ITSS GENOTYPES AND BLOOD DISSEMINATION DURING ACUTE PCP

The presence of P. carinii DNA in serum and in Peripheral Blood Mononu clear Cells (PBMC) during acute phase of PCP in AIDS patients was prev iously demonstrated by several authors using different specific primer s [1,2,3]. Amplification by ITSs nested PCR followed by TSO hybridizat ion of P. carinii isolates [4] derived from BAL and blood samples allo ws to compare genotypes involved in the disease and genotype-related d ynamics of Pc-DNA clearance from blood during therapy. Different virul ence charateristics among P. carinii genotypes could explain the vario us spectrum of clinical presentation (pulmonary and extrapulmonary) an d susceptibility to classic antipneumocystic drugs during PCP. MATERIA LS AND METHODS. We analysed by TSO hybridization 26 BAL, 60 sera and 1 2 PBMC collected from 25 AIDS patients with morphological diagnosis of PCP. Briefly, blood was collected by Vacutainer venopuncture in hepar inized (for cells) or in clotting (for serum) 7 ml tubes and processed by centrifugation at 4 C within few hours from withdrawn. PBMC were s eparated in 4/1 RPMI/Ficoll gradient. 250 mu l of each sample were dig ested with proteinase K and used to prepare DNA by phenol/clorophorm e xtraction followed by ethanol/sodium acetate precipitation. To purify DNA from buffer salt, TE resuspended samples were spin-dyalized throug h S200 Microspin columns (Pharmacia Biotech). 10 mu l of template were used for Pc ITSs nested PCR with 1724F-TTS2R primers for the first st ep and ITS1F-ITS2R1 for the second step. All PCR products, after denat uration with 0.4N NaOH, were blotted on Nytran paper, prehybridized O/ N at 37 C with 100 mu g/ml salmon testes DNA (SIGMA Chemicals) in GX S SC, 5X Denhart's solution, 0.5% SDS, 0.05%Ppi and then hybridized O/N at 37 C by using 1A, 1B, 2a, 2b, 2c biotynilated probes at high string ency conditions. The signal detection was obtained by using Phototope- Star Detection Kit (BioLabs) followed by exposition to AR Kodak film. RESULTS AND DISCUSSION. Alter TSO hybridization, considering 28 episod es of PCP occurred in 25 AIDS patients (25 prime episodes and 3 relaps es), in 16/28 cases we detected the presence of a single P. carinii ge notype in BAL, in 12/28 the presence of more than one P. carinii genot ypes, suggesting a coinfection. In 4/12 coinfections observed, there w as perfect correspondence between P. carinii genotype detected on BAL compared to the one on serum. In 8/12, P. carinii genotype detected on serum only partially matched with the one present on BAL. During foll ow up, we observed a different genotype-specific clearance from blood, suggesting virulence and drugs susceptibility related to genotype. Du ring PCP relapses, genotype analysis revealed either endogenous (same genotype) or exogenous reinfection (genetic switch). The recent findin g of further variation among ITSs sequences could explain the lack of hybridization of some samples and underlines the necessity of sequenci ng for P. carinii typing.