Quality control of multidrug resistance assays in adult acute leukemia: correlation between assays for P-glycoprotein expression and activity

We have compared multiple assays for the P-glycoprotein (Pgp/MDR1) phenotype in fresh and thawed adult acute leukemia to validate and quantitate measures for the expression and function of Pgp. The results are related to the Pgp-expressing KB8 and KB8-5 call lines. The most sensitive assay was the measurement of modulation of the rhodamine 123 (R123) fluorescence by 2 micromol/L PSC833, followed by the modulation of the probe calcein-AM. We also found a good intralaboratory and interlaboratory correlation between the values of the R123/PSC833 assay for fresh as well as thawed samples. In addition, the affects of PSC833 on 3H-daunorubicin (DNR) accumulation, DNR fluorescence, and 3H-vincristine accumulation were very similar. The correlation between the DNR/PSC833 and R123/PSC833 test was r = .86 (N = 51). The modulation of drug accumulation by 8 micromol/L verapamil was the some as the PSC833 effect for DNR (117%, N = 21), but was higher for vincristine in every single case (161% v 121%, N = 22; P< .001), indicating additional verapamil effects, not related to Pgp. The correlation of the staining of viable cells for Pgp with the monoclonal antibody MRK16 was r = .77 (N = 52) for the R123/PSC833 functional test and r = .84 (N = 50) for the DNR/PSC833 test. From these results it could be calculated that a maximal increase of the mean DNR accumulation of about 50% can be achieved by blocking Pgp pump activity with PSC833 in leukemic blast samples with the highest mean Pgp expression. Subpopulations of blast calls with higher Pgp activity are likely to be present. Their relevance has to be studied further. The methods outlined here allow the reliable, quantitative monitoring of the Pgp/MDR1 phenotype in leukemias in multicentered, clinical Pgp modulation studies.     

An outbreak of Burkholderia (formerly Pseudomonas) cepacia respiratory tract colonization and infection associated with nebulized albuterol therapy

OBJECTIVE: To investigate an outbreak of Burkholderia (formerly Pseudomonas) cepacia respiratory tract colonization and infection in mechanically ventilated patients. DESIGN: A retrospective case-control and bacteriologic study. SETTING: Veterans Affairs medical center. PATIENTS: 42 mechanically ventilated patients who developed respiratory tract colonization or infection with B. cepacia and 135 ventilator-dependent controls who were not colonized and did not develop infections. MEASUREMENTS: Clinical and demographic data; benzalkonium chloride concentrations and pH levels in albuterol sulfate solutions; repetitive-element polymerase chain reaction (PCR)-mediated molecular fingerprinting on eight patient isolates and three environmental B. cepacia isolates that were available for study. RESULTS: 42 patients had B. cepacia respiratory tract colonization or infection. Observation of intensive care unit and respiratory care personnel showed faulty infection control procedures (for example, the same multiple-dose bottle of albuterol was used for many mechanically ventilated patients). More case patients (39 [92.9%]) than controls (95 [70.4%]; P = 0.006) received nebulized albuterol, and case patients (67.5 treatments) received more treatments than controls (18 treatments; P < 0.001). In-use albuterol solutions had pH values that were unstable, and benzalkonium chloride concentrations declined over time to levels capable of supporting bacterial growth. Medication nebulizers and in-use bottles of albuterol harbored B. cepacia. Molecular fingerprints of patient isolates and environmental B. cepacia isolates were identical using repetitive-element PCR. No further isolates of B. cepacia were identified after institution of appropriate infection control procedures. CONCLUSIONS: Multiple-dose medications and reliance on benzalkonium chloride as a medication preservative provide a mechanism for nosocomial spread of microorganisms, particularly if infection control procedures are not carefully followed. Repetitive-element PCR is a useful fingerprinting technique for molecular epidemiologic studies of B. cepacia.     

Impact of ganciclovir prophylaxis on heart-lung and lung transplant recipients

BACKGROUND: Cytomegalovirus infection threatens pulmonary allograft survival and function. This retrospective study details the experience of ganciclovir prophylaxis against cytomegalovirus infection and its sequelae. METHODS: Eight-nine lung and heart-lung transplant recipients with positive cytomegalovirus serology were analyzed. The 37 recipients who underwent transplantation before September 1989 received no prophylaxis. The 52 subsequent recipients received ganciclovir prophylaxis. RESULTS: Thirty-six non-prophylaxed versus 42 prophylaxed patients had cytomegalovirus events with cumulative incidences of 100% and 86% (p < < 0.01), and median onsets of 37 +/- 21 versus 85 +/- 35 days, respectively (p < < 0.01); 22 non-prophylaxed versus 27 prophylaxed patients had cytomegalovirus pneumonitis with cumulative incidences of 60% and 55% (p < < 0.01), and median onsets of 34 +/- 14 and 84 +/- 26 days, respectively (p < < 0.01). Respiratory failure caused by cytomegalovirus pneumonitis developed in nine of the non-prophylaxed versus two of the prophylaxed patients (p < < 0.01). The significant estimated survival benefit in patients who received prophylaxis (p = 0.04) was not apparent when reanalysis was performed after exclusion of patients with respiratory failure (p = 0.36). Ganciclovir prophylaxis produced a significant delay in the development of obliterative bronchiolitis with a median time to onset of 1072 +/- 280 days versus 432 +/- 189 days for the non-prophylaxis cohort (p < < 0.01). CONCLUSIONS: Ganciclovir prophylaxis (1) improves recipient survival by reducing the severity of disease and essentially eliminating respiratory failure caused by cytomegalovirus pneumonitis, (2) reduces the incidence and delays the onset of cytomegalovirus events and pneumonitis, and (3) delays the onset of obliterative bronchiolitis.