Pneumocystis carinii in pleural fluid. The cytologic appearance

We describe the cytologic appearance of Pneumocystis carinii in pleural fluid of a patient with acquired immunodeficiency syndrome and a rapidly accumulating pleural effusion. The diagnosis of P carinii infection was made by examination of air-dried, Diff-Quik-stained Cytospin preparations of the pleural fluid. The diagnostic appearances of P carinii organisms stained by this method and by the Papanicolaou stain are reviewed. The unusual predominance of the trophozoite forms of the organism in this case made Diff-Quik an ideal special stain for identifying the organisms. Furthermore, this case illustrates a novel presentation of P carinii infection and suggests that P carinii should be considered an etiologic agent in the differential diagnosis of pleural effusion in an immunocompromised host.     

Cytology of extrapulmonary Pneumocystis carinii infection in the acquired immunodeficiency syndrome.

Rare cases of extrapulmonary Pneumocystis carinii (EPPC) have been seen in patients with acquired immunodeficiency syndrome (AIDS). We report seven such diagnoses of nonpulmonary P carinii (PC) from four AIDS patients between 1986 and 1989. The specimens included fine needle aspirate of liver, spleen, periarticular tissue and pleura as well as ankle fluid, pleural fluid and ascites. In some, but not all, cases the patients had concurrent or previous episodes of PC pneumonia. In all cases the typical granular, eosinophilic aggregates of PC cysts were noted on routine Papanicolaou staining, leading to the definitive detection of PC cysts with Grocott silver stain. In most cases, evidence for granulomalike and neovascularized tissue reaction was present in cytologic material. One specimen demonstrated concurrent acid fast bacilli. In the setting of AIDS, cytology of effusions and masses should include an evaluation for EPPC.     

Proliferation patterns of latent Pneumocystis carinii in rat organs during progressive stages of immunosuppression

Pneumocystis carinii is known to proliferate mainly in the lung of an immunocompromised host. In AIDS and other immune disorders sporadic extrapulmonary presence of this organism has been documented. Occasionally, P. carinii does not appear to infect the lung. These observations have been based on the detection of P. carinii by conventional staining techniques. We have sought to determine the extent of these infections by the polymerase chain reaction (PCR) in a rat model. Harlan Sprague-Dawley rats weighing between 110 and 130 g were immunosuppressed with dexamethasone (1.2 mg/l) in drinking water. During progressive stages of immunosuppression 2 rats were sacrificed at 2, 3, 4 and 5 wk, and the lung, liver, kidney, spleen and bone marrow were taken. Sonicated crude extracts of the tissues were used as template DNA for the amplification of the dihydrofolate reductase (DHFR) gene of P. carinii. All the PCR products were analyzed by Southern hybridization with radiolabelled DHFR DNA. These analyses revealed a general trend of P. carinii proliferation first in bone marrow at 2 wk, followed by liver at 3 wk, and lung at 5 wk on immunosuppression. Kidney and spleen infections were infrequent. Although P. carinii appears to proliferate in the lung at later stages of immunosuppression, the degree of proliferation is several-fold greater than in extrapulmonary organs. The extrapulmonary proliferation of P. carinii, however small, may possibly suppress hematopoietic stem cell differentiation in bone marrow, and may also contribute to the pathology present in various organs.     

Inhibitory effect of human natural yeast killer toxin-like candidacidal antibodies on Pneumocystis carinii.

BACKGROUND: Human natural antibodies have been found that owe their candidacidal action to the mimicry of a yeast killer toxin produced by the yeast Pichia anomala (PaKT). Candidacidal human natural antibodies (KTAb) are elicited by and bind to a KT receptor (PaKTR) present on the cell surface of infectious PaKT-sensitive microorganisms. Because of the recognized susceptibility of Pneumocystis carinii organisms to PaKT upon the occurrence of specific PaKTR, we examined whether human natural KTAb could also bind to and inhibit P. carinii. MATERIALS AND METHODS: Immunoaffinity-purified KTAb from the vaginal fluid of patients affected by candidiasis were tested and compared with PaKT for their ability to inhibit rat-derived P. carinii attachment to epithelial lung cells as well as infectivity to nude rats. Immunofluorescence studies were also performed by biotinylated PaKT in competition with human KTAb to establish their specific binding to PaKTR on the surface of rat-derived and human P. carinii organisms. RESULTS: Human natural candidacidal KTAb exerted a strong, specific inhibitory activity against rat-derived P. carinii organisms that are susceptible to PaKT itself. The antimicrobial activity of human KTAb was abolished by adsorption with a specific PaKT-neutralizing mAb KT4. Immunofluorescence studies of competition with PaKT showed that human KTAb efficiently bind to the specific PaKTR on the surface of rat-derived and human P. carinii organisms. CONCLUSIONS: The results strongly suggest that human KTAb, elicited by a common transphyletic receptor of different pathogenic microorganisms during infection, may play a role in antibody-mediated cross-immunity and, if properly engineered, as functionally equivalent recombinant antibodies they could exert a therapeutic activity against pneumocystosis in vivo.     

