Carinii Cysts Research Articles

Expression and characterization of a cDNA clone encoding an immunodominant surface glycoprotein of Pneumocystis carinii.

Most studies of antigens of Pneumocystis carinii have focused on an abundant, immunogenic 95- to 140-kDa surface glycoprotein referred to as gpA. Expression cloning of gpA from P. carinii obtained from ferrets resulted in isolation of colinear fragments of gpA cDNA encoding approximately 87 kDa of the core protein. Northern hybridization detected an abundant, single species of gpA-specific mRNA of 3600 nucleotides. Southern hybridization revealed gpA-specific bands only in P. carinii-infected lung genomic DNA, suggesting that gpA cDNA did not result from induction of a host lung gene. Antiserum raised against a fragment of recombinant gpA detected P. carinii cysts and isolated native P. carinii gpA, indicating retention of epitopes between the nonglycosylated recombinant gpA and glycosylated native gpA. The deduced amino acid sequence is hydrophilic and contains 12 potential N-linked glycosylation sites and 47 cysteine residues, consistent with the surface orientation of gpA on the organism and other known characteristics of the native molecule.

Treatment of experimental pneumocystosis: review of 7 years of experience and development of a new system for classifying antimicrobial drugs.

Over a 7-year period, we analyzed 261 dose regimens of antimicrobial drugs in the treatment and prevention of Pneumocystis carinii pneumonia in an immunosuppressed rat model. These compounds ranged from drugs in clinical use to newly synthesized agents. Drug efficacy was expressed as the magnitude of the reduction in median P. carinii cyst or nucleus counts on a scale ranging from inactive (less than 5-fold) to very markedly active (greater than or equal to 1,000-fold). The classification system was reproducible and allowed drugs studied at different times to be compared with each other. The system demonstrated a hierarchy of anti-P. carinii activity not only among classes of compounds but also among individual members of a drug class. Sulfonamides, sulfones, and diamidines were the most active agents; some purine nucleosides and nitrofurans also showed promising activity; and most antiparasitic, antifungal, antibacterial, and antiviral drugs were inactive. We conclude that this classification system represents a simple, quantitative method of comparing the activities of antimicrobial drugs against P. carinii. Information gained from this system should be helpful in developing new anti-P. carinii compounds and establishing standard procedures for their evaluation.

Elution mode of Pneumocystis carinii cysts in gravitational field-flow fractionation

The simplest field-flow fractionation technique, i.e. gravitational, was used in an attempt to purify a Pneumocystis carinii cyst suspension. This parasite is an opportunistic invader in immunocompromised patients, especially those suffering from AIDS. The cyst stage is spherical and 5 μm in diameter. Unexpected retention times, not systematically related to the size and the density of the parasite, were obtained under various experimental conditions. When silicone-coated walls were used, Pneumocystis carinii cysts were eluted in the void volume, whereas when uncoated walls were used with a sodium dodecyl sulphate-enriched carrier phase, retention was observed. These phenomena are probably related to the high degree of hydrophobicity of these micrometre-sized biological particles; this degree can be easily determined. The use of the gravitational field-flow fractionation technique can be of a great interest for the development of new methods for diagnostic purposes. Particle—wall interactions and their modifications due to the carrier phase or to the wall treatment can be employed in the search for new bronchoalveolar lavage solutions.

Correlation of Pneumocystis carinii cyst density with mortality in patients with acquired immunodeficiency syndrome and pneumocystis pneumonia

Fifteen percent to 20% of patients with the acquired immunodeficiency syndrome and pneumocystis pneumonia do poorly despite early intervention. It is not known what distinguishes those who die, despite early intervention and aggressive therapy, from those who readily respond to therapy. We used image analysis to determine the relative abundance of cysts within aggregates of Pneumocystis carinii found in induced sputa (21 patients) and bronchoalveolar lavage fluid (14 patients) from 35 patients with pneumocystis pneumonia. We calculated a cyst density (number of cysts per area of aggregate) for each aggregate and a mean cyst density for all of the aggregates on the smear. Six patients died within 2 weeks of diagnosis; four of these six patients who had autopsies all had residual P carinii. The mean cyst density for those who died was 9.7 ± 3.9 (range, 5 to 15 × 10 −3). The 29 patients who survived beyond 2 weeks had a mean cyst density of 18.4 ± 8.7 (range, 5 to 35 × 10 3; P = .01). Mean cyst density was not influenced by the number of aggregates present in the smear, the variation in cyst density among aggregates in a smear, or the episode of pneumocystis pneumonia. Cyst density determinations alone should not be used to predict outcome for individuals with P carinii pneumonia until further study is completed. Nevertheless, the current study suggests that a low cyst density specimen, which may indirectly indicate a greater proportion of trophozoites compared with a high cyst density specimen, may be associated with an unfavorable outcome in acquired immunodeficiency syndrome-associated pneumocystis pneumonia.

