The biology of Pneumocystis carinii.

Critical Reviews in Microbiology

ISSN: 1040-841X (Print) 1549-7828 (Online) Journal homepage: http://www.tandfonline.com/loi/imby20

The Biology of Pneumocystis Carinii A. George Smulian & Peter D. Walzer To cite this article: A. George Smulian & Peter D. Walzer (1992) The Biology of Pneumocystis Carinii, Critical Reviews in Microbiology, 18:3, 191-216 To link to this article: http://dx.doi.org/10.3109/10408419209114558

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Critical Reviews in Microbiology, 18(3): 19 1-216 (1992)

The Biology of Pneumocystis carinii A. George Smulian and Peter D. Walzer University of Cincinnati College of Medicine, Department of Internal Medicine, Division of Infectious Diseases, 231 Bethesda Avenue, Cincinnati, OH 45267; and Veteran's Affairs Medical Center, Cincinnati, OH 45220

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KEY WORDS: Pneurnocysris carinii, pneumonia, AIDS,opportunistic infection.

1. INTRODUCTION Pneumocystis carinii was first described in the lungs of guinea pigs during studies of experimental American trypanosomiasis by Chagas in 1909.' The organism was thought to represent a variant in the sexual life cycle of Trypanosoma cruzi. Carini found the organism in trypanosomeinfected rats, but it was not until 1912 that the organisms were recognized as belonging to a new genus and assigned the name of Pneumocystis carinii when Delanoes identified identical forms in the lungs of rats which had not been infected with trypanosomes.Z The organism attained medical significance when, in 1952, Vanek and Jirovec implicated it as the cause of interstitial plasma cell pneum ~ n i aThis . ~ was a disease of high mortality affecting premature and debilitated infants in central and eastern Europe characterized by interstitial plasma cell ix~filtrate.~Interstitial plasma cell pneumonia gradually disappeared from Europe with improved socioeconomic conditions, but it still occurs in parts of the world where poor health care, overcrowding, and malnutrition exist.5 P . carinii assumed new importance when it emerged in the 1960s as an important cause of pneumonia in the immunocompromised host. Patients included children with primary immunodeficiencies and patients of all ages receiving corticosteroids and other immunosuppressive

drugs for the treatment of cancer, organ transplantation, and other disorders.'j The disease in these individuals was characterized histologically by the lack of any significant host inflammatory response. This form of pneumocystosis was largely controlled in the 1970s by the use of trimethoprim-sulfamethoxazole as therapy and prophylaxis in individuals at risk.' The availability of safe and effective treatment for P . carinii pneumonia and the formidable technical problems of working with P . carinii in the laboratory had discouraged research on the organism. As a result, the basic biology of P . carinii was poorly understood. The emergence of the acquired immunodeficiency syndrome (AIDS) thrust P . carinii to the forefront in the 1980s. P . carinii pneumonia became recognized as the most common opportunistic infection in AIDS patients and a leading cause of morbidity and mortality.* This has stimulated basic research on P . carinii which has enhanced our understanding of the organism. This review summarizes current concepts of the basic immunobiology and clinical significance of P . carinii.

11. TAXONOMY

Since the days when Chagas misidentified P . carinii as a schizogonial stage of trypanosomes, its phylogeny has been uncertain. Because P .

1040-8411\092/$.50 0 1992 by CRC Press, Inc.

