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 pneumonia: detection of parasites in sputum and bronchoalveolar lavage fluid by monoclonal antibodies.

Diagnosis of pneumocystis pneumonia is based on identifying Pneumocystis carinii cytochemically in material from the lung. The silver methenamine staining methods most commonly used are technically difficult and lack specificity. The diagnostic value of immunocytological identification of the parasite was evaluated by using mouse monoclonal antibody 3F6, specific for human pneumocystis, to identify P carinii in bronchoalveolar lavage fluid and sputum by immunofluorescence and was compared with that of other variables. Bronchoalveolar lavage was performed on 25 patients positive for HIV antibody with clinically suspected pneumocystis pneumonia and 40 patients negative for HIV antibody who presented with interstitial disorders of the lung. Lavage fluid showed pneumocystis only in the patients positive for antibody, the parasite being detected in 19 by immunofluorescence and in 17 by a modified silver methenamine staining method. Chest x ray films obtained at the time of bronchoscopy showed interstitial or alveolar shadowing in 17 of the 19 patients, but clinical symptoms and the presence of antibodies to pneumocystis did not seem to be predictive. Sputum samples were collected during 43 episodes of clinically suspected pneumocystis pneumonia in patients positive for HIV antibody. Pneumocystis was detected consistently more commonly by immunofluorescence than the silver strain in sputum collected routinely and induced by inhalation of saline. In 17 patients bronchoalveolar lavage followed sputum collection, and the sensitivity of detection of pneumocystis in immunofluorescence in sputum compared with lavage fluid was 57% (8/14). Immunofluorescence was suitable for specimens fixed in ethanol and seemed highly specific and more sensitive than the standard cytochemical methods for identifying pneumocystis.     

Population structure of rat-derived Pneumocystis carinii in Danish wild rats

The rat model of Pneumocystis carinii pneumonia is frequently used to study human P. carinii infection, but there are many differences between the rat and human infections. We studied naturally acquired P. carinii in wild rats to examine the relevance of the rat model for human infection. P. carinii DNA was detected in 47 of 51 wild rats and in 10 of 12 nonimmunosuppressed laboratory rats. Evidence for three novel formae speciales of rat-derived P. carinii was found, and these were provisionally named Pneumocystis carinii f. sp. rattus-secundi, Pneumocystis carinii f. sp. rattus-tertii, and Pneumocystis carinii f. sp. rattus-quarti. Our data suggest that low-level carriage of P. carinii in wild rats and nonimmunosuppressed laboratory rats is common and that wild rats are frequently coinfected with more than one forma specialis of P. carinii. We also examined the diversity in the internally transcribed spacer (ITS) regions of the nuclear rRNA operon of Pneumocystis carinii f. sp. carinii by using samples from wild rats and laboratory rats and spore trap samples. We report a lack of variation in the ITS1 and ITS2 regions that is consistent with an evolutionary bottleneck in the P. carinii f. sp. carinii population. This study shows that human- and rat-derived P. carinii organisms are very different, not only in genetic composition but also in population structure and natural history.     

Pneumocystis carinii,Toxoplasma gondii,Cytomegalovirus and the Compromised Host

Pneumocystis carinii and Toxoplasma gondii are the two major parasitic protozoan pathogens in the immunocompromised host. Both organisms cause latent infection in humans and many animals. Cats are the definitive hosts for toxoplasmosis; the animal vector for pneumocystis (if any) has not been defined. Toxoplasma is an obligate intracellular parasite, whereas the available evidence suggests that Pneumocystis carinii exists primarily extracellularly. In compromised hosts, pneumocystis infection usually involves only lungs, whereas toxoplasma causes a generalized infection with encephalitis being the principal clinical manifestation. Both types of infection are treated with combinations of folate antagonists (trimethoprim or pyrimethamine with sulfonamide). Both parasites are associated with cytomegalovirus infection in immunosuppressed hosts, an association which may be due to symbiosis between parasites, or to an additive immunosuppressive effect of dual infection on the hosts.     

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.     

Ultrastructural morphology and staining characteristics of Pneumocystis carinii in situ and from bronchoalveolar lavage

The ultrastructure of Pneumocystis carinii obtained from rats by bronchoalveolar lavage (BAL) was compared with organisms in situ. All developmental forms of the organism as seen in situ were present in the lavage fluid. Trophozoites in situ were adhered to type I epithelium, had smooth surfaces, and were interdigitated with the underlying epithelium. Nonadherent trophozoites in situ and trophozoites in lavage fluid were more pleomorphic and irregular in shape with tubular projections extending from all surfaces. Microtubular and nuclear details not reported elsewhere were observed. To enhance the ultrastructural detail of P. carinii obtained by lavage, phosphotungstic and tannic acid fixation, uranyl acetate en bloc staining, and acid phosphatase staining were performed. These techniques enhanced the visibility of membranes, mitochondria, nuclei, and vacuoles. With tannic acid, increased contrast of the organism's cell coat was obtained and differences in staining intensity and thickness related to developmental stages were observed. In lavage samples with few pneumocystis organisms or those specimens heavily contaminated with macrophages, erythrocytes, or other cellular debris, tannic acid allows for easier recognition as other lung materials do not show the same distinctive staining reaction. Lung sections observed after BAL showed intact but damaged epithelial surfaces devoid of organisms. No intracellular organisms were observed. BAL removes organisms from the alveolar lumen as well as adhered organisms and is a useful method for concentrating the various morphologic forms of P. carinii.     

