Carinii Strains Research Articles

Mutations in dihydropteroate synthase gene of Pneumocystis carinii in HIV patients with Pneumocystis carinii pneumonia

The purpose of this study was to determine whether dihydropteroate synthase gene (DHPS) mutations were associated with the failure of sulpha/sulphone drugs used as prophylaxis agents in HIV infected patients. Results suggested that DHPS mutations were significantly associated with failure of anti- Pneumocystis carinii sulphone prophylaxis ( P=0.031). An increasing number of mutant P. carinii strains have been isolated from patients no longer having prophylaxis. There was no statistically significant difference in severity or outcome of the pneumonia caused by wild-type or mutant DHPS. Moreover, two of the three patients with mutant P. carinii pneumonia (PCP) were successfully treated with sulpha drugs. We think that P. carinii drug-resistance could be an emerging problem for immunocompromised patients including those with HIV infection.

Survival of patients with AIDS, after diagnosis of Pneumocystis carinii pneumonia, in the United States.

To examine survival after diagnosis of Pneumocystis carinii pneumonia (PCP) and factors associated with early death (during the month of or the month after diagnosis of PCP), data were analyzed from the Adult and Adolescent Spectrum HIV Disease project. Among 4412 patients with 5222 episodes of PCP during follow-up (1992-1998), survival at >1 month after diagnosis was 82%, and survival at > or =12 months after diagnosis was 47%; 12-month survival increased from 40% in 1992-1993 to 63% in 1996-1998. By multiple logistic regression analysis, early death was associated with history of PCP (odds ratio [OR], 1.4), age 45-59 years (OR, 1.9) or > or =60 years (OR, 3.7), and CD4 cell count of 0-24 cells/microL (< or =5 months before PCP; OR, 1.8) or 25-49 cells/microL (OR, 1.4) (P<.05). Concurrent prescription of combination antiretroviral therapy (OR, 0.2) and other antiretroviral therapy (OR, 0.4) was associated with surviving the early period. This study shows improved survival after diagnosis of PCP in recent years, despite emergence of antibiotic-resistant mutant P. carinii strains.

Pneumocystis carinii pneumonia: a review of current issues in diagnosis and management.

Pneumocystis carinii pneumonia: a review of current issues in diagnosis and management.

Identification, characterization, and expression of the BiP endoplasmic reticulum resident chaperonins in Pneumocystis carinii.

We have isolated, characterized, and examined the expression of the genes encoding BiP endoplasmic reticulum (ER) resident chaperonins responsible for transport, maturation, and proper folding of membrane and secreted proteins from two divergent strains of Pneumocystis carinii. The BiP genes, Pcbip and Prbip, from the P. c. carinii (prototype) strain and the P. c. rattus (variant) strain, respectively, are single-copy genes that reside on chromosomes of approximately 330 and approximately 350 kbp. Both genes encode approximately 72.5-kDa proteins that are most homologous to BiP genes from other organisms and exhibit the amino-terminal signal peptides and carboxyl-terminal ER retention sequences that are hallmarks of BiP proteins. We established short-term P. carinii cultures to examine expression and induction of Pcbip in response to heat shock, glucose starvation, inhibition of protein transport or N-linked glycosylation, and other conditions known to affect proper transport, glycosylation, and maturation of membrane and secreted proteins. These studies indicated that Pcbip mRNA is constitutively expressed but induced under conditions known to induce BiP expression in other organisms. In contrast to mammalian BiP genes but like other fungal BiP genes, P. carinii BiP mRNA levels are induced by heat shock. Finally, the Prbip and Pcbip coding sequences surprisingly exhibit only approximately 83% DNA and approximately 90% amino acid sequence identity and show only limited conservation in noncoding flanking and intron sequences. Analyses of the P. carinii BiP gene sequences support inclusion of P. carinii among the fungi but suggest a large divergence and possible speciation among P. carinii strains infecting a given host.

Typing of Pneumocystis carinii strains with type-specific oligonucleotide probes derived from nucleotide sequences of internal transcribed spacers of rRNA genes

We have recently developed a method for typing Pneumocystis carinii strains that infect humans. The method takes advantage of nucleotide sequence variations in internal transcribed spacers (ITSs) of the rRNA genes of P. carinii. To date, two types of nucleotide sequences (designated types A and B) have been found in the ITS1 region, and three types of nucleotide sequences (designated types a, b and c) have been found in the ITS2 region. Of the six potential combination types, we have detected four, designated types Ac, Bb, Ba, and Bc. To simplify typing, we have designed five oligonucleotide probes, probes 1-A, 1-B, 2-a, 2-b, and 2-c, which are specific to ITS1 type A and type B and ITS2 type a, type b, and type c, respectively, of P. carinii strains that infect humans. We also have designed an oligonucleotide which reacts specifically with P. carinii strains that infect rats. The ITS region were amplified by PCR, and the PCR products were then probed with these type-specific oligonucleotide probes. Typing with the type-specific oligonucleotide probes was found to be effective with specimens containing only one type of P. carinii. These methods are rapid and simple to perform and will be useful for studying the epidemiology of P. carinii infections.

