randomly assigned to receive placebo. Although vancomycin had no effect on the presence or absence of documented fungal infection, patients treated with vancomycin received empiric amphotericin B for recurrent or persistent fever later (mean of 14 days after initial antibiotic coverage was begun) than did patients receiving placebo (mean of 9.9 days; p less than 0.005), and thus received fewer total days of empiric amphotericin B therapy (mean of 16.3 days) than did patients given placebo (mean of 24.6 days; p less than 0.01). These data demonstrate that empiric use of vancomycin reduces the morbidity of gram-positive infections following intensive antileukemic therapy and decreases the need for empiric use of toxic amphotericin B.

Viral Sensitivity Testing in Patients With Cytomegalovirus Retinitis Clinically Resistant to Foscarnet or Ganciclovir

PURPOSE Resistance to antiviral therapy is a potential cause of progression of cytomegalovirus retinitis in patients with the acquired immunodeficiency syndrome. We investigated the results of viral sensitivity testing in a series of patients with clinically resistant retinitis who had positive results of blood or urine cytomegalovirus cultures. METHODS All patients with newly diagnosed cytomegalovirus retinitis between January 1990 and December 1991 were prospectively studied. Blood and urine cultures for cytomegalovirus were obtained in a nonrandomized subgroup of this group. The results of in vitro sensitivity to foscarnet and ganciclovir, determined by a DNA hybridization assay, were then analyzed in seven patients with clinically resistant cytomegalovirus retinitis and whose blood or urine culture results, or both, were positive for cytomegalovirus while on a treatment regimen. RESULTS Foscarnet-resistant cytomegalovirus (ID50 > 300 microM) was isolated from two patients, one of whom was being treated with foscarnet. Ganciclovir-resistant cytomegalovirus (ID 50 > 6.0 microM) was isolated from four patients, three of whom were being treated with ganciclovir. Foscarnet- and ganciclovir-resistant cytomegalovirus occurred with previous ganciclovir therapy in one patient. Clinical improvement occurred in three patients whose change in therapy was based on viral sensitivity testing. In general, prolonged therapy with one drug was associated with a progressive increase in the ID 50 for that drug. CONCLUSIONS Viral resistance to foscarnet or ganciclovir may explain refractory cytomegalovirus retinitis in some patients.

Clearance of adenoviral hepatitis with ribavirin therapy in a pediatric liver transplant recipient

Adenovirus (AdV) infections, localized or disseminated, are being increasingly recognized in immunocompromised patients including bone marrow and solid organ transplant recipients and HIV-1-infected patients. AdV infection in pediatric liver transplant recipients may present as hepatitis in the transplanted liver and is usually diagnosed by immunohistochemical staining of liver tissue. Recommended therapies have included withdrawal of immunosuppressive agents and in some cases intravenous ribavirin. There has been no previous report of successful clearance of AdV hepatitis with ribavirin in a pediatric liver transplant recipient. We report the clearance of AdV hepatitis in a child who received two liver transplants, developed AdV hepatitis and was successfully treated with intravenous ribavirin. Diagnosis and monitoring of her AdV infection were enhanced through the use of a newly described polymerase chain reaction for AdV.

Developing a sustainable process to provide quality control materials for genetic testing

Purpose: To provide a summary of the outcomes of two working conferences organized by the Centers for Disease Control and Prevention (CDC), to develop recommendations for practical, sustainable mechanisms to make quality control (QC) materials available to the genetic testing community.Methods: Participants were selected to include experts in genetic testing and molecular diagnostics from professional organizations, government agencies, industry, laboratories, academic institutions, cell repositories, and proficiency testing (PT)/external Quality Assessment (EQA) programs. Current efforts to develop QC materials for genetic tests were reviewed; key issues and areas of need were identified; and workgroups were formed to address each area of need and to formulate recommendations and next steps.Results: Recommendations were developed toward establishing a sustainable process to improve the availability of appropriate QC materials for genetic testing, with an emphasis on molecular genetic testing as an initial step.Conclusions: Improving the availability of appropriate QC materials is of critical importance for assuring the quality of genetic testing, enhancing performance evaluation and PT/EQA programs, and facilitating new test development. To meet the needs of the rapidly expanding capacity of genetic testing in clinical and public health settings, a comprehensive, coordinated program should be developed. A Genetic Testing Quality Control Materials Program has therefore been established by CDC in March 2005 to serve these needs.