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carinii cannot be grown in continuous culture, taxonomic studies have relied on histological and ultrastructural analysis of infected tissues. Most investigators have classified it as a protozoan based on the amoeboid appearance of the trophic form of P . carinii, the presence of filopodia, the structural similarities of the mitochondria, and cell pellicle/wall with analogous structures in protozoa. 12,9-15 Other features supporting it being a protozoan are its inability to grow on fungal media, the lack of ergosterol in the cell wall, its insensitivity to antifungal agents (e.g., amphotericin B), and its sensitivity to antiprotozoal agents including trimethoprim-sulfamethoxazole, sulfonamides, pentamidine isethionate, and a-difluoromethylornithine. Other investigators have regarded it as a fungus supported by the similaritiesbetween the sporogenous state of P . curinii and ascospore formation in yeast, and the poorly developed mitochondria and the paucity of other organelles. 11*19-21 Ultrastructural studies have shown that the mitochondria of P. carinii have lamellar cristae in common with most fungi, whereas most protozoa have tubular cristae. IZ In addition, the cyst wall stains with methenamine silver and other fungal stains. Unfortunately, none of these criteria can provide a definitive classification of the organism. Comparison of ribosomal RNA sequences has become a powerful tool for resolution of phylogenic origin^.^^.^^ Recent work by a number of groups has supported the fungal origins of P. carinii. Analysis of a cloned 16s-like small subunit rRNA revealed it to closely resemble Saccharomyces cerevisiue and Neuorspora crassa and to be distinct from that of T. b r u ~ e iDi.~~ rectly sequenced portions of the small subunit of rRNA showed P . carinii to be closer to the ascomycetes S. cerevisiae and N . crassa than to basidiomycetes such as Cryprococcus difiens.25 Analysis of the 5s ribosomal RNA sequence suggested that P . carinii was associated with the RhizopodalMyxomycotalZymycota group of “Protista fungi” but distinct from the more common fungi such as the ascomycetes and basidiomycetes.26This molecular evidence, corroborated by multiple groups using distinct but similar techniques, strongly favors a fungal origin of Pneumocystis.

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Other molecular genetic features also support this taxonomic classification of P. carinii. The thymidylate synthase and dihydrofolate reductase genes have been sequenced and both have been found to be more similar to their counterparts in S. cerevisiae than to any other known seq u e n ~ e . ~ These ’ * ~ ~ genes were also found not to be linked but rather to reside on different chromosomes. This contrasts with the situation in many protozoa where these two enzymatic activities are encoded on a single t r a n ~ c r i p t . ~ ~ . ~ ~ Taken together, these findings strongly support placement of Pneumocysris within the fungal kingdom. This classification will stimulate many aspects of basic research such as the cultivation in modified fungal media and the search for additional life cycle stages (e.g., spores) and environmental sources typically associated with fungi. It will also stimulate the development of newer therapeutic options modeled on fungal diseases such as the use ‘of glucan synthetase inhibitor^.^^

111. STRUCTURE AND COMPOSITIONAL ANALYSIS Studies of the life cycle of P. curinii have relied mainly on histochemical and ultrastructural analysis of organisms found in human and rat lung sections. These studies have identified a variable number of stages and forms throughout the life cycle. 13*32-36 However, all investigators have consistently identified two major forms of P . carinii: a trophic stage (or trophozoite) and a cyst (Figure 1). An intermediate stage, a precyst, is also commonly identified. Since the life cycle is not fully resolved, the designation of this morphological form as a specific life cycle stage is perhaps premature. Some of the differences reported in morphology, ultrastructure, and developmental stages are related to suboptimal fixation techniques and will be resolved as these techniques improve and become standardized. The trophic stage is the smallest form with a size range of between 1 to 4 p,m. The most prominent feature of this stage is the single nucleus in which an electron-dense nucleolus can sometimes be identified.36The nucleus is surrounded by a double nuclear envelope with eight


FIGURE 1. Clusters of Pneumocystis carinii showing various developmental forms. Clusters of P. carinii were removed from tissue culture and examined using Nomarski interference contrast microscopy. Cyst forms of the organism containing daughter forms or intracystic bodies are clearly evident (solid arrow). Trophic forms are also present in the cluster (open arrow). (Figure provided by Dr. M. T. Cushion.)

nuclear pores per square micron.3s Few organelles are seen within the cytoplasm of the organisms, although this might represent suboptimal fixation. Those organelles identified include a single mitochondrion, rough endoplasmic retic-

ulum, occasional vacuoles, and glycogen granules. I 3 The mitochondria were originally reported as being rounded with tubular cristae resembling those of protozoa. l4 More recent ultrastructural analysis utilizing more optimal fixation have de-

