Pneumocystis carinii ITS Typing: Doubtful Evidence of Genotype-Related Virulence
Pneumocystis carinii is an opportunistic pulmonary pathogen particularly associated with HIV-infected patients. Controversial results have been reported on the possible occurrence of genotype-related virulence [1–2] based on Internal Transcribed Spacers (ITS) typing. Wide ITS variability has been identified, and the locus is described as a single copy gene, thus allowing accurate description of genotypes during P. carinii pneumonia (PCP) episodes [3,4]. Based on paired clinical and molecular analysis of ITS sequences, we examined if there is a correlation between these two parameters among P. carinii isolated from patients in Italy.
MATERIALS AND METHODS
115 bronchoalveolar lavage (BAL) fluid samples from HIV-infected patients with PCP collected in our hospital from 1994 to 1999 were designated positive for P. carinii by microscopic analysis of morphology. These samples were analyzed by ITSs nested PCR followed by type-specific oligonucleotide typing (TSO) procedures. Alternatively these were analyzed by direct sequencing (Perkin Elmer Big Dye Terminator) using the Cromas® software analysis of sequences. When co-infections occurred, the TA cloning kit was used to discriminate sequences of 5 different clones.
The following features of patients were considered for statistical analysis: risk factors, CD4 count, final outcome, previous P.carinii prophylaxis, lowest measured arterial pO2, highest measured lactate dehydrogenase (LDH), maximum extent of respiratory support, and treatment with corticosteroids. Statistical analysis was performed by the Prism ® software, using the Fisher exact test and the non parametric MannWithney test.
RESULTS AND DISCUSSION
The main features of the 115 patients are summarized: 94 males and 21 females, mean ages were 38.7 and 34, respectively;. Risk factors for HIV infection were: intravenous drug use (52%), homosexuality (15.6%), and heterosexuality (32.4). Final outcome: recover (100), death (12) and PCP relapse (3). The mean lowest measured arterial pO2 was 61.5 (range 36–105); the highest serum LDH was 910 U (range 418 U-2,380 U). The maximum respiratory support used was none (25), oxygen mask 3 1/min (31), Venturi mask 15 1/min (31), positive pressure (5 to 10 cm H2O) mask (21) and mechanical ventilation (5). After ITS nested PCR, positive products from 115 BAL samples were first typed by TSO. This method allowed complete genotyping of only 69 isolates from 54/115 (46.9%) patients (due to doubtful or incomplete hybridization of the ITS1 or ITS2 probes). The following genotypes were found: Ab (7.2%), Ac (23.2%), Ba (13.9%), Bb (26.1%) and Bc (30.4%). The attempt of correlating this limited number of genotypes to clinical parameters (CD4 counts, highest LDH, age, lowest pO2, corticosteroid treatment, etc) showed no statistically significant differences (p>0.05). With evidence for multiple variants of ITS, direct sequencing of positive nested PCR products was performed. Coinfections prevented direct sequencing in 31/115 (26.9%) of examined samples. Thus, cloning was required in order to determine the genotypes involved. Forty different genotypes were found in 78 BALs (single infections), which were fully typed (forward and reverse), and repeatedly confirmed (Table 1). Interestingly, the genotypes identified in non-invasive samples collected from individual patients (garglings, blood) did not always match the genotype identified in the BAL sample from the same patient (data not shown).
| Number | ITSs Genotypes |
|---|---|
| 9 | Eg |
| 7 | Kf |
| 5 | Ac, Bi, Eb, Em |
| 3 | Bu |
| 2 | Bc, Eo, Ec, Ed, Ef, Gf, Gi |
| 1 | Af, Ai, Ba, Bf, Cc, Cf, Ea, Ec, Eh, El, Fg. Fm, Gc, Gh, Gu, Hc, He, Hf, Hg, Ih, Jf, Jg, Iu, Ne, Of |
We analyzed single infections in an attempt to attribute “virulence” to single genotypes by analyzing clinical parameters. However, there were no statistically significant correlation between genotype and LDH, sex, age, CD4 count, previous prophylaxis, use of corticosteroids, lowest value of arterial oxygen (p>0.05), co-pathologies, differences in prophylaxis regimens (cotrimoxazole oral dose or aerosol pentamidine), intolerance to first choice therapy (intravenous cotrimoxazole, intravenous clindamycin, oral primaquine, oral atovaquone). The interference by DHPS mutation (<10% in our sample) complicated the analysis of clinical outcome (we could not distinguish between true microrganism virulence or drug resistance). This was true for both TSO type and for the sequence of the isolates. Although Eg genotype was frequently associated with the clinical need for aggressive respiratory support, this correlation was not statistically significant. We conclude that TSO is not as reliable a method as sequencing for ITS genotypes. A consensus in defining nomenclature for specific ITSs would allow easier comparison of results in different study groups.
ACKNOWLEDGMENTS
This study was supported by a grant Istituto Superiore Sanità (ISS 50C.2)
