[RNA amplification from low numbers of bacteria in human blood to solve low sensitivity problem of conventional PCR amplification]

Blood culture from a febrile patient is often unsuccessful when a number of bacteria is lower than 10 CFU/ml in circulation. Theoretically, PCR amplification of bacteria in this low bacterial level in human blood is beyond the sensitivity of PCR method. RNA based Amplification (NASBA) method is more sensitive than PCR base assay because living bacteria carries several hundred to thousand RNA molecules in a cell. Increasing evidences also revealed another problem that human blood stream is often contaminated with bacterial genome. This often cause false positive results and bring difficulty of the interpretation to determine causative agent. Direct DNA amplification should be limited to severe causative agent such as Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitides, and etc. and also applied only for slow growing and fastidious pathogens such as mycoplasmae, chlamydiae, bartonellae, mycobacteria, and etc. Most cases blood culture should be carried out first and genetic amplification should be selected as a next step to detect pathogens from a blood culture bottle, because in these cases, patients are not in life threatening stages. These attempts also decrease the chance to detect circulating bacterial DNA fragment in blood stream.