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tected lamellar cristae more in keeping with fungal origins. I2 Recently, a Golgi-like apparatus was identified within the cytoplasm of rabbitderived P . curinii.37*38 The trophic stage is pleomorphic, depending on the method of fixation. These forms in lung sections appear ameboid, while in unfixed preparations they appear as oval bodies resembling clusters of grapes. 15.39 Each individual organism is defined by a cell wall measuring approximately 20 to 30 nm across what consists of three layers: an outer unit membrane covered by an electrondense layer; a thin electron-lucent layer; and an inner trilaminar unit membrane characteristic of a p l a ~ m a l e m m a . ~The . ~ ~electron-lucent middle layer is poorly developed in the trophic form but becomes more prominent in precyst and cyst forms. Intramembranous particles (IMP),which are integral proteins located in the hydrophobic domain of the membrane lipid bilayer, have been identified by freeze fracture techniques on both the P face (protoplasmic face) and the E face (exoplasmic face) of the membrane.35The density of the IMPS is greater on the P face of the membrane in all developmental stages, with the greatest density being found in the trophic forms, less in the precyst, and least in the cyst forms. This suggests that membrane activity decreases as the parasite proceeds in its development from a trophozoite to a cyst. On the surface of mature trophic forms, tubular expansions or filopodia are found. 15.41 These polymorphic membranous vesicles have been seen to contain a unit membrane associated with electron-dense material. The function of these vesicles is unknown, but it is postulated that they function in organism nutrition and transport of enzymes to the environment. The intermediate stage between the trophic and the cystic forms, the precyst, demonstrates considerable morphological variation. The precyst ranges in size from 4 to 6 pm and is characterized by an oval shape. The cell wall now shows thickening to approximately 100 nm, with the electron-lucent middle layer becoming more pronounced than in the trophic form. Within the cytoplasm, the precyst contains one or more nuclei, mitochondria often in clumps adjacent to the nucleus, free ribosomes, glycogen granules, vacuoles, and microtubules. Endoplasmic reticulum, while reported, rarely appears in this stage.

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Ultrastructural studies have identified three subtypes of the precyst stage, characterized by progressive thickening of the cell wall. The early precyst features a single nucleus within which synaptonemal complexes have been observed.33 This structure conducts the alignment of homol.ogous chromosomes and is therefore used as an indicator of meiotic division and hence a sexual cycle of replication. Investigators have also reported forms with a nucleus twice the normal size.39Following this in later precyst stages, they noted a reduction in nuclear size with concomitant increase in the number of nuclei, also suggestive of meiotic division. However, synaptonemal complexes have been observed in haploid nuclei of higher plants and in nonmeiotic ascospores of some fungi.42-44 Cytoplasmic inclusions unique to the intermediate precyst stage include spindle microtubules which appear to originate from a dense body termed the nucleus-associated organelle (NAO). Two to four nuclei are seen in this stage, each associated with a clump of mitochondria. In the late precyst, four or more rounded nuclei are found in association with NAOs. The cyst is the largest and most easily recognized stage of the organism and measures between 5 and 8 pm in size. It is spherical and surrounded by a trilaminar cell wall 70 to 160 nm in thickness. The cyst wall is we11 stained by fungal stains such as cresyl echt violet, toluidine blue, and methenamine silver. It has been shown that the silver particles are deposited only on the electron-lucent middle layer of the cell wall.45 This explains the lack of staining of the trophic forms where the electron-lucent layer is thin or absent. Extensive work has been done to characterize the cell wall of the cyst stage. The thick cyst wall consists of a thick electron-dense outermost layer, the electron-lucent middle layer, and the inner cell membrane. It has been shown that treatment of P . curinii cysts with zymolyase, a p-1-3 glucanase, disrupts the cyst wall and liberates the surface By a combination of transmission and freeze-fracture electron microscopy, zymolyase treatment, and membrane labeling with fluorescent lipid analogs, the outermost layer has been shown to contain a bilayer membrane (Figure 2).47 It has also been shown that intracystic


