The development of a typing method for an uncultivable microorganism: the example of Pneumocystis jirovecii

The development of a molecular typing method for the uncultivable fungus Pneumocystis jirovecii is described. The method consists of the amplification by PCR of four variable regions of P. jirovecii genome, followed by the detection of the polymorphisms by the single-strand conformation polymorphism technique. About 70% of the patients harbored two or rarely three alleles of at least one of the four genomic regions. This was shown to most probably be due to co-infections with several P. jirovecii types. Each combination of four alleles of the four genomic regions defines a type. Analysis of the alleles and their abundance allows identification of the co-infecting types in about 65% of the specimens co-infected with two types. The method has been validated by the evaluation of several criteria. The main advantage of the method is that it is relatively cheap and that up to 50 specimens can be analysed at the same time. Its main disadvantage is that about 30% of the specimens cannot be typed because of the complexity of the alleles configuration. This multitarget typing method has been used for several epidemiological purposes.