Pneumocystis carinii infection in corticosteroid-treated cats

Corticosteroids were administered to produce Pneumocystis carinii infection in cats. Six of 10 cats, injected intramuscularly for 97-141 days with 2 mg/cat twice weekly of betamethasone sodium phosphate, developed a light infection with P. carinii. Six of 7 cats, injected intramuscularly for 11-168 days with 10-25 mg/cat weekly of prednisolone acetate, also developed a light infection with P. carinii. There was no significant difference in the infection rate between the sexes and ages of the cats. Using Giemsa staining and Gomori's methenamine silver nitrate stain, P. carinii organisms were indistinguishable morphologically from human and rat P. carinii. The cysts and trophozoites were usually present singly or in small groups, and they always were adhering to the periphery of alveoli. The inflammatory changes were inconspicuous except for the fact that alveolar macrophages often were seen. Corticosteroid-treated cats should be useful in the study of experimental P. carinii infection. This is the first reported case of experimentally induced P. carinii infection in cats.     

Pneumocystis carinii: immunoblotting and immunofluorescent analyses of serum antibodies during experimental rat infection and recovery

Serum antibodies to Pneumocystis carinii were measured in rats by the indirect fluorescent antibody and immunoblotting techniques. Serum IgG and IgM antibodies developed with environmental exposure to P. carinii, were low or absent during immunosuppression to induce P. carinii pneumonia, and rose when immunosuppression was withdrawn. The IgG and IgM antibodies formed at the same time, but the titers of each antibody varied in individual rats. Serum IgG antibodies by immunoblotting recognized bands of 45, 50, and 116 kDa as the major reactive moieties of P. carinii. The bands were detected with sera from all rat groups in a temporal pattern which closely paralleled antibody formation by indirect immunofluorescence. The pattern of immunoblotting reactivity varied among individual rats, particularly with immunosuppression. Additional bands were detected with prolonged exposure to P. carinii. Thus, the rat makes both IgG and IgM antibodies to P. carinii, and specific P. carinii antigens identified in this immune response might be targeted for future serologic studies.     

Direct Fluorescent-Antibody Method for the Diagnosis of Pneumocystis carinii Pneumonitis from Sputa or Tracheal Aspirates from Humans

A direct fluorescent antibody (DFA) method was applied to sputum or tracheal aspirate from 68 patients with clinical or radiological evidence suggesting Pneumocystis carinii pneumonitis, and to 50 control patients. P. carinii was detected by DFA in specimens from 33 of the 69 clinical cases and 3 of the 50 controls. Specimens of lung from 11 of 33 DFA-positive cases were examined histologically, and 9 were positive. Four of 35 DFA-negative cases were examined histologically, and all were negative. Sputa or tracheal aspirates from 6 patients who were positive by both DFA and histological examination were examined also by methenamine silver staining; none could be diagnosed conclusively by this method. The results indicate that the DFA method is a sensitive and dependable procedure for the laboratory diagnosis of P. carinii pneumonitis in man.     

An ELISA method for quantitation of Pneumocystis carinii in culture and lung.

Numbers of Pneumocystis carinii in cultures or tissues traditionally have been determined by counting organisms on Giemsa-stained slides. For cultures, 10 microliters of culture supernatants have been sampled and counted on days 1, 3, 5 and 7. Infectivity scores of P. carinii-infected animal lung have been determined by three examiners scoring lung impression smears stained with Giemsa using a roughly logarithmic scale. Both counting procedures are tedious and time consuming. We have developed an enzyme-linked immunosorbent assay (ELISA) system which uses culture supernatants (in vitro) or homogenized animal lung (in vivo) as antigen, convalescent rat sera as primary antibody, and goat anti-rat alkaline phosphatase-conjugated immunoglobulin G as secondary antibody. The ELISA method shows good correlation with manual counts of Giemsa stains and allows a more rapid, more efficient method for quantitating P. carinii in both culture and infected lung.     

Decreased yield of Pneumocystis carinii from cortisonized rats

Thirty-five male Sprague-Dawley rats were divided into 3 groups, including 1 control and 2 experimental groups, in order to compare the efficacy of using cortisone acetate alone or in addition to intranasal inoculation of Pneumocystis carinii organisms for the purpose of inducing acute P. carinii pneumonia. The presence of P. carinii was monitored in nasal secretions on a weekly basis and in lungs at autopsy. Titers of IgG antibody were also monitored by enzyme-linked immunosorbent assay. No rat receiving cortisone acetate injections alone and only 2 of the rats receiving both cortisone and intranasal inoculation of P. carinii organisms showed Pneumocystis organisms in the lungs. However, Pneumocystis cysts did appear in the nasal secretions of 3 of the 5 control rats, all 8 rats receiving cortisone acetate injections only, and 12 of 18 rats receiving both cortisone acetate injections and an intranasal inoculum. IgG titers of both cortisonized groups remained less than 1:4 throughout the course of the experiment. The titer of the control group increased from negative to 13 (geometric mean).     

