1 Service de Parasitologie, Mycologie et Médecine des Voyages, Hôpital Sud, Centre Hospitalier Universitaire, Université de Picardie Jules Verne, 1 rue Laennec, 80054 Amiens, Paris, France
2 Centre Hospitalier Universitaire Saint Antoine, Paris, France
Correspondence
Anne Totet
totet.anne{at}chu-amiens.fr
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ABSTRACT |
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INTRODUCTION |
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New detection tools, such as PCR assays, have revealed that Pneumocystis infections can give rise to a broad spectrum of clinical manifestations, of which PCP in immunocompromised patients may represent only a small part, while other presentations may constitute the major part. It has been shown that pulmonary colonization with P. jirovecii occurs frequently in immunocompromised patients (Leigh et al., 1993; Nevez et al., 1999
) and less frequently in apparently immunocompetent persons suffering from lung diseases (Armbruster et al., 1997
; Sing et al., 1999
). Moreover, it has recently been established that P. jirovecii is detectable in immunocompetent infants at risk of primary Pneumocystis infection who developed acute respiratory syndromes (Nevez et al., 2001
; Vargas et al., 2001
). The existence of similar genomic characteristics among P. jirovecii isolates from patients developing different forms of infection would provide arguments implicating these patients in a common human reservoir for the fungus. The aim of the present study was to identify P. jirovecii genotypes at two loci in adults who developed PCP, in adults colonized by P. jirovecii and in immunocompetent infants infected with the fungus.
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METHODS |
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P. jirovecii dihydropteroate synthase (DHPS) genotyping.
The P. jirovecii DHPS locus was examined using PCR-RFLP analysis. The DHPS sequence was first amplified by a nested-PCR assay. The first PCR round was performed with primer pair AHUM (5'-GCG CCT ACA CAT ATT ATG GCC ATT TTA AAT C-3')/BHUM (5'-CAT AAA CAT CAT GAA CCC G-3') (Lane et al., 1997) by using a touch-down programme. The second PCR round was performed with primer pair CPRIM (5'-CCC CCA CTT ATA TCA-3')/DPRIM (5'-GGG GGT GTT CAT TCA-3') (Demanche et al., 2001
). To monitor and prevent contamination, identical measures were applied as described above. The RFLP assay was performed with two restriction enzymes, AccI and HaeIII, according to the manufacturer's recommendations (Promega). Part of the second-round PCR products was digested with AccI and another part with HaeIII, making possible the detection of mutations at nucleotide positions 165 and 171, respectively (Diop Santos et al., 1999
). The restriction profiles were visualized by electrophoresis of digested products on a 1·5 % agarose gel with ethidium bromide.
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RESULTS |
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DISCUSSION |
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Applying the typing score described by Tsolaki et al. (1996, 1998) for ITS genotyping, we identified 14, six and nine ITS genotypes in groups 1, 2 and 3, respectively. The polymorphism of genotypes was close in the three patient groups. Furthermore, similar main genotypes (B1a3 and B2a1) as well as mixed infections were observed. However, the rate of mixed infections was lower in infants than in the two groups of adults. This may partially be due to the lower number of clones sequenced for infant samples (mean, 2·6 clones per sample). It may also be related to the relative low efficiency of detection of minor genotypes in samples recovered by non-invasive means, such as nasopharyngeal aspirates.
Three alleles which we designated A, b and b1 were considered to be new alleles, since they present a combination of substitutions (at scoring positions) that was not reported by Tsolaki et al. (1998). However, applying the typing score described by Lee et al. (1998)
, allele A is the same as allele G (GenBank accession no. AF013812). Alleles b and b1 remain with no correspondence (Lee et al., 1998
) and are described in this study for the first time (Matos et al., 2003
; Nimri et al., 2002
). To the extent that each of these two alleles has only been detected in one patient, the hypothesis that they actually represent new alleles remains to be confirmed.