FIGURE 2. Visualization of the outer .membrane of Pneurnocystis carinii cyst wall by transmission electron microscopy (TEM) and freeze-fracture electron microscopy. (a) Untreated cysts, shown by TEM, exhibited the familiar trilaminar wall configuration. The upper left insert depicts the cyst wall enlarged four times (bar = 0.1 pm). The wall consists of an inner membrane (l), a middle electron-lucent space (2), and an outer electron-dense region (3). Arrow points to the dark wavy line, the putative outer bilayer membrane. The corresponding freeze-fracture electron micrograph of untreated P. carinii is depicted in panel (d). (b) Cysts treated for 60 min with zymolyase lOOT (in solution with PMSF and p-mercaptoethanol) lacked the outer two cyst wall layers as seen by TEM; only the inner membrane (IM) remained after this treatment. The corresponding freeze-fracture electron micrograph is shown in panel (e). (c) Cysts treated for 30 min with the zymolyase solution showed an outer membrane (arrow) by TEM. The corresponding freeze-fracture electron micrograph is shown in panel (9. (d) The cyst wall of untreated P. carinii fractured through the inner membrane, leaving the protoplasmic fracture attached to the electron-lucent and electron-dense layers (arrow). (e) Freeze-fracture electron micrographs of 60-min zymolyase-treated cysts exhibited a single exoplasmic fracture of the inner membrane (arrow). (f) Freeze-fracture electron microscopy of 30-min treated cysts exhibited four fracture planes: 1 = exoplasmic fracture of inner membrane; 2 = exoplasmic surface of inner membrane; 3 = exoplasmic fracture of outer membrane: and 4 = exoplasmic surface of outer membrane. (Reprinted from DeStefano, J. A., Cushion, M. T., Sleight, R. E., and Walzer, P. D., J. Profozool., 37, 428, 1990. With permission.)

bodies and young trophic forms possess an outer membrane, and it thus appears that this might be present in all developmental stages. Other or-

ganisms in which two bilayer membranes are found include Gram-negative bacteria and bluegreen algae.48.49These membranes may function

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as selective permeability barriers, as regulators of uptake and transport of macromolecules, and as anchors for surface antigens. The periplasmic space between these membranes might also be a metabolically important region. Studies to characterize the composition of the cyst wall have been performed. Lectin studies, ultrastructural analysis, and reaction with specific glycosidases have shown that carbohydrates are well represented on the surface of P. carinii cysts and t r o p h o ~ o i t e sThe . ~ ~lectin ~ studies have shown the surface to be rich in glucosyl/mannosyl, N-acetyl-D-glucosamine (GlcNAc), and galactose/N-acetyl galactosamine (GaUGalNAc) residues. More recently, gas chromatography coupled with mass spectrometry (GUMS) studies have shown that carbohydrates constitute about 8% of the outer cyst wall of rat-derived P. carink5’ Glucose is the most abundant sugar, with smaller quantities of mannose, galactose, and GlcNAc detected. Cyst protoplasts and trophic forms were similar to the cyst in carbohydrate composition, but the percentage of carbohydrate was markedly lower at 0.5%. It is thought that these cyst wall carbohydrates could be in the form of glucan because of the abundance of glucose, the effectiveness of zymolyase digestion, and the effects of glucan synthetase inhibitors on clinical disease.” This has been substantiated by the staining of the cyst wall with aniline blue, a stain which is highly specific for p-1,3 glucan and by binding of lectins specific for GldMan residues to the electron-lucent middle layer.54 Carbohydrate compositional studies on human-derived P. carinii surface antigen by high performance liquid chromatography (HPLC) have shown similar sugar composition, although differences in the relative proportions of the individual sugars were noted.55 Lipids constitute 50% of zymolyase-cleaved cyst wall preparations. Within this preparation, palmitate (16:O) is the most abundant fatty acid; however, other fatty acids such as oleate are also present in significant amounts.56 These studies are similar to analysis of total lipid saponifiable (ester-linked) and sphingolipid (amide-linked) fatty acid of rat-derived P. carinii organisms. Approximately 50% of the saponifiable and 75% of the sphingolipid fatty acids are saturated, and few differences have been noted between the or-