A comparison of the antigenic characteristics of rat and human Pneumocystis carinii by immunoblotting

The antigenic characteristics of rat Pneumocystis carinii obtained from infected lungs and grown in tissue culture were compared with the properties of human P. carinii obtained from the lungs of AIDS and non-AIDS patients by the immunoblotting technique, using different sources of antibody. Major immunoreactive bands of 45, 50, and 116 kd were found in both lung and tissue culture-derived rat P. carinii, suggesting the organism retains its antigenic characteristics in short-term culture. The principal immunoreactive bands in human P. carinii included a band of 40 kd, and to a lesser extent, a band of 66 kd; these antigens were found in the lungs of six and seven AIDS patients but in only one of eight non-AIDS patients with pneumocystosis. The rat and human P. carinii antigens reacted with sera from immunized rabbits, from rats with pneumocystosis and prolonged environmental exposure to the organism, from AIDS and non-AIDS P. carinii patients, and from healthy blood donors. Reactivity of these antigens could be removed by adsorption of antisera with P. carinii-infected lungs but not with normal lungs or lungs infected with bacteria and fungi. We conclude that rat and human P. carinii have shared, as well as species-specific, antigenic determinants, which should be useful for a variety of studies with this organism.     

Immunization with recombinant Pneumocystis carinii p55 antigen provides partial protection against infection: characterization of epitope recognition associated with immunization

Many therapeutic options exist for the treatment of Pneumocystis carinii pneumonia, a common fungal opportunistic pulmonary pathogen, but treatment is often complicated by side effects and toxicity and, more recently, markers of drug resistance have been described. The development of immunotherapetic modalities such as active immunization or passive immunotherapy may play an increasing important role in the prevention and treatment of infection. Passive immunotherapy with polyclonal anti-P. carinii reagents, such as serum or T cells, and monospecific reagents reactive with the major surface glycoprotein (MSG or gpA), such as monoclonal antibodies or MSG primed T cells, reduce the severity or eradicate infection. Active immunization with whole P. carinii, P. carinii extracts or MSG has afforded partial protection against the subsequent development of P. carinii pneumonia in some animal models. Identification of additional antigens with protective benefits will aid in the development of vaccines or other reagents. The p55 antigen of rat-derived P. carinii is well recognized by animals following natural exposure to the organism. This 414 amino acid residue antigen found within the cell wall of P. carinii contains 7 repeats of a glutamic acid-rich motif in the carboxyl portion of the molecule. Both humoral and cellular immune responses reactive with this repeated domain are present following natural infection while, the amino terminal portion of the molecule is immunologically silent. In this study, immunization with recombinant p55 elicited significant humoral and cellular immune responses which persisted during 10 weeks of immunosupression in corticosteroid treated rats; rp55 immunization resulted in a significant reduction in organism burden, improved histological score, lower lung weight to body weight ratio (a marker of infection or lung inflammation) and improved survival (P < 0.01). Greater protection was afforded by immunization with a peptide containing amino acid residues 1-200, than by the entire rp55 molecule. Epitope recognition by serum from animals immunized with rp55 differed from that of naturally exposed animals with oligoclonal responses to residues 22-92 and residues 196-218. This study demonstrates that protection against P. carinii can be afforded by immunization with antigen preparations other than whole extracts of P. carinii or the major surface antigen, MSG. This antigen moiety will likely be most useful as a vaccine candidate in combination with other immunogens which provide similar partial protection.     

