Demonstration by a nested PCR for Mycoplasma pneumoniae that M. pneumoniae load in the throat is higher in patients hospitalised for M. pneumoniae infection than in non-hospitalised subjects

A nested PCR protocol to detect Mycoplasma pneumoniae DNA in throat specimens was developed. An amplification control (AC) template, which is amplified by the same primers as the M. pneumoniae target sequence, was constructed. The assay allowed highly specific and sensitive detection of M. pneumoniae DNA. In all, 305 throat samples, 62 from hospitalised patients and 243 from non-hospitalised subjects, were analysed by the nested PCR. Inhibition of the PCR was observed in 20% of the samples, but was abolished after a 1 in 10 dilution. Throat samples from 5 (8%) of the hospitalised patients and from 7 (3%) of the non-hospitalised subjects were positive for M. pneumoniae DNA. To investigate the relationship between M. pneumoniae load and the severity of disease, the M. pneumoniae load in 10 throat samples from M. pneumoniae-positive subjects was assessed semi-quantitatively by application of the nested PCR to a series of limiting dilutions of nucleic acid extracted from these throat samples. The calculated M. pneumoniae load varied from 20 to 3830 cfu/ml of throat sample. The mean M. pneumoniae load in samples from the hospitalised patients was significantly higher than that in samples from the non-hospitalised subjects. The nested PCR is a useful tool to detect M. pneumoniae DNA in the throat and to study the relationship between the load of M. pneumoniae in throat samples and severity of disease due to M. pneumoniae infection.