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ganisms and lung controls. Steryl esters, triglycerides, free fatty acids, free sterols, and monoglycerides are present in both P. carinii and lung controls. The organism, however, does contain more esterified sterols, suggesting that the organism is capable of altering some lipids to those ,unique from its host. Ergosterol has not been detected in these studies or in previous freeze fracture studies using a filipin Cholesterol, on the other hand, is found in abundance in both the host lung controls and the organism preparations. The lack of ergosterol can explain the resistance of P . carinii to sterol synthesis inhibitors, such as amphotericin B and ketoconazole, but this is, perhaps, surprising given its phylogenetic relationship to common fungi. Within the cyst, eight daughter forms or intracystic bodies are commonly noted. The intracystic bodies are spherical bodies about 1 to 2 pm in diameter, surrounded by a double unit membrane. They contain a single nucleus, a single mitochondrion, and abundant endoplasmic reticulum and ribosomes. Microtubules, vacuoles, and (rarely) glycogen granules can be found within the intracystic bodies. The intracystic bodies have been reported to be attached to each other and/or the cyst wall by a thread or stalk-like structure. Within the confines of the cyst wall, glycogen granules, mitochondria, rough endoplasmic reticulum membrane, and free ribosomes are also found. Other stages of P . carinii that have been identified include thin-walled cysts filled with irregularly shaped daughter forms, empty or “ghost” cysts, and crescent-shaped form^.^' The exact position of these forms within the life cycle of P . carinii remains controversial. Identification of the above-mentioned developmental forms both in laboratory and clinical specimens relies predominantly on tinctorial methods. Stains designed for identification of fungal elements, such as Gomori’s methenamine silver stain, toluidine blue 0, cresyl echt violet, and calcofluor white, have all been successfully used to identify the cyst stage of the organism.39*152,161 The major advantages of cyst-wall stains are that they can be analyzed rapidly and do not require a great deal of expertise for proper interpretation. Giemsa and modified Giemsa stains (e.g., Diff-Quik) stain trophozoites, intra-

cystic bodies, and all intermediate These stains that stain the nuclei of all life cycle stages allow for more accurate quantitation of organism burden but require greater expertise for accurate interpretation. In addition to tinctorial methods, direct and indirect fluorescent antibody stains using monoclonal antibodies are used to diagnose infection in clinical


IV. LIFE CYCLE A number of different life cycles for P. carinii have been proposed following the initial description by Carini and Maciel in 1916.11.13.17,32.33.41 These are based solely on forms identified by ultrastructure and light microscopy and have increased in complexity as additional developmental stages have been identified. Two themes are common to most cycles: (1) replication of the trophic form by an asexual process such as binary fission and (2) a pathway leading to the development of cysts. Based on studies performed on rat-derived P. carinii obtained from infected lung homogenates and tissue culture, a life cycle that includes both an asexual and a sexual phase of development has been proposed (Figure 3).32The trophic $oms, the predominant forms in active infection, appear to replicate by binary fission. Evidence for nuclear division in trophic forms has been shown by transmission electron microscopy and by the estimation of DNA content of organisms stained with DAPI.57Initiation of a sexual cycle usually begins with gametic fusion, but since micro- and macrogametic forms of P. carinii have not been identified, some trophic forms may function as “isogametes”. This gametic fusion is followed by nuclear fusion to a diploid zygote considered an early precyst. Synaptonemal complexes indicative of meiosis have been identified in this early precyst stage. This supports the postulate that at least one mechanism of cyst formation is via a sexual cycle. This meiotic division and subsequent mitosis occur within the cyst, as the organism differentiates from precyst to mature cystic form. Various cystic stages, based on morphological changes of the intracystic bodies, have been identified, ranging from ellipsoidal forms to very elongated forms which might represent forms prior to excystation.

The process of excystation and the mechanism of release of the daughter forms from the cyst form to give rise to the trophic forms have not been well documented. Another element which has not been delineated within the life cycle scheme is the infective form. Using the immunosuppressed rat model of P. carinii pneumonia, workers have shown that the most likely means of transmission was via an airborne particle. It has also been shown that the majority of the human population develops antibodies to P. carinii by the age of three, suggesting that this infective particle is ubiquitous within the envir~nment.~*-@ It remains unknown as to whether these particles are disseminated by a reservoir host, are produced in an enviromental cycle, or arise directly from an infected host. Most of the currently proposed life cycles are based on static observations with little kinetic data available because of the lack of adequate in vitro cultivation. These proposed life cycles may require extensive revision as a result of the recent taxonomic and molecular information suggesting a fungal origin of P. carinii.