Cloning and sequence of a β-tubulin cDNA from Pneumocystis carinii: possible implications for drug therapy

This work describes the isolation and characterization of a full-length cDNA clone encoding beta-tubulin from the pathogen Pneumocystis carinii. P. carinii contains a single gene encoding beta-tubulin. The complete sequence of this cDNA has been determined and its inferred amino acid sequence compared with the beta-tubulins from other organisms. This analysis augments the data indicating that P. carinii should be classified as a fungal organism. Further comparisons between the P. carinii beta-tubulin and those of fungal beta-tubulins resistant to benomyl, a beta-tubulin-binding drug, indicate a difference which may be exploited in the development of a new drug therapy for P. carinii pneumonitis. These results suggest that, theoretically, a drug presently administered for treatment of nematode worm infections may be an effective agent against P. carinii, without being toxic to the mammalian host. This possibility is currently being investigated.     

Structure, Expression and Phylogenetic Analysis of the Gene Encoding Actin I in Pneumocystis Carinii

Actin is a major component of the cytoskeleton and one of the most abundant proteins found in eukaryotic cells. Comparative sequence analysis shows that this essential gene has been highly conserved throughout eukaryotic evolution making it useful for phylogenetic analysis. Complete cDNA clones for the actin-encoding gene were isolated and characterized from Pneumocystis carinii purified from immunosuppressed rat lungs. The nucleotide sequence encodes a protein of 376 amino acids. The predicted actin protein of P. carinii shares a high degree of conservation to other known actins. Only one major actin gene was found in P. carinii. The P. carinii actin sequence was compared with 30 other actin sequences. Gene phylogenies constructed using both neighbor-joining and protein parsimony methods places the P. carinii actin sequence closest to the majority of the fungi. Since the phylogenetic relationship of P. carinii to fungi and protists has been questioned, these data on the actin gene phylogeny support the grouping of P. carinii with the fungi.     

Genetic heterogeneity of Pneumocystis carinii from rats of several regions and strains

Pneumocystis carinii is a major opportunistic pathogen which has been found in the lungs of a wide variety of mammalian host species, and the fact suggests the possibility of intraspecific variation. Until now, P. carinii from different mammalian species are differentiated as subspecies, and the rats are known to be infected by two subspecies. The present study investigated genetic heterogeneity of P. carinii isolates from two strains of rats in Korea and China by molecular karyotyping, RFLP and sequencing analysis. Karyotypes of P. carinii were grouped into three, two from two strains of rats in Korea and one from rats in China. However RFLP of PCR product of ribosomal and MSG gene of the P. carinii isolates showed same pattern. The sequence homology rates of alpha-tubulin DNA of the P. carinii isolates were 96% in Seoul Wistar rats, 93% in Seoul Sprague-Dawley rats, and 85% in Chinese Sprague-Dawley rats. The present finding confirmed that P. carinii from rats in Korea are grouped into two karyotype strains which are different from that of P. carinii from rats in China. The Chinese isolate shows a little different sequences of alpha-tubulin DNA.     

Indicators of Pneumocystis carinii viability in short-term cell culture.

Growth of P. carinii in culture has been difficult to document in the absence of reliable methods for distinguishing live from dead organisms. We studied three markers of cell function in P. carinii during the course of short-term cell culture, and correlated these with the number of P. carinii present in culture supernatants. The markers were glucan synthase activity, esterase activity and cell membrane integrity. The last two were assessed by double staining with fluorescein diacetate and propidium iodide followed by analysis of fluorescence using flow cytometry. The rise in P. carinii number after 5 to 7 days in culture was associated with increased glucan synthase activity. Flow cytometry analysis of day-6 P. carinii cultures confirmed that over 80% of the organisms catalyzed the conversion of fluorescein diacetate to fluorescein and excluded propidium iodide. The demonstration of three indices of metabolic activity in an expanding P. carinii population has confirmed the efficacy of a culture system as a means of sustaining the continued activity, albeit short-lived, of viable P. carinii.     