Isolation of Pneumocystis carinii cysts by flow cytometry.

Pneumocystis carinii cysts were separated as enriched populations from suspensions of lung homogenate obtained from infected rats containing all developmental stages of this organism. Isolation of cyst populations was achieved by incubating filtered lung homogenate with the fluorescent phospholipid analog 1-palmitoyl-2-C6-NBD-phosphatidylcholine. Whereas cysts did not fluorescence as a result of outer-wall restraint of lipid integration, trophozoites and young intermediate stages readily incorporated the fluorescently labeled lipid analog into their outer membrane. The two distinct labeling patterns displayed by cysts and other developmental phases of P. carinii constitute a novel, easy, and reproducible means of isolating cysts from infected lung homogenate by flow cytometry.

Study on the therapeutic effects of interferon and gamma-globulin in experimental Pneumocystis carinii pneumonia

This study was performed to observe the therapeutic effects of interferon-gamma(IFN-gamma) and gamma-globulin(gamma-globulin) in experimental Pneumocystis carinii pneumonia of immune suppressed mice. After 9 weeks, trimethoprim-sulfamethoxazole(TMP-SMZ; 10-50 mg/mouse/day), mouse IFN-gamma(5 x 10(4) units/mouse/day) and mouse gamma-globulin(20 mg/mouse/day) were administered to the mice for 3 weeks by the experimental group. The therapeutic efficacy was evaluated by body weights, histopathologic and electron microscopic findings of the lungs, and number of P. carinii cysts by Gomori's methenamine silver stain. Body weights of the mice were significantly increased in the group of combination therapy of TMP-SMZ with IFN-gamma or gamma-globulin, and in the group of TMP-SMZ treatment (p < 0.05), however, little effect was found in the group of gamma-globulin alone. Histopathologic findings of P. carinii pneumonia were much improved in the group of combination therapy of TMP-SMZ with IFN-gamma. Treatment with either TMP-SMZ or IFN-gamma significantly reduced the number of cysts in the P. carinii pneumonia, but gamma-globulin alone was ineffective. In electron microscopic findings of P. carinii pneumonia, the number of trophozoites and cysts were reduced by treatment with either TMP-SMZ or IFN-gamma, and most of the cysts were empty or containing one or two intracystic bodies. The present results suggested, that combination therapy of TMP-SMZ with IFN-gamma had synergistic effects in treatment of P. carinii pneumonia in experimental mice.

Quantification and assessment of viability of Pneumocystis carinii organisms by flow cytometry

Analysis of drug efficacy in animal models of Pneumocystis carinii pneumonia requires an accurate method of quantification of organisms, as well as a means of assessing viability. Lung homogenates were prepared from a colony of athymic nude F344 rats experiencing a spontaneous outbreak of P. carinii pneumonia. With the fluorescent nucleic acid stain propidium iodide, flow cytometric analysis was able to quantify P. carinii cysts and trophozoites reproducibly. As this stain is excluded by living cells, this method was also used to assess the viability of organisms. Application of this technique to analysis of bronchoalveolar lavage specimens was demonstrated.

Antifungal/Echinocandin B Derivatives: Novel Fungal Cell Wall Glucan Synthetase Inhibitors

SummarySummaryNovelty: New antibiotic substances produced by the cultivation of Zalerion arboricola are described. The compounds are cyclic lipopeptides and are claimed as potential agents for Pneumocystis carinii, Candida albicans and Candida parapsilosis infections.Biology: The tested compound has an IC50 = 0.5μg/ml against Candida albicans (MY 1055), 16-128 μg/ml against Candida parapsilosis (MY 1009). One compound gave 66% reduction of Pneumocystis carinii cysts in C3H/He mice at 2 mg/kg (given for seven days).Chemistry: 1-[N2-(10,12-dimethyl-1-oxotetradecyl)ornithine]-4-[4-hydroxyphenyl)-2-aminobutanoic acid]-5-(3-hydroxyglutamine) echinocandin B is one exemplified compound.

Examination of Induced Sputum In the Diagnosis of Pneumocystis Carinii Pneumonia

The results of the examination of sputum induced by the inhalation of nebulized hypertonic saline in the diagnosis of Pneumocystis carinii pneumonia (PCP) are presented. In suspected cases of PCP in patients who were either HIV antibody positive or were receiving immunosuppressive therapy, 46 induced sputum specimens were stained using both Grocott's modified Gomori methenamine silver nitrate (GMS) and immunofluorescence staining. In 12 specimens P. carinii cysts were detected by both methods, in four specimens by GMS staining only and in five specimens by immunofluorescence only. The sensitivity of induced sputum examination in the detection of P. carinii cysts was increased by using both of these staining methods on each sputum specimen and the need for more invasive methods of diagnosis was reduced.