V. MOLECULAR BIOLOGY Molecular studies of P. carinii are only in their infancy, with results only having become available during the past 3 years. Nevertheless, we now have a fairly clear picture of the size and complexity of the P. carinii genome. Studies on bulk DNA have been difficult because of the difficulty in preparing large quantities of pure P. carinii DNA. Early attempts to measure the amount of DNA in P. carinii by biochemical analysis appear to have been overestimates because of host DNA contamination. The genome of P . carinii is now estimated between 7 X lo6 and 1 X lo7 bp, based on summation of the molecular sizes of P. carinii chromosomes in pulse field gel electrophoretic studies.6’.62This genetic material in rat-derived P. carinii is arranged as 13 to 19 chromosomes between 295 and 710 kb in size (Figure 4). While larger bands have been reported, these have been shown by two groups to hybridize with a rat cell total DNA probe and not with P.carinii-specific probe^.^^*^ These karyotype patterns have shown heteroge-

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L

t F G

H

0

h

4

FIGURE 3. Diagramatic representation of the developmental cycles of P. carinii, in vifro. The asexual cycle. (A'), trophic form; (W), mitotic replication of trophic form; (Cl), trophic forms, products of binary fission. The sexual cycle. A and B, isogametic forms; C, karyogamy; D, early precyst, diploid zygote; E, intermediate precyst, beginning of mitotic replication of nuclei; F, intermediate precyst, four nuclei; G, intermediate precyst, completion of nuclear replication, eight nuclei; H,late precyst, migrationof nuclei to periphery; I, late precyst, initiationof compartmentation of daughter forms; J, early cyst, completed separation of daughter forms; K, mature cyst, eight rounded daughter forms within a thick wall; L, cyst containing ellipsoidal daughter forms; M, cyst containing elongated daughter forms; N, cyst containing thin, very elongated daughter forms; 0, collapsed excysted cyst with trophic form. The progressive elongation of the daughter forms within the cyst stages L to N may represent the process required for excystation. These forms have been seen repeatedly in culture and in infectedrat lung homogenates, although the actual process of excystation was not observed. (Reprinted from Cushion, M. T., Ruffolo, J. J., and Walzer, P. D., Lab. Invest., 58, 324, 1988. With permission.)

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FIGURE 4. Pneumocystjscarinii chromosomes resolved by field inversion gel electrophoresis (FIGE). Multiple lanes of a 1% agarose gel were loaded with rat-derivedP. carinii SD(3), subjected to FIGE, and the DNA transferred to nylon membrane. Strips of nylon membrane were hybridized to probes made from Rp 3-1, a repeated DNA sequence, in lane 2; a synthetic 25-base oligonucleotide designed to be specific for 18s rRNA sequences of P. carjnji in lane 4; and the rRNA gene in lane 6. Ethidium bromide-stained gel prior to the transfer to respective nylon strips is shown in lanes 1, 3, and 5.The genome appears to be composed of 15 to 19 chromosomes between 300 and 700 kb. (Adapted from Reference 62.)