Identification of antigens and antibodies specific for Pneumocystis carinii

To increase understanding of Pneumocystis carinii and its interaction with its hosts, Ag specific for rodent and human P. carinii were identified by the immunoblot method after PAGE of P. carinii organism extracts. The m.w. of the major Ag of rat P. carinii were 45,000, 110,000, and a broad band of 49,000 to 64,000, and of human P. carinii were 22,000, 24,000, and a broad band of 35,000 to 45,000 daltons. Human and rat pneumocystis were not antigenically identical. Specific antibodies against rat P. carinii Ag were found in 18 of 79 rats by the immunoblot method. Specific antibodies against human P. carinii Ag were found in 32 of 33 adult human sera, but in only 1 of 8 sera from infants and children. Specific antibodies were found in sera of 13 of the 14 adults with no history of P. carinii pneumonia, and all 19 patients with recently diagnosed P. carinii pneumonia, including 9 patients with P. carinii pneumonia associated with AIDS. The results of this study support previous suggestions that a large proportion of adults have been exposed to P. carinii and provide a basis for the further investigations of host-P. carinii interactions.     

Pneumocystis carinii--animal production perspective.

Pneumocystis carinii is an important cause of pneumonia in immunocompromised human patients. The organism is also found as a saprophyte in the lungs of many species of animals. Animal models have been used as a source of P. carinii organisms for study of the disease. The rat model has been especially useful. Initially, the infection was latent in most colonies, and P. carinii pneumonia readily developed when animals were immunosuppressed. Today, many barrier raised rodent colonies are free of adventitious viruses, bacteria, Mycoplasma sp., and parasites, including P. carinii. Variability is now seen in the rat model. The use of cultured organisms to experimentally infect rats and mice prior to immunosuppression has met the need for some investigators, however, latent-infected, barrier-raised and isolator-raised rodents are still required. Colonies specifically infected with P. carinii can provide latent-infected animals and are better protected from potentially interfering organisms than barrier-raised animals. The development of these colonies is feasible as investigators and animal producers work together to define and develop this resource.     

Pneumocystis carinii f. sp. carinii synthesizes de novo four homologs of ubiquinone

Ubiquinone, coenzyme Q, plays a pivotal role in electron transport and is a target for chemotherapy against a number of eukaryotic infectious agents, including Pneumocystis carinii. Coenzyme Q10 was previously identified as the major ubiquinone homolog in P. carinii isolated and purified from rat lungs; CoQ9 was also present. In contrast, CoQ9 and CoQ8 (but not CoQ10) were detected in the lungs of uninfected rat controls. These observations suggested that the pathogen synthesizes CoQ10, and perhaps CoQ9 as well. In the present study, CoQ biosynthesis in P. carinii was examined in greater detail. Radiolabeled mevalonate, a precursor of the CoQ polyprenyl chain, was incorporated in vitro into P. carinii ubiquinones. Incorporation of radiolabeled mevalonate into P. carinii CoQ was not enhanced by treating cells with lovastatin, suggesting that the cells did not transport the drug, or that a lovastatin-insensitive pathway for de novo synthesis of isoprenoids may also function in this organism. Radiolabeled precursors of the ring moiety, including shikimic acid, p-hydroxybenzoic acid, and tyrosine were also incorporated into P. carinii CoQ. Unexpectedly, it was found that not only CoQ9 and CoQ10, but also CoQ7, and CoQ8, were metabolically radiolabeled by all the precursors tested, indicating that the organism synthesizes CoQ7, CoQ8, CoQ9, and CoQ10. Metabolic radiolabeling of ubiquinones in rat lung controls was not detected in experiments using either radioactive mevalonate or p-hydroxybenzoate. Thus the incorporations measured using purified P. carinii preparations were due to the enzymes of the organism.     

Detection of Pneumocystis carinii in serum of AIDS patients with Pneumocystis pneumonia by the polymerase chain reaction.

Amplification of DNA by the polymerase chain reaction (PCR) offers a highly sensitive and specific method for detecting DNA sequences in biological samples. We applied this technology to develop an assay for the P. carinii dihydrofolate reductase (DHFR) gene. This assay was found to be sensitive enough to detect as little as 1 organism-'equivalent' of DHFR DNA. In rats with experimentally-induced P. carinii pneumonia, DHFR DNA amplification demonstrated the presence of pulmonary P. carinii 2 wk prior to the onset of histopathological changes. When rat serum was analyzed by PCR, serum P. carinii DNA was found in 5 of 14 experimental rats. Finally, P. carinii DNA was detected in the serum of 7 of 18 patients (39%) with AIDS and active P. carinii pneumonia. These results suggest that circulating serum P. carinii DNA can be detected frequently in the course of pulmonary infection and may represent a blood-borne phase of infection. The PCR detection of P. carinii DNA provides a useful tool to study the natural history of P. carinii infection and may offer a non-invasive diagnostic procedure in some patients with P. carinii pneumonia.     

