Quantitative pp65-antigenemia assay for the prediction of human cytomegalovirus disease in HIV-infected patients.

OBJECTIVE: To evaluate the ability of a quantified pp65-antigenemia assay to predict the development of human cytomegalovirus (HCMV) disease in patients with an advanced HIV infection. DESIGN: A prospective longitudinal study between March 1993 and December 1996. Blood samples for the pp65-antigenemia assay were drawn at 2-3 month intervals. SETTING: AIDS department of an institutional tertiary care centre. PATIENTS: A total of 101 HIV-infected patients with CD4 lymphocyte counts of 100/mm3 or less were enrolled. Ninety-seven patients were eligible for analysis. All patients gave informed consent. MAIN OUTCOME MEASURES: The development of HCMV disease. RESULTS: Of the 97 patients, 24 developed HCMV disease after a median follow-up of 10.6 months. Three months before the development of HCMV disease, an increase in the median number of pp65-antigen-positive leukocytes was observed. The highest combination of sensitivity (45%) and specificity (94%) for the development of HCMV disease within the next 3 months was found when an assay cut-off level of 48/10(5) pp65-antigen-positive leukocytes was applied, with a positive predictive value (PPV) for the development of HCMV disease of 75%. The Kaplan-Meier estimate of HCMV disease-free survival after patients reached 48/10(5) or more antigen-positive leukocytes on longitudinal follow-up was a median 3.7 months [95% confidence interval (CI), 2.5-8.5]. The hazard ratio (HR) of this threshold level for the development of HCMV disease was 9.6 (95% CI, 4.2-21.8). CONCLUSION: Longitudinal follow-up using the pp65-antigenemia assay of HIV-infected patients with a low CD4 lymphocyte count improves the identification of patients who will develop HCMV disease in the foreseeable future, and should be considered for the selection of patients who may benefit from pre-emptive HCMV treatment.

Microbiological aspects of heat sterilization of drugs. III. Heat resistance of spore-forming bacteria, isolated from large-volume parenterals.

In order to calculate the minimum sterilization process conditions to obtain the generally accepted sterility level (less than 1.10(-6) probability of microbial survival), we determined the bioburden and its heat resistance of 500 ml large-volume parenteral bottles over a period of 5 years. For the bioburden determination 1,832 bottles were examined by the membrane filtration method. Mean bioburden was 9.36 colony-forming units/bottle. Of the colony-forming units isolated 118 were heat resistant (0.69%). These were spore-forming Bacillus species. Of the isolated Bacillus species heat resistance was determined in 5% glucose, 0.9% sodium chloride and 8% amino acids solution. D values greater than 1 min at 105 degrees C were found for 2, 5 and 4 different Bacillus species in glucose 5%, sodium chloride 0.9% and amino acids 8%, respectively. 2 Bacillus species showed a D value over 2 min at 105 degrees C in all three media. D values at 110 degrees C in sodium chloride 0.9% for these 2 Bacillus species were 1.8 and 2.6 min and in amino acids 8% 0.9 and 1.7 min, respectively. The minimum sterilization process time at 110 degrees C, calculated with the experimentally determined bioburden and D values is less than 25 min. When introducing reduced exposure times/temperatures, each individual manufacturer should assess the bioburden. The time-consuming determination of the heat resistance of bioburden isolates is not always necessary. By dividing the isolated colony-forming units in a 'heat-resistant' group and a 'not-heat-resistant' group, changing from standard overkill sterilization procedures to processes with lower F0 values is possible.

Microbiological aspects of heat sterilization of medicines. II. A method for the determination of the effectiveness of a sterilization process using the bioburden and the bioburdens heat resistance.

In order to verify whether the sterilization process of 60 min at 100 degrees C for invert sugar 20% is sufficiently effective to attain the generally accepted probability of survival of maximum 1 X 10(-6), we determined the bioburden and the bioburdens heat resistance for this product. We examined 98 bottles by the membrane filtration method and found 84 bottles with 0 colony forming units (CFU's) and 14 bottles with 1-9 CFU's. Because none of the isolated CFU's was heat resistant (Bacillus species), we isolated heat resistant CFU's from the environment and determined the heat resistance in invert sugar, water and NaCl solution 0.9% of four different Bacillus species. The results in invert sugar for the most heat resistant Bacillus species were a D-value of 0.92 min at 100 degrees C. For the determination of the D-value the end-point method is the most practical one, and the D-value calculation with the most probable number method is sufficiently accurate. Because of unavoidable inaccuracies in the experimentally determined D-value, safety margins of 100% have to be taken into account in the sterilization process calculations in which these D-values are used. Hence, in our case, we have to use a D-value of 2 X 0.92 min in the sterilization process calculation for invert sugar 20%. The maximum bioburden in the examined 98 test bottles was 9 CFU's. The maximum heat resistant bioburden which must be used in sterilization process calculations may be safely fixed at 10% of the total bioburden, therefore we have to use 0.9 micro-organisms in our calculation.(ABSTRACT TRUNCATED AT 250 WORDS)