Pulmonary Calcification Caused by Pneumocystis carinii Pneumonia

Pulmonary microcalcifications were identified in 13 patients with Pneumocystis carinii pneumonia (PCP) and acquired immune deficiency syndrome (AIDS). Several patterns of calcification were noted including bubbly, plate-like, elongate, and conchoidal forms. All calcifications contained P. carinii cysts within them as demonstrated by Grocott's methenamine silver stain. In eight of the 13 cases, the typical intra-alveolar exudate of P. carinii was absent. Therefore, the calcifications were the only histopathologic indication of prior P. carinii infection. The above-mentioned calcifications are important because in five cases they occurred without a prior diagnosis of PCP, subsequent therapy, or prophylaxis. This indicates that in some patients there is destruction of P. carinii organisms with subsequent calcification, and this can occur without therapy.

Cytology of treated and minimal Pneumocystis carinii pneumonia and a pitfall of the grocott methenamine silver stain

To clarify the role of foamy alveolar casts (FACs) in the diagnosis of Pneumocystis carinii pneumonia (PCP), we retrospectively reviewed Papanicolaou (Pap)- and Grocott methenamine silver (GMS)-stained slides from 205 bronchial specimens submitted for suspected opportunistic lung infection. FACs containing sporozoites were seen in 86 cases, all with positive GMS. FACs were absent in 119 cases with negative GMS. In patients previously treated for PCP, Pap showed FAC-like material which lacked sporozoites. GMS demonstrated clumped degenerated Pneumocystis carinii (PC) cysts. In 17 GMS-positive cases there were no FACs on Pap due to sampling error and low burden of organisms. Morphologic features of macrophages in these cases can suggest the presence of PC. When FACs are lacking, a special stain is required. When GMS is used, the control must contain PC to avoid a false negative GMS. The inconsistent uptake of GMS by PC is a pitfall which is distinct from sampling error and which to our knowledge has not been previously reported.

Analysis of Pneumocystis carinii Cyst Wall. II. Sugar Composition

Pneumocystis carinii cysts are capable of resisting host defenses and antimicrobial drugs and are therefore thought to be responsible for relapses of P. carinii pneumonia in AIDS and other immunocompromised patients. The interaction of P. carinii with its host, and other P. carinii, might be mediated by molecules which form the outer surfaces of this organism. Carbohydrates are known to play many roles in cell-cell adhesion, and have been detected on the surface of P. carinii by lectin labeling experiments. In this study P. carinii cyst wall material was obtained from Zymolyase treatment. Alditol acetate derivatives of neutral and amino sugars or trimethylsilyl derivatives of methyl glycosides were prepared from the monosaccharides released from the sample by acid hydrolysis. Analyses were done by a combination of gas chromatography and mass spectrometry. Glucose was found to be the major sugar constituent. Mannose and galactose were present in equal ratios. A lesser amount of N-acetyl-D-glucosamine, and trace amounts of ribose and sialic acid were present in the cyst wall samples analyzed. These sugars may mediate P. carinii-host interaction and play an important protective role by creating a permeability barrier around the cyst.

Analysis of Pneumocystis carinii Cyst Wall I. Evidence for an Outer Surface Membrane

It has long been thought that the cyst form of Pneumocystis carinii, which can resist host defenses and antimicrobial drugs, is responsible for relapses of P. carinii pneumonia. The thick wall of the cyst is immunogenic and rich in glucosyl/mannosyl and N-acetyl-D-glucosamine residues. In this study we have demonstrated the presence of a hitherto unreported outer membrane in the cyst wall of P. carinii. This membrane was detected by a combination of techniques, including transmission electron microscopy, freeze-fracture electron microscopy, and membrane labeling with fluorescent lipid analogs following treatment of P. carinii cysts from infected rats for 30 min with Zymolyase, a beta-1-3 glucanase. As in gram-negative bacteria and blue-green algae, this 2nd membrane may have an important role in osmoregulation and nutrient utilization; it may also mediate the interaction of P. carinii with its host and serve as a target for drug therapy.

