A rapid and simple sterility test method based on solid culture medium containing blood.

Current sterility test performed for most biological products takes 14 days. We evaluated solid medium, containing 5% blood for use in the membrane filtration (MF) and direct inoculation (DI) sterility test. Representative microorganisms prepared in a sample matrix at approximately 0.1, 1, 10 and 100 colony forming units were tested for growth by compendial MF sterility test using fluid thioglycolate medium and tryptic soy broth and also on the Schaedler blood agar (SBA). Sterility test performed on SBA was significantly more sensitive and faster in detecting various microorganisms than the compendial method, particularly for sample matrix containing 0.01% thimerosal (p < 0.05). SBA detected all microorganisms within 7 days. To implement solid medium in the DI sterility test, multiple BA plates were inoculated with the sample. All representative microorganisms were detected within 5 days. The sterility test using solid medium required 3 different incubation conditions, 30-35 °C aerobically and anaerobically to detect bacteria, and at 20-25 °C aerobically to detect mold and yeast. To eliminate aerobic incubation of solid medium at 20-25 °C, we evaluated representative species of mold and yeast for their growth at 30-35 °C and 20-25 °C in the sterility test performed on solid medium. Penicillium chrysogenum could not be detected at 30-35 °C consistently within 7 days. Sterility test performed on solid medium without any additional technology could be completed in 7 days, as compared to the 14 days required for the current compendial method.

Supportive Care of the First 2 Ebola Virus Disease Patients at the Monrovia Medical Unit.

We describe the first 2 patients admitted to the Monrovia Medical Unit, a facility established to treat Liberian and international response workers with suspected or known Ebola virus disease (EVD). Their recoveries illustrate the value of local point-of-care diagnostics, parenteral therapies, and electrolyte replacement in EVD supportive care.

Evaluation of growth based rapid microbiological methods for sterility testing of vaccines and other biological products.

Most biological products, including vaccines, administered by the parenteral route are required to be tested for sterility at the final container and also at various stages during manufacture. The sterility testing method described in the Code of Federal Regulations (21 CFR 610.12) and the United States Pharmacopoeia (USP, Chapter <71>) is based on the observation of turbidity in liquid culture media due to growth of potential contaminants. We evaluated rapid microbiological methods (RMM) based on detection of growth 1) by adenosine triphosphate (ATP) bioluminescence technology (Rapid Milliflex(®) Detection System [RMDS]), and 2) by CO(2) monitoring technologies (BacT/Alert and the BACTEC systems), as alternate sterility methods. Microorganisms representing Gram negative, Gram positive, aerobic, anaerobic, spore forming, slow growing bacteria, yeast, and fungi were prepared in aliquots of Fluid A or a biological matrix (including inactivated influenza vaccines) to contain approximately 0.1, 1, 10 and 100 colony forming units (CFU) in an inoculum of 10 ml. These preparations were inoculated to the specific media required for the various methods: 1) fluid thioglycollate medium (FTM) and tryptic soy broth (TSB) of the compendial sterility method (both membrane filtration and direct inoculation); 2) tryptic soy agar (TSA), Sabouraud dextrose agar (SDA) and Schaedler blood agar (SBA) of the RMDS; 3) iAST and iNST media of the BacT/Alert system and 4) Standard 10 Aerobic/F and Standard Anaerobic/F media of the BACTEC system. RMDS was significantly more sensitive in detecting various microorganisms at 0.1CFU than the compendial methods (p<0.05), whereas the compendial membrane filtration method was significantly more sensitive than the BACTEC and BacT/Alert methods (p<0.05). RMDS detected all microorganisms significantly faster than the compendial method (p<0.05). BacT/Alert and BACTEC methods detected most microorganisms significantly faster than the compendial method (p<0.05), but took almost the same time to detect the slow growing microorganism P. acnes, compared to the compendial method. RMDS using SBA detected all test microorganisms in the presence of a matrix containing preservative 0.01% thimerosal, whereas the BacT/Alert and BACTEC systems did not consistently detect all the test microorganisms in the presence of 0.01% thimerosal. RMDS was compatible with inactivated influenza vaccines and aluminum phosphate or aluminum hydroxide adjuvants at up to 8 mg/ml without any interference in bioluminescence. RMDS was shown to be acceptable as an alternate sterility method taking 5 days as compared to the 14 days required of the compendial method. Isolation of microorganisms from the RMDS was accomplished by re-incubation of membranes with fresh SBA medium and microbial identification was confirmed using the MicroSEQ Identification System. BacT/Alert and BACTEC systems may be applicable as alternate methods to the compendial direct inoculation sterility method for products that do not contain preservatives or anti-microbial agents.