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neity between different isolates. 62 Systematic study of this heterogeneity revealed that the karyotype varied between rats from different colonies but remained stable within any colony over time (Figure 5). It also suggested the possibility of coinfection by at least two strains of P. carinii because one colony of rats produced a complex pattern which comigrated with those of other patterns. These karyotypic differences are being used as a marker in studies to clarify the transmission of P. carinii pneumonia. The karyotype of human-derived P. carinii appears different from that of rat-derived organisms but has not been well characterized.62 Studies on the base composition of the P. carinii genome have revealed a high content of adenine and thymine. Thermal denaturation studies on bulk P. carinii DNA indicated an average guanine plus cytosine (G+C) content of only 33%.65This has been supported by the finding in the thymidylate synthase and dihydrofolate reductase genes of G + C content of 30 and 29%, respectively.27*28 Sequence data of these genes also revealed strong codon bias, with 78 to 87% of codons containing an A or T in the third position. Three genes have been cloned from rat-derived P. carinii. These are the nuclear small ribosomal subunit 18s RNA gene, dihydrofolate reductase gene, and the thymidylate synthase gene.B+27.28 The 18s P . carinii gene has been found to contain 2194 bp. This sequence appears to have a 390-bp intron located 23 bp from the putative 3' end of the RNA. This intron has the structural characteristics of a group 1 intron which has been described in nuclear ribosomal genes of protozoa, chloroplast DNA, and in fungal mitochondria. The P. carinii rRNA intron has also been shown to undergo self-splicing in the absence of catalytic proteins, following in v i m transcription. The rRNA locus appears to be organized with 18S, 5.8S, and 26s genes arranged head to tail in tandem array on a chromosome of approximately 535 kb. 61.62 The thymidylate synthase coding sequence spans 1087 bp including four short introns resulting in an 891 nucleotide open reading frame. This gene has been mapped to a 330-kb chro-

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mosome by hybridization to PFGE bands. The dihydrofolate reductase gene, located on a 520kb chromosome, is 663 bp long including a 43bp intron. This encodes a 206 amino acid enzyme, which is slightly larger than the mammalian and bacterial enzyme but similar in size 'to that of S. cerevisiae. The actin and topoisomerase genes have been mapped as chromosomespecific markers but have not been cloned.61 Repeated elements of DNA have been identified within the genome of P. carinii. The best characterized of these belongs to a family demonstrating a high degree of polymorphism with respect to restriction enzyme cleavage sites and appears to be repeated approximately 100 times per genome.62,66 This repeat is more than 10 kb in length and is found on each of the chromosomes resolved by field inversion gel electrophoresis. The function of this repeated DNA is not known. Studies on the ribosomal RNA of P. carinii have shown its similarity to ribosomal RNA sequences of other fungi, as previously mentioned. The major ribosomal species migrate at sizes of 3.7 and 1.9 kb intermediate between the size of rat rRNA (4.4 and 1.9 kb) and Escherichia coli rRNA (2.85 and 1.45 kb) but similar in size to rRNA of S. cerevisiae (3.5 and 1.85 kb).64.65-67 The small subunit rRNA has been referred to as 18s- or 16s-like by different authors. A portion of the large subunit of mitochondrial ribosomal RNA has also been characterized in both rat- and human-derived P. carinii using polymerase chain reaction to amplify the sequences .68.69 This sequence apparently demonstrates significant homology to fungal sequences. Consistent but limited differences have been found in the sequence of this gene between rat- and human-derived specimens. These studies emphasize the power and versatility of PCR techniques which will undoubtably contribute to our further molecular understanding of P. carinii and its epidemiology.

VI. GROWTH AND METABOLISM Most aspects of P. carinii research have been


FIGURE 5. Variation of band patterns among P. carinii samples from different rat colonies and stability of the pattern from a given colony over time. Chromosomes of P. carinii organisms isolated from four physically distinct immunosuppressed rat colonies (A-D) were resolved by FlGE and visualized by ethidium bromide staining. The organisms were obtained from Sprague-Dawley rats (SD) obtained from Harlan Sprague-Dawley, Madison, WI, Lewis rats (L)from Charles River Breeding Laboratories, Inc., Wilrnington, MA, or through an interagency contract with the National Cancer Institute.The date the organisms were harvested are shown on top of the figure. Lanes 2-4 exhibit the banding pattern “ N B ” ; lanes 5-7 show the “C” pattern and demonstrate the stability of a given karyotype over time; the “D” pattern is shown in lanes 8 and 9. Sc, S. cerevisiae. (Adapted from Reference 62.)