Cell surface protease PRT1 identified in the fungal pathogen Pneumocystis carinii

The subtelomeric regions of the chromosomes of many organisms contain gene families that allow adaptation to a changing environment. In a number of parasites, these subtelomeric gene families encode cell surface proteins that undergo antigenic variation. Proteases are another important virulence determinant in pathogenic microorganisms. We report the localization of the PRT1 protease of the pathogenic fungus Pneumocystis carinii sp. f. carinii, encoded by a subtelomeric gene family, to the cell surface of both the trophozoite and the cyst phase of the organism. Using anti-PRT1 antiserum, we demonstrated specificity to P. carinii sp. f. carinii in sections of infected rat lungs and, using immunofluorescence, we showed that the PRT1 protease has the characteristic distribution of a surface protein. The anti-PRT1 antiserum showed cross-reactivity with a number of P. carinii sp. f. carinii proteins migrating between 185 kDa and 28 kDa, the majority migrating between 42 kDa and 52 kDa, a region that has been shown by serological studies to contain important immunodominant P. carinii proteins. Cross-reactivity was also observed with P. carinii sp. f. hominis proteins. We have also cloned a portion of the catalytic domain of PRT1 from P. carinii sp. f. hominis, P. carinii sp. f. muris and P. carinii sp. f. rattus. Our data suggest that the PRT1 protease plays an important role in the pathogenicity of P. carinii.     

Cloning and Identification of Arp1, an Actin-Related Protein from Pneumocystis carinii

ABSTRACT. The complete Pneumocystis carinii Arp1 gene has been sequenced from two cDNA clones. The gene encodes a protein 385 bp in length with an estimated size of 45,000 kD. The A + T% for the Arp1 gene and a 900-bp sequence upstream of the gene were 63.7% and 70.3%, respectively. These values are consistant with A + T codon preference displayed by P. carinii and are similar to values reported for other P. carinii genes. The predicted amino acid sequence of the P. carinii Arp1 protein had a similarity of 87.6% with Neurospora crassa Arp1, 82.1% similarity with vertebrate centractin, and 71.2% similarity with the Saccharomyces cerevisiae Act5p. Expression of Arp1 mRNA in P. carinii was detectable via synthesis of cDNA and subsequent PCR amplification. Affinity purified antibodies against S. cerevisiae Act5p, and canine centractin recognized both the recombinantly expressed protein and a 45,000 kD protein in P. carinii nuclear extracts. The Arp1 gene is the second member of the actin multigene family that has been identified in P. carinii .     

Yeast glucan in the cyst wall of Pneumocystis carinii

Ultrastructurally, the cyst wall of Pneumocystis carinii consists of an electron-dense outer layer, an electron-lucent middle layer, and an innermost plasmalemma. This is similar in appearance to the cell wall of some yeasts, e.g. Saccharomyces cerevisiae, which consists of an outer dense layer of mannan, a middle lucent layer of beta-1,3-glucan (yeast glucan) and an innermost plasmalemma. The cyst wall of P. carinii, as well as the cell wall of S. cerevisiae, can be labeled by a variety of methods which stain polysaccharides, such as Gomori's methenamine silver (GMS) and by Aniline blue, a dye which selectively stains beta-1,3-glucan. The treatment of P. carinii cysts with Zymolyase, which the key enzyme is beta-1,3-glucan laminaripentaohydrolase, results in lysis of the outer 2 layers of the cyst wall and the loss of positive staining by both GMS and Aniline blue. The lysis of elements of the cyst wall of P. carinii is achieved under the same conditions and concentration at which Zymolyase lyses the outer 2 layers of the cell wall of viable cells of S. cerevisiae. These observations indicate that a major component of the cyst wall of P. carinii is beta-1,3-glucan.