Extraction buffer contaminated bacterially as a cause of invalid HIV-1 viral load results on the NucliSens EasyQ...

Shortly after starting to use the NucliSens EasyQ HIV-1 V1.1 system for HIV-1 RNA load testing, the number of invalid tests per assay run gradually increased. Within five days, approximately 50% of tests showed a total lack of amplification of the calibrator and in most cases also of the HIV-1 template. According to the manufacturer's specifications, the lysis buffer and three extraction buffers remain on the automated NucliSens easyMAG extraction system between assay runs. Therefore possible microbial contamination of these buffers was investigated, after they had been on the automated system for approximately one week. The NucliSens easyMAG extraction buffer 2 yielded bacterial growth identified as Acinetobacter baumannii. After regular decontamination of the machine's tubing system with 70% alcohol and storage of the buffers at 4 degrees C between assay runs were commenced, invalid results due to failed internal calibrator signal occurred no longer. It is likely that bacterial contamination of the buffer was the cause of assay failure, probably due to ribonuclease (RNase) activity. Bacterial contamination of PCR systems should be added to the list of potential hazards in diagnostic virology. This experience underlines the necessity of state-of-the-art assay design incorporating adequate internal controls and calibrators.