Lee et al. reported on the nucleotide sequence variation of the two internal transcribed spacers (ITSs), ITS1 and ITS2, of the rRNA operon of Pneumocystis carinii f. sp. hominis (3). Thanks to their work, the number of polymorphic nucleotide positions recognized has increased and the number of recognized alleles has passed from 2 to 15 for ITS1 and from 3 to 14 for ITS2. Also, the number of different combinations of the alleles of the two loci, each defining a P. carinii f. sp. hominis type, has increased from 4 to 59. This great diversity of types will undoubtedly be useful for epidemiological studies. Accordingly, Lee et al. concluded that nucleotide sequence determination of PCR products from these regions is the method of choice for typing P. carinii f. sp. hominis isolates.
However, typing of P. carinii f. sp. hominis is complicated by the fact that coinfections of single patients are likely to occur (for a review, see reference 1). To approach this problem, Lee et al. used as a screening step five oligonucleotides hybridizing specifically to only two alleles of ITS1 and three alleles of ITS2. A coinfection was assumed if, for one locus, the PCR product from a specimen hybridized to more than one oligonucleotide. However, (i) this method does not allow the differentiation of polymorphisms located outside the annealing site of the oligonucleotide. Also, (ii) an oligonucleotide may fail to hybridize to alleles with unrecognized polymorphisms at the annealing site. According to the data of Lee et al., the first possibility probably occurred for both ITSs and the second possibility occurred for only ITS2. Thus, the presence of more than one allele, revealing possible coinfection events, may be missed by their screening step.
Given these considerations, we feel that only efficient detection of potential coinfections, either by direct subcloning of the PCR product from the ITSs followed by sequencing of numerous clones or by a multiple-target approach using single-strand conformation polymorphism (2), will help the progress of P. carinii f. sp. hominis epidemiology.
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