It was suggested in the literature that the high polymorphism of the ITS regions was related to an intrinsic instability, which may generate changes from a single P. jirovecii ITS genotype (Helweg-Larsen et al., 2001). However, in the fungal kingdom, the mean DNA substitution rate at ITS regions has been estimated at 1·4±1·3x109 substitutions per site per year (Kasuga et al., 2002
). Change at this rate would not present a problem for P. jirovecii ITS genotyping in epidemiological studies. Furthermore, previous reports have shown that the most frequent P. jirovecii ITS genotypes in patients from different regions of Europe or the United States were identical [B2a1 and B1a3, corresponding to Ne and Eg as described by Lee et al. (1998)
]. This identity of main genotypes among non-epidemiologically linked isolates pleads in favour of ITS sequence analysis as a valuable method for P. jirovecii genotyping.
Overall, our results of ITS genotyping make it possible to establish that in our three patient groups (i) a similar degree of genotype diversity and identical main genotypes can be observed and (ii) mixed infections occur. These shared features suggest that fungus acquisition may result from common sources. These data are compatible with the hypothesis that the three patient groups make up a common reservoir for the fungus.
The other target that we examined for genotyping was the DHPS locus. The absence of correlation between ITS and DHPS genotypes observed in this study is consistent with previous reports concerning immunocompromised patients with PCP (Ma & Kovacs, 2001; Matos et al., 2003
).
Analysis of the DHPS locus can be used as a marker for studying the potential circulation of the fungus within the human reservoir (Beard et al., 2000; Huang et al., 2000
). Indeed, prior sulfonamide treatment has been identified as a predictor of mutant genotypes (Huang et al., 2000
; Kazanjian et al., 2000
; Ma et al., 1999
). However, the city of patient residence has also been identified as an independent risk factor, a factor that supports the hypothesis that P. jirovecii can be transmitted from infected treated patients to susceptible untreated patients, either directly or through hypothetical environmental sources. In our study, mutants were detected in patients from the three groups, although none of them had prior exposure to sulfonamide drugs. Because of the young age of the infants and consequently their short medical history, it was easy to ascertain that none had been subjected to prior sulfonamide exposure. Conversely, this exposure in adults throughout their lifetime cannot strictly be ruled out. These difficulties have previously been evoked by Huang et al. (2001)
, who have pointed out the need for standardization of the definition of exposure to sulfonamides. In particular, the period during which sulfonamides have not been used, preceding patient sampling, to define the absence of selective pressure, varies according to the experience of each medical team. At any rate, in the present study, no adults were treated with sulfonamides in the 3 months preceding specimen retrieval. For these reasons, the presence of mutants in our patients, which included an infant group, may be related to an incidental acquisition of the micro-organism, directly or indirectly, from individuals treated with sulfonamides.
Besides mutant presence in each patient group, other common genomic characteristics at the DHPS locus were observed in P. jirovecii organisms from the three patient groups. The most frequent P. jirovecii DHPS genotype was the wild genotype. Mutant genotypes have only been detected within mixed infection occurrences. This community renders it compatible with a circulation of P. jirovecii between infected infants and adults with either PCP or colonization.
Until recently, investigations of P. jirovecii transmission using genotyping have been based mainly on analyses of clustered cases of PCP occurring among severe immunocompromised patients (Helweg-Larsen et al., 1998; Latouche et al., 1997
; Miller et al., 2002
; Olsson et al., 2001
). Few have considered the potential role of other human populations developing other forms of Pneumocystis infection and therefore contributing to the circulation of the fungus. Miller et al. (2001)
have suggested that immunocompetent asymptomatic health-care workers in close contact with PCP patients may have a potential role in further circulation or transmission of the fungus. This hypothesis was prompted by the results of Dumoulin et al. (2000)
, who have shown that immunocompetent and apparently asymptomatic mice transiently parasitized by Pneumocystis after a brief contact with SCID mice with PCP were able to transmit the fungus to other susceptible mice. Thus, experimental results have established that Pneumocystis organisms are transmissible between hosts developing diverse forms of Pneumocystis parasitism, and medical studies have suggested a similar P. jirovecii transmission in humans.
Considering that the patient groups we studied came from a restricted geographical region, our results of multilocus genotyping are compatible with the hypothesis that they make up a common human reservoir in which the fungus may circulate. Because of the potential transmissibility of P. jirovecii, all individuals parasitized by the fungus, whatever their risk factor for infection and the form of parasitism they presented, may play a role in circulation of the fungus within human communities.
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ACKNOWLEDGEMENTS |
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Received 18 November 2003;
revised 23 February 2004;
accepted 23 February 2004.
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