Typing of Pneumocystis carinii strains that infect humans based on nucleotide sequence variations of internal transcribed spacers of rRNA genes

Small portions of the 18S and the 26S rRNA genes, the entire 5.8S rRNA gene, and internal transcribed spacers ITS1 and ITS2 (located between the 18S and 5.8S rRNA genes and between the 5.8S and 26S rRNA genes, respectively) of Pneumocystis carinii that infect humans were cloned and sequenced. The nucleotide sequences of the 18S, 5.8S, and 26S rRNA genes determined in the study were approximately 90% homologous to those of P. carinii that infect rats, while the sequences of ITS1 and ITS2 of P. carinii from the two different hosts were only 60% homologous. The 18S, 5.8S, and 26S rRNA gene sequences of P. carinii from 15 patient specimens were determined and were found to be identical to each other, whereas the ITS sequences were found to be variable. With the observed sequence variation, it was possible to classify the ITS1 sequences into two types and the ITS2 sequences into three types. P. carinii strains that had the same type of ITS1 sequence could have a different type of ITS2 sequence. On the basis of the sequence types of the two ITS regions, P. carinii from the 15 patients were classified into four groups. P. carinii from three patient specimens were found to contain two different ITS sequence patterns. More surprisingly, one additional specimen was found to have one ITS sequence typical of P. carinii isolates that infect humans and another typical of P. carinii isolates that infect rats. The studies indicate that it is possible to type P. carinii strains on the basis from one patient, suggesting that coinfection with more than one strain of P. carinii may occur in the same patient.

Genetic stability and diversity of Pneumocystis carinii infecting rat colonies.

There is increasing molecular and antigenic evidence that Pneumocystis carinii organisms isolated from humans, ferrets, and rats are different species. In contrast, little is known about the extent of genetic diversity among P. carinii strains found within a single mammalian species. In the present study, electrophoretic karyotypes were obtained from P. carinii prepared from 10 chronically immunosuppressed rat colonies to investigate diversity at the chromosomal level. Most organism preparations produced patterns with 13 to 15 bands, but as many as 24 bands were observed in a few preparations. All bands separated between 700 and 300 kbp. Four distinct karyotype forms emerged from among the 13- to 15-band karyotypes of the 10 colonies sampled. Form 1 was shared by five rat strains from two vendors; form 2 was shared by two rat strains from the same vendor; and forms 3 and 4 were unique to their vendor colonies. Within a given rat colony, most rats harbored the same P. carinii karyotype. A survey of selected rat colonies showed that the karyotype within a vendor colony could remain stable over a period of 2 to 3 years. Hybridization of the blotted karyotypes with a repetitive DNA element isolated from rat-derived P. carinii and with single-copy gene probes showed that every chromosome in the karyotypes contained some repetitive DNA, and there was a general size concordance among the chromosomes carrying the unique gene loci. Differences in gene sequences, electrophoretic karyotypes, and hybridization profiles suggested that the immunosuppressed rats were infected by genetically distinct P. carinii strains. A provisional system of nomenclature for P. carinii that will permit differentiation of P. carinii organisms from the same mammalian host is discussed. These data show that all rats were not infected by a single type of P. carinii, that pulsed-field gradient electrophoresis can detect sufficient genetic diversity among the organism preparations to allow for characterization of the organisms, and that the genome of the organism within the rat host is relatively stable over time.

Nucleotide sequence variation in Pneumocystis carinii strains that infect humans.

The nucleotide sequences of a portion of the large-subunit mitochondrial rRNA gene of Pneumocystis carinii derived from 12 patients were examined. Five sequences were found to be identical to the prototype sequence reported by Sinclair et al. (K. Sinclair, A. E. Wakefield, S. Banerji, and J. M. Hopkin, Mol. Biochem. Parasitol. 45:183-184, 1991). Six sequences differed from the prototype sequence at one to three positions. The remaining sequence was markedly different from the prototype sequence and appeared to be a hybrid of the human and rat P. carinii sequences. The results of this study indicate the existence of multiple P. carinii strains infecting humans and suggest that coinfections of animal and human P. carinii strains in the same host are possible.

Improved rat model of Pneumocystis carinii pneumonia: induced laboratory infections in Pneumocystis-free animals.

An immunosuppressed rat model of Pneumocystis carinii pneumonia is described that utilizes simple, noninvasive intratracheal (i.t.) inoculation of cryopreserved parasites and results in development of severe P. carinii pneumonia within 5 weeks. This is an improvement over the most commonly used models of P. carinii pneumonia that rely on immune suppression to activate latent P. carinii infections and that often require 8 to 12 weeks to produce heavy infections of P. carinii. It is also less labor intensive than more recent models requiring surgical instillation of parasites. Our report describes a series of preliminary studies to select an appropriate strain of rat; to determine suitable methods for inducing uniform immunosuppression, P. carinii inoculation, and laboratory maintenance of P. carinii; and to determine effective animal husbandry methods for maintaining animals free from serious secondary infections. Results of our more detailed studies demonstrate that animals receiving two or three i.t. inoculations of approximately 10(6) cryopreserved P. carinii organisms have a predictable course of disease progression which includes moderate P. carinii infections within 3 weeks, severe P. carinii pneumonia in 5 weeks, and a high percentage of mortality due to P. carinii pneumonia in 6 weeks. Parasites were distributed evenly between the right and left lungs, regardless of the number of P. carinii inoculations administered. Non-P. carinii-inoculated immunosuppressed control rats maintained in microisolator cages remained free of P. carinii, thus providing an important control that is missing from many P. carinii pneumonia models. Most non-P. carinii-inoculated control animals and P. carinii-inoculated rats treated with trimethoprim-sulfamethoxazole that were housed in open caging in the same room containing heavily infected animals had no detectable infections after 5 to 6 weeks of immunosuppression; however, some had a small number of P. carinii in their lungs. Because heavy, reproducible infections are achieved 5 weeks after i.t. inoculation, because few animals are lost to secondary infections, and because animals can be maintained as noninfected contemporaneous controls, this animal model is useful for the maintenance of P. carinii strains, for studies of the transmission and natural history of P. carinii, for the production of large numbers of organisms for laboratory studies, and for the evaluation of potential anti-P. carinii drugs.