Detection and identification of Pneumocystis carinii-DNA : emphasis on laboratory diagnosis and occurrence in air

Abstract

Pneumocystis carinii is a fungal opportunistic pathogen, which causes serious pneumonia (P. carinii pneumonia - PCP) in patients with AIDS, leukaemia, and other immunodeficient conditions. The laboratory diagnosis is dependant on microscopic demonstration of the organism. Increased sensitivity and subsequent genotyping can be achieved by the use of the polymerase chain reaction (PCR). The infectious route of PCP is airborne, based on animal studies and epidemiological observations. In outbreaks of PCP among immunosuppressed patients in hospitals, a horizontal transmission of P. carinii has been suggested.

The primary aim of this thesis was to establish a diagnostic PCR assay, based on the thymidylate synthase (TS) gene of rat P. carinii, and evaluate the method on induced sputum (IS) and bronchoalveolar lavage (BAL) samples from immunosuppressed and non-immunosuppressed patients. To make the method more adapted for routine use, a rapid DNA preparation method was evaluated, preceding a nested PCR assay, based on the same gene of rat P. carinii. The method was shown to be of predictive value for HIV infected patients without PCP symptoms.

A second aim was to study the transmission of P. carinii using air filtration and subsequent PCR amplification of the filter eluates, as well as P. carinii carriership in hospital staff, caring for AIDS patients. Amplified DNA of the TS gene of rat P. carinii was demonstrated in the proximity air of a P. carinii infected rat colony. The TS-PCR assays and P. carinii serology were used to test induced sputa and blood from the hospital staff. No P. carinii carriership in immunocompetent subjects was demonstrated.

Amplification of nucleic acids of the gene coding for the mitochondrial large sub-unit rRNA (mt LSU) of P. carinii f.sp. hominis (human form) was used to identify genotypes, present in the air of PCP patient rooms in hospitals. The results showed a presence of the same P. c. hominis genotypes in the air of the PCP patient room and the adjacent corridors, as those identified in the patient. P. c. hominis was also detected in ward units without past or present PCP patients. This suggests that a risk of person-to-person transmission of P. c . hominis between PCP patients and susceptible individuals cannot be excluded. A similar matching of P. carinii genotypes was also shown in an experimental study of the air surrounding P. carinii infected rabbits and rats, using the genotypes of internal transcribed spacer (ITS) regions and the mt LSU rRNA locus of P. carinii f. sp. oryctolagi and P. carinii f.sp. carinii. Moreover, an immunosuppression and time dependent increase in host-specific P. carinii-DNA was demonstrated in the lungs of the infected rats and in air.

The mt LSU DNA sequence markers for human P. carinii were used to study possible horizontal transmission of P. c. hominis among renal transplant recipients and patients with haematological malignancies in hospitals. Taking into account a postulated incubation period and comparing P. c. hominis genotypes isolated from BAL samples, the data do not support a putative person-to-person transmission among the PCP patients in the outbreaks studied.