Oligonucleotides for polymerase chain reaction amplification and hybridization detection of Epstein-Barr virus DNA in clinical specimens

We designed synthetic oligonucleotide primers and hybridization probe for use in polymerase chain reaction (PCR) amplification and hybridization detection of Epstein-Barr virus (EBV) nucleic acid sequences. Primer sequences were chosen from the coding region for the Epstein-Barr virus nuclear antigen-1 (EBNA-1). PCR amplification and hybridization with these oligonucleotides was carried out on standard laboratory cell lines including African Burkitts lymphoma and infectious mononucleosis derived cell lines, as well as cell lines recently established from clinical EBV isolates from bone marrow transplant recipients. All EBV cell lines tested were positive. No false-positives were detected with uninfected cell lines, human placental DNA or with other viruses. The sensitivity of the detection procedure was such that four copies of the EBV genome could consistently be detected in a background of 1 microgram of placental DNA. EBV was detected in DNA extracts from the peripheral blood mononuclear cells of two patients with infectious mononucleosis and one patient with viral-associated hemophagocytic syndrome. Three of 18 EBV seropositive patients without known ongoing EBV-associated illness undergoing ambulatory surgery also had EBV detected in DNA extracts from their peripheral blood mononuclear cells. EBV was detected in DNA extracts from lymphoma tissue from two patients with post-transplant lymphomas and two AIDS patients with primary CNS lymphomas. EBV was not detected in 12 B-cell lymphoma specimens from patients without history of immunocompromise. DNA extracts from formalin-fixed paraffin-embedded Hodgkins tissues previously shown to be EBV positive by Southern blot were also demonstrated to be EBV positive by PCR. Thus, with the oligonucleotides described, PCR is applicable to the detection of EBV in a spectrum of clinical isolates. The broad specificity of these oligonucleotides for all strains of EBV tested is probably a function of the highly conserved sequence of the EBNA-1 DNA binding domain.

Antibiotic-resistant bacteria in surveillance stool cultures of patients with prolonged neutropenia.

The value of stool surveillance for antibiotic-resistant gram-negative bacteria was analyzed in 86 neutropenic bone marrow transplant patients. Twice-weekly specimens were inoculated onto culture medium containing gentamicin plus carbenicillin. The recovered organisms were identified to the species level and tested for antibiotic susceptibility. Forty-eight resistant organisms were recovered from 35 patients. Thirteen isolates persistently colonized patients. Escherichia coli (29%) and Pseudomonas aeruginosa (19%) were the most frequently recovered organisms. Although most organisms were recovered while patients were on antibiotics, 15 isolates, including eight of nine resistant P. aeruginosa, were detected before antibiotics were initiated. The duration of antibiotic use was longer for patients persistently colonized than for those not colonized (P = 0.03). Of the 15 resistant organisms which caused infection, 12 were detected in the surveillance cultures. Infections by antibiotic-resistant organisms occurred more frequently in patients colonized than in those not colonized (P = 0.006) and more frequently in patients persistently colonized than in those colonized only once (P = 0.01). The absence of colonization or persistent colonization correlated well with the absence of infection (negative predictive values of 94 and 91%, respectively).

Viral resistance and CMV retinitis: design and methods of a prospective study.

A prospective study following a cohort of patients with newly diagnosed, previously untreated cytomegalovirus (CMV) retinitis is being conducted to study drug resistant CMV. Prior to initiation of treatment, patients undergo a baseline eye examination, fundus photography, and blood and urine culture for presence of CMV, and drug susceptibility testing against positive isolates. Patients are followed monthly with a detailed eye examination to diagnose progression of retinitis, and for fundus photography. Cultures are repeated at 1 and 3 months after enrollment, every 3 months thereafter, and at the time of treatment reinduction for the progression of retinitis. This study was designed to determine the prevalence and incidence of drug resistant CMV, as well as risk factors for the development of resistant CMV. It also will determine the correlation between clinical outcome, as measured both by eye examination and fundus photography, and viral resistance.