Typing of Pneumocystis carinii f.sp. hominis in Patients with or without Pneumocystosis

Over the past decade, the polymerase chain reaction (PCR) has proved to be the most sensitive technique for detection of Pneumocystis carinii (P. carinii) in pulmonary specimens from patients developing P. carinii pneumonia (PCP) [9]. Despite this high sensitivity, P. carinii detection by PCR on post-mortem lungs from immunocompetent individuals failed to give positive results. For this reason, long term pulmonary carriage of P. carinii in healthy immunocompetent subjects has been reevaluated. Thus, PCP is now frequently considered to result from de novo infection rather than from reactivation of latent infection [4]. Nonetheless, use of PCR assays has led to detection of low numbers of P. carinii organisms, which were undetectable by microscopy, in bronchoalveolar lavage (BAL) fluid specimens from patients who showed alternative diagnosis of acute PCP. These low levels of P. carinii organisms were considered to reflect pulmonary colonization or carriage. These cases were observed mainly in symptomatic patients with underlying pulmonary diseases [5]. There is little data concerning genotypes of P. carinii carried by colonized patients [1,3] whereas several P.carinii isolates from patients with acute PCP have been typed [2, 6–8]. The aim of the present study was to identify the genotypes of Pneumocystis carinii f. sp. hominis (human derived P. carinii) obtained from patients colonized by the fungus. These genotypes were compared with those obtained from a patient population developing acute PCP who was admitted to the same hospital at the same time.

PATIENTS AND METHODS

All patients enrolled in the study were admitted and followed up in the same hospital (University Hospital, University of Picardy, Amiens, France) between March 1996 and March 1999. Six specimens were obtained from 6 HIV-negative patients colonized by P.carinii. The patients underwent a BAL to investigate pulmonary symptoms (abnormal chest X-Ray, cough) or fever. The underlying conditions were myeloma, sarcoidosis associated with panhypopituitarism, renal transplantation, chronic obstructive pulmonary disease, systemic lupus erythematosus associated with pulmonary lymphoma, and chronic lymphoid leukemia. Microscopic detection of P.carinii in BAL fluid samples was negative whereas P. carinii DNA was detected using a PCR assay. After a rapid DNA extraction on a portion of BAL sediment (GeneReleaser®, BioVentures, Murfreesboro, TN, USA) a heminested-PCR assay was performed using the specific primers pAZ102E & pAZ102H (first PCR round) and pAZ102L2 & pAZ102E (second PCR round) [9]. These positive results were controlled by a second experiment of PCR. PCR results were not used to determine patient management, but patients were followed up to detect the occurrence of PCP. As clinical improvement was obtained despite the absence of specific treatment for P.carinii, the patients were considered to be merely colonized by the fungus. The remaining sediment from BAL samples was also stored at - 20 °C for further analysis.

Ten other specimens were obtained from 10 patients diagnosed with PCP according to criteria described for HIV-infected patients by the CDC. The underlying conditions were HIV-infection (8 patients), systemic lupus erythematosus (one patient) and hepatic granulomatosis (one patient). P.carinii was detected in BAL fluid samples by Giemsa staining and immunofluorescence with an anti-P. carinii cyst monoclonal antibody. Specimens were stored at 20°C for further analysis.

The 16 P. carinii isolates from both patient population (colonized patients and patients with PCP) were typed by sequence analysis of the internal transcribed spacers (ITS 1 and ITS 2) of the nuclear rRNA operon. DNA extraction of all specimens was performed using a commercially available kit (QIAamp DNA MiniKit®, Qiagen, Santa Clarita, CA, USA). A nested PCR was performed with the two pairs of primers N18SF and N26SRX (first PCR round) and ITSF3 and ITS2R3 (second PCR round), specific of P. carinii f.sp. hominis (P.c.hominis) [6–8]. The PCR products of the second PCR round were cloned into pGEMT Vector® System II (Promega Corporation, Madison, WI, USA) and sequenced from the two strands. The sequences were compared with those reported by Tsolaki et al. [6–8]. P. carinii ITS types are defined by a combination of the alleles of the two ITS1 and ITS2 loci.

RESULTS AND DISCUSSION

The results are summarized in Table 1. Five new P.c.hominis ITS types (new ITS1 and ITS2 types and/or new allele combinations) were found in the patient population with PCP (types “A” a3, A2a4, A2 “b”, B2bl, and A3a2). Except for one (type A3a3), all P.c.hominis types found in patients colonized by the fungus were previously described in AIDS patients with PCP [6–8]. A total of 12 different types were detected in our 16 patients. Eight of these 12 types were only detected in the patient population with PCP, another type was only observed in the colonized patient population and the 3 remaining types were found in both patient populations. More than one type was detected in 5/10 of patients with PCP and in 2/6 of the colonized patients, showing that mixed infections may occur in both patient populations.

The results show that ITS 1 and ITS 2 sequence analysis enables the typing of P.c.hominis organisms carried by colonized patients. There is a high diversity of P.c.hominis organisms in colonized patients as well as in patients with PCP. This high diversity of P.c.hominis genotypes, as well as the occurrence of mixed infections, render it quite difficult to clearly establish a correlation between genotypes and clinical profiles (colonization vs. PCP). Nonetheless, the results suggest that several types could be present only in patients who develop PCP. Conversely, similar types can be found in both patient populations suggesting that P.c.hominis acquisition could result from a common source of the fungus. As PCP in man is now considered to be an anthroponosis, the hypothesis that all patient populations infected with P.c.hominis could play a role as a reservoir must be investigated.