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.     

Variation of antigenicity and serological reaction to Pneumocystis carinii in Korea.

The present study observed the variation of antigenicity of Pneumocystis carinii and serum IgG antibody reaction to the antigens from different localities in Korea. Antigens of rat P. carinii and sera of inhabitants were collected at Chunchon. Chungju, Kwangju, and Seoul during 1995-1996. Enzyme-linked Immunosorbent Assay and immunoblot were used for immune reaction. Absorbance of 1,294 human sera ranged between 0.01 and 0.93. Sera from Chunchon showed higher absorbances than those from other areas. Immunoblotting revealed IgG antibody reactions to 116, 100, and 45-55 kDa antigenic bands of rat P. carinii, but the frequencies of positive reaction to individual bands were variable by localities. Total 62.6% of the sera showed the reaction to 116 kDa band while 37.7% reacted to 100 kDa band and 32.0% to 45-55 kDa bands. For the reaction to 116 kDa, the reaction rate was 60.0% to 82.6% by localities. It is found that the reaction rates of the human sera to rat P. carinii antigen are variable according to the localities. Also, the high molecular antigen of 116 kDa of rat P. carinii is the most frequent antigenic band reacting to human sera.     

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.     

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.     

Pneumocystis carinii of the Common Shrew, Sorex araneus, Shows a Discrete Phenotype

ABSTRACT. We carried out an immunohistological and morphological study on Pneumocystis carinii originating from the common shrew, Sorex araneus . Immunologic properties were studied by applying two commercially available immunofluorescence staining kits with differing developmental form specificity to a lung homogenate. The cyst form-specific staining kit reacted with cysts originating from S. araneus . Ultrastructurally this particular antigen epitope specifically deposited on the electron-lucent middle layer of the cyst pellicle. The immunohistochemical staining kit reacting with both cyst and trophozoite forms from human and rat origin did not react with any developmental forms of P. carinii originating from S. araneus . Both kits demonstrated P. carinii in the lung homogenate of a field vole, Microtus agrestis. In morphologic examination, the methenamine silver-stained cyst forms of P. carinii from S. araneus and from M. agrestis differed in size from each other and from those originating from laboratory rats. Ultrastructurally P. carinii from S. aruneus did not differ from organisms of rat origin.     

Zymolyase Treatment Exposes p55 Antigen of Pneumocystis carinii

Rats exposed to Pneumocystis carinii mount antibody responses to a broad band migrating on western blot with an apparent molecular weight of 45-55 kDa. One antigen within this band, designated p55, is uniformly recognized by P. carinii exposed rats. Although the gene encoding the p55 antigen had been previously cloned, the location of this antigen within the organism was unknown. Prior attempts to localize the protein were unsuccessful. A monospecific polyclonal antiserum raised against a carboxyl-terminai 15-oligomer peptide yielded specific reactivity with a single 55 kDa band on a western blot of P. carinii. Using this antiserum, little to no reactivity could be detected with P. carinii organisms by immunofluorescence assay (IIFA). However, zymolyase treatment of P. carinii dramatically increased the intensity and proportion of organisms reactive by IFA. Zymolyase, an enzyme with β-1,3 glucanase activity, has previously been shown to remove the electron dense outer layer of the P. carinii cell wall, exposing an electron lucent layer. Immunoelectron microscopy performed on zymolyase treated organisms showed the majority of labeling occurs within the cell wall.     

Differential splicing of Pneumocystis carinii f. sp. carinii inosine 5'-monophosphate dehydrogenase pre-mRNA

Inosine 5'-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in guanine nucleotide metabolism that has drawn attention as a drug target in several organisms. Pneumocystis carinii f. sp. carinii IMPDH mRNA (GeneBank Accession No: U42442) previously identified from cultured organisms yielded a predicted amino acid sequence about 70 amino acids shorter at the amino terminus than IMPDH from other species. Recent research has shown that the amino terminal region is important for enzyme activity, suggesting that the previous putative P. carinii IMPDH might not represent full length, functional enzyme. To test this hypothesis, RT-PCR was performed with total RNA isolated from P. carinii f. sp. carinii. Three IMPDH splicing variants were found and splicing preference was observed: P. carinii isolated from infected rat lung contained primarily splicing variant one (introns two and four deleted), but organisms from spinner flask culture contained primarily splicing variant three (all four introns deleted). Importantly, splicing variant one (GeneBank Accession No: AF196975) contained an open reading frame for 529 amino acids, a size comparable to that of other eukaryotic IMPDH forms. The other variants contained the same open reading frame (454 amino acids) previously reported. Sequence analysis and complementation studies suggest variant one represents the full length, catalytically active form of P. carinii IMPDH. The differential splicing of the enzyme may reflect a mechanism by which the organism regulates the expression of IMPDH in response to environmental stresses.