Typing of Pneumocystis carinii f.sp. hominis Isolates from Nasopharyngeal Aspirates of Immunocompetent Infants with Bronchiolitis by Dihydropteroate Synthase Gene Analysis

Sulfa and sulfone drugs are widely used in Pneumocystis carinii pneumonia (PCP) prophylaxis and treatment. Their enzymatic target is the dihydropteroate synthase (DHPS). Point mutations have been described in Pneumocystis carinii f.sp. hominis (P.c.hominis) DHPS gene, mainly at positions 165 and 171 [4]. Mutant P.c.hominis DHPS genotypes have been detected in patients developing PCP with or without prior sulfa or sulfone prophylaxis [3,5]. In a recent study, it was shown that P.c.hominis infected 45 immunocompetent infants diagnosed with bronchiolitis (Nevez et al. IWOP-7 2001, abstract PO2). The fungus was detected in nasopharyngeal aspirates (NPAs) using a nested PCR assay amplifying a portion of the mitochondrial large sub-unit rRNA (mtLSUrRNA) gene [6]. None of these infants received sulfa or sulfone drugs before and after P.c. hominis DNA detection. The aim of this study was to identify wild and mutant P.c.hominis DHPS genotypes in this infant population.

PATIENTS AND METHODS

The forty five NPA specimens positive for P.c.hominis obtained from 45 infants diagnosed with bronchiolitis were stored at -20 °C for further typing. The typing of P.c.hominis was performed by amplification of DHPS gene and restriction length polymorphism (RFLP) analysis. A nested PCR assay was performed with Ahum and Bhum primers [4] for the first round and Cprim and Dprim primers (S. Latouche, unpubl.) for the second round. The amplification products were analyzed using electrophoresis in agarose gel to detect a specific 269 bp band. The positive PCR products were divided into two-parts. One part was digested by the restriction enzyme AccI, the second part was digested by the enzyme HaeIII [2]. AccI and HaeIII lead to detect mutations at positions 165 and 171 respectively. After digestion, the PCR products were analyzed by electrophoretic migration.

RESULTS AND DISCUSSION

P.c. hominis was initially detected in 45 NPAs (45 infants) by the first PCR assay amplifying the mtLSUrRNA gene, whereas the fungus was detected by DHPS gene amplification in only 30 NPAs. This discrepancy of sensitivity between the 2 PCR assays has been previously observed concerning immunocompromised patients diagnosed with acute PCP (S. Latouche, unpubl.). A P.c.hominis wild type DHPS was detected in 27 NPAs. A mutant type was associated with a wild type in the three remaining NPAs, suggesting mixed infections. In one NPA, the mutant type had a mutation at position 165. In two other NPAs, the mutant types had a mutation at position 171.

Thus, P.c. hominis mutant types are present early in childhood. However, no infant was infected with a mutant singly nor with a double mutant, unlike results previously reported concerning immunocompromised adults developing PCP [1,3,5].

Since no infant had a past history of sulfa or sulfone treatment, the results show that mutant types can be present among a population of P.c. hominis organisms not submitted to selection pressure. Presence of these mutant types in this infant population could result in an incidental acquisition of the fungus. These results are consistent with those previously observed in adult patients diagnosed with PCP. Indeed, mutant types have also been detected casually in these patients despite the absence of sulfa or sulfone treatment [3,5].

These observations suggest that transmission cycles of P.c.hominis in infants with bronchiolitis and adults with PCP could not be strictly independent. The acquisition of P.c.hominis in immunosuppressed patients and immunocompetent infants developing different clinical forms of P.c.hominis infection could result from a common exogenous (human or non-human) source of the fungus.

ACKNOWLEDGMENTS

This study was partially supported by the PRFMMIP (Programme de Recherche Fondamentale en Microbiologie et Maladies Infectieuses et Parasitaires), French Ministry of Education, Research and Technology.