Evaluation of calcofluor white stain for detection of Pneumocystis carinii

A rapid calcofluor white (CFW) stain for detecting Pneumocystis carinii was evaluated prospectively. Eighty-nine bronchoalveolar lavage (BAL) specimens, 21 open-lung biopsy (OLB) tissues, 2 induced sputums, 1 expectorated sputum, 2 tracheal secretions, and 1 bronchial secretion from 102 patients were examined for P. carinii cysts by both the CFW stain and a modified methenamine silver (MS) stain. Twenty episodes of P. carinii pneumonia were detected: 19 of these episodes were detected by CFW stain, and 16 of those episodes were detected by MS stain. Discrepancies between the two staining methods were resolved by review of the clinical histories and, in one case, by testing an OLB specimen. Neither staining procedure gave false-positive results with any specimen. More cysts were detected in CFW-stained specimens than in MS-stained specimens (p = 0.05). CFW stain is a simple, rapid, and inexpensive method for detecting P. carinii in clinical specimens and is at least as sensitive as MS stain.

Rapid detection of Pneumocystis carinii in bronchoalveolar lavage samples by using Cellufluor staining

Cellufluor (Calcofluor white) has been found to be a useful, rapid chemofluorescent stain for detection of Pneumocystis carinii cysts in bronchoalveolar lavage samples. When compared with toluidine blue O and Giemsa stains on 45 specimens (22 positive and 23 negative), the sensitivity and specificity of the Cellufluor stain were 95 and 100%, respectively.

An electrophoretic karyotype and assignment of ribosomal genes to resolved chromosomes of Pneumocystis carinii.

Pulsed-field gel electrophoresis was used to generate a molecular karyotype of chromosomes from the opportunistic AIDS pathogen, Pneumocystis carinii. P. carinii cysts and trophozoites were isolated from immunosuppressed rats, lysed in situ in agarose blocks, and subjected to orthogonal-field gel electrophoresis (OFAGE) and contour-clamped homogeneous-field gel electrophoresis (CHEF). OFAGE and CHEF gels resolved, respectively, 16 or 20 chromosome bands ranging in size from 0.32-1.5 megabase pairs. Summation of the estimated sizes of these chromosomes suggested a total genome complexity for P. carinii of 8-16 megabase pairs. Homologous probes for the genes encoding the 18S, 5.8S, and 5S ribosomal RNAs were hybridized to filter blots of the pulsed-field gels to map these genes to specific P. carinii chromosomes.

Cresyl Violet

Over a three-month period at the pathology laboratory of Jackson Memorial Hospital, 110 sputum samples from 62 hospitalized patients with suspected AIDS were examined for Pneumocystis carinii. Sputum specimens were either expectorated spontaneously (most patients) or expectorated after the inhalation of small amounts of nebulized normal saline. Each sputum sample was cytocentrifuged onto two slides. One slide was stained with Gomori methenamine-silver (GMS) and the other with cresyl violet (CV). Among the 62 study patients, 18 were proven to have no histologic evidence of P carinii pneumonia. Of the remaining 44 patients, P carinii organisms were found by GMS stain in 14 (32%) and by CV stain in 18 (41%). Among those with a positive CV stain, the diagnosis was made on the first sputum specimen in 14 patients and on the second specimen in the remaining four patients. CV stain is at least as sensitive as GMS in detecting P carinii cysts in the sputum of AIDS patients with P carinii pneumonia, and its diagnostic sensitivity may exceed 40% under field conditions. Further, CV stain is much simpler to prepare than GMS and much simpler to interpret than Giemsa. It could be easily adapted for general use to expedite the diagnosis and treatment of P carinii pneumonia.

Pneumocystis carinii is not a major cause of pneumonia in HIV infected patients in Lusaka, Zambia

The clinical occurrence of Pneumocystis carinii and Mycobacterium tuberculosis was investigated in patients infected with human immunodeficiency virus (HIV) who had clinical pneumonia of unknown aetiology in Lusaka, Zambia. The results were compared with a similar group of patients in Stockholm, Sweden. Induced sputum samples were stained for Pneumocystis by indirect immunofluorescence using monoclonal antibody 3F6 and toluidine blue O. Mycobacterial culture and acid fast stain were performed on the specimens from Lusaka. P. carinii cysts were detected in none of 27 Lusaka patients, compared to 10 of 33 Stockholm patients. M. tuberculosis was identified in 11 of 22 Lusaka patients tested. In conclusion, P. carinii could not be incriminated as the aetiological agent of HIV-associated pneumonia in Zambia in contrast to the situation in Sweden, where Pneumocystis is the dominating aetiological agent.

Yeast Glucan in the Cyst Wall of Pneumocystis carinii

ABSTRACTUluastraclurally, 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 β‐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 β‐l,3‐glucan. The treatment of P. carinii cysts with Zymolyase, which the key enzyme is β1‐l,3‐glucan laminari‐pentaohydrolase, 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 β‐l,3‐glucan.

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.