Development and evaluation of a real-time PCR assay for detection of Pneumocystis jiroveci.

Pneumocystis jiroveci is an important agent of pneumonia in immunocompromised hosts. Usually, this pathogen is detected by Giemsa or direct fluorescence stains of bronchoalveolar lavage (BAL) fluids. Microscopic methods, however, have 2 disadvantages. P. jiroveci is not stable outside the human body, which means that slow sample transport might result in false-negative results. Additionally, exact quantification, which is needed for therapy monitoring, is not possible. In this study, we developed a real-time polymerase chain reaction assay for the LightCycler. Two Pneumocystis-specific TaqMan systems, one based on the sequence of the 5.8S ribosomal gene and another one targeting the dihydrofolate reductase gene were evaluated. Additionally, the amount of human DNA in the sample was measured by a TaqMan assay based on the human albumin gene, allowing assessment of sample quality and quantification normalized on sample concentration. For clinical evaluation, 69 BAL specimens from 26 positive patients as well as 60 negative controls were tested. Both systems were able to detect all proven cases of Pneumocystis pneumonia. Differentiation of carriage, asymptomatic reactivation, and clinical infection as well as normalized quantification by calculating the ratio of Pneumocystis DNA to human DNA are discussed.

Successful preemptive cidofovir treatment for CMV antigenemia after dose-reduced conditioning and allogeneic blood stem cell transplantation.

BACKGROUND: Cidofovir (CDV) is a nucleotide analogue with proven in vitro effects against cytomegalovirus (CMV) and adenovirus and has been successfully used in the treatment of CMV retinitis in AIDS patients. METHODS: We performed a prospective study to evaluate the efficacy of CDV in 17 patients with hematological malignancies after allogeneic blood stem cell transplantation from related (n=3) and unrelated (n=14) donors. Dose-reduced conditioning (DRC) regimen consisted of busulfan (Bu)/fludarabine (Flu) (n=9) and idarubicin/cytosine arabinoside/Flu (n=1). Myeloablative conditioning (MC) was performed with Bu/cyclophosphamide (Cy)/etoposide (Eto) (n=4), Bu/Cy (n=2), and total body irradiation (TBI)/Cy/Eto (n=1). Antithymocyte globulin (ATG) was used in seven patients with DRC and in six patients with MC. In all patients, either the donor, host, or both were CMV IgG positive pretransplant. Indication for therapy was preemptive treatment of primary CMV antigenemia defined as two consecutive positive tests of pp65 antigenemia assay after transplant. In case of response with a decreasing number of pp65-positive leukocytes, CDV was scheduled in a dosage of 5 mg/kg body weight once a week for 2 weeks followed by maintenance therapy every 2 weeks in an outpatient setting. All patients received probenecid and prehydration as recommended. Patients were monitored using an immunostaining assay for pp65 antigen and a qualitative and quantitative CMV polymerase chain reaction (PCR). Success of treatment was defined as negativity for the pp65 antigen. RESULTS: After DRC, nine of ten patients (90%) showed a response with seven of nine revealing a complete clearance of the virus (pp65 negative, qualitative PCR negative). In the remaining two responders, treatment was changed to ganciclovir because of either renal impairment or slow clearance of antigenemia. Only one of seven patients in the MC group experienced a temporary clearance of pp65 antigen. After MC, two patients experienced CMV disease. Treatment-related toxicity rate was moderate with four patients developing reversible renal impairment (creatinine 133-180 micromol/L); one patient with proteinuria and three patients with complaints of nausea and vomiting. CONCLUSION: Our data suggest the feasibility of CDV administration in patients after allogeneic transplantation. In the recommended dose, it might be used successfully for low-risk patients, e.g., after DRC or organ transplantation, in an outpatient setting.

[DNA-bound proteins mediate attachment to the nuclear skeleton of transcriptionally active DNA fraction]. / Prochno sviazannye s DNK belki oposreduiut prikreplenie k iadernomu skeletu transkriptsionno aktivnoi fraktsii DNK.

The proteins tightly bound to DNA are non-randomly distributed along the DNA chain and are concentrated within the transcriptionally active areas. The distribution of tightly bound proteins along the DNA reflects the type of cell differentiation and does not depend directly on transcription. In functionally active nuclei, the DNA-tightly bound proteins complexes are fixed at the nuclear matrix which is therefore associated with the whole transcriptionally active DNA fraction.