[Application of polymerase chain reaction in medical microbiology]

An overview is presented of different nucleic acid amplification techniques, particularly the polymerase chain reaction (PCR). The possible applications of the PCR in medical microbiology and the different phases of the PCR are discussed. Four PCR applications, as elaborated and used in the authors' laboratory are presented: the diagnoses of Mycoplasma pneumoniae infections, herpes simplex virus encephalitis and Toxoplasma gondii encephalitis, and the detection of Mycobacterium leprae. For the first and the last of these, original internal controls to detect the presence of inhibitors were elaborated. The target amplicon was inserted in a plasmid and the central part modified, either by insertion of a sequence of foreign DNA (Mycoplasma pneumoniae) or excision of a sequence (Mycobacterium leprae). The addition of a small amount (in order to avoid inhibition of the diagnostic reaction) of these plasmids to the reaction mixture, results in coamplification of this modified DNA with the same primers as the target DNA. During three successive winter periods, nasopharyngeal aspirates from children presenting with respiratory infections at the Universitair Ziekenhuis Antwerpen, were examined for viruses and for M.pneumoniae by the PCR. The PCR for herpes virus in cerebrospinal fluid is a powerful diagnostic technique. Five cases could be documented, one of them being the second known case of herpes virus encephalitis of the brain stem. The sensitivity of the PCR for Toxoplasma gondii encephalitis as performed in our laboratory on cerebro-spinal fluid probably needs to be improved. The detection of M. leprae by a PCR for the 16S rRNA can detect as little as 30 organisms, which is insufficiently sensitive for the diagnosis of paucibacillary leprosy in skin biopsy specimens. The technique allowed for the first time the detection of M. leprae DNA in nose mucosae of patients and some of their contacts. The PCR is very promising to elucidate the etiology of diseases the responsible agent of which is present in too small numbers to be detected by traditional techniques, as well as for the diagnosis of agents difficult or impossible to cultivate or to identify still unknown agents.