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hampered by the absence of a method of continuous culture. Successful culture of the organism as yeast-like colonies was reported on solid fungal media in 1953 and 1954 but workers have subsequently been unable to reproduce these rep o r t ~ . Since ~ ~ . the ~ ~ 1970s, multiple groups of workers ’have attempted to culture P . carinii in primary and continuous cell lines and axenic media. Successful culture can be obtained with multiple different cell lines but is limited in many aspects. 18.71-75 Passage of cultures has been limited to 3 to 4 cycles with a 10- to 15-fold increase in organism number over the period. Evaluation of in vitro culture has been complicated by a lack of standardized criteria among investigators for organism quantitation, viability, and growth. This laboratory has utilized Diff-Qwik stain (a modified Wright-Giemsa stain) for counting P . carinii because it stains the nuclei of all life cycle ~tages.~’ Cyst stains (e.g., cresyl echt violet or toluidine blue) are unable to differentiate live from empty cysts and thus can lead to errors in quantitation. Enumeration of trophic forms does not take into account possible excystation, where release of up to eight daughter forms could be interpreted as replication. For these reasons, nuclear counting with Diff-Qwik stain is the preferred technique. While many cell lines have been identified as supporting growth, there appears to be little specificity of these systems. Various laboratories have utilized cell lines as diverse as chick embryonic epithelial lung cells, Chang human liver epithelial-like cells, and MCF7 human epitheliallike breast carcinoma cells. The most successful and widely used cell lines are A549, an epithelial cell line derived from human lung carcinoma; WI-38, a human fibroblast-likelung cell line; and a mink lung cell line (Mv 1 Lu). These cell lines support growth in the presence of 10 to 20% serum and other nutritional supplementation. Clusters of organisms made up of many mixed developmental stages are found floating in the supernatant with attachment of part of the cluster to the cell monolayer. Trophic forms appear to account for most of the replication observed in in vitro c~1tux-e.~’ These culture systems have been used to provide some information on the life cycle and metabolism of the organism. They have also been

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used as an intermediate step to remove contamination by host lung products in immunological and metabolic They are used by some groups for in vitro drug susceptibility testing, although the reliability of this system is not fully confirmed. The use of the in vitro culture to provide information on the metabolism of the organism has been limited by the difficulty of differentiating P . carinii metabolism from that of the cell monolayer. For this and other reasons, axenic culture would offer many advantages. Utilizing the recent molecular information suggesting that P . carinii was closely related to fungi, formulations designed for growth of some common and fastidious fungi were reevaluated. Media composed of neopeptone and the amino sugar Nacetylglucosamine at low pH have been able to support an eight- to tenfold increase in organisms in single pa~sage.~’ These organisms were shown to be metabolically active during 7 days of growth, incorporating radiolabeled methionine into specific proteins. This system offers the potential of more detailed metabolic studies and a method to evaluate drugs without cell monolayer interference. Other workers have also been able to support the growth of P . carinii in cell-free media consisting of Dulbecco’s MEM supplemented with fetal bovine serum, 2-mercaptoenthanol, bathocuprine sulfate, and c y ~ t e i n e . ~ ~ Some metabolic studies have been performed despite the lack of an adequate culture system, but must be interpreted with caution since the organism preparations may have been contaminated with host cells and other microbes. Using nonreplicating organisms, some workers have been able to define certain metabolic activities using radiolabeled compounds. It has been reported that P . carinii is able to metabolize I4Cglucose and L-pyruvate to COz, synthesize proteins from ’H-amino acids, and incorporate 3Huridine into ribonucleic a ~ i d . Inhibitors ~ ~ . ~ ~of these pathways can effectively block these reactions, suggesting specificity of these findings. P . carinii is able to incorporate choline and ethanolamine into some phospholipids, but does not appear to synthesize dipalmitoyl phosphatidylcholine, the major phospholipid of surfactant In axenic culture, P . carinii incorporates radiolabeled methionine, isoleucine, cysteine,


GlcNAc, and hypoxanthine into acid-insoluble products. 77 The ability to incorporate the purine nucleotide, hypoxanthine, suggests the capacity for purine salvage like many protists (e.g., Plasmodia). This ability to salvage purines might explain its sensitivity to purine nucleoside analogs.82 P. carinii also possess the ability to synthesize its own folates. This has been shown by the detection of 3H-para-aminobenzoic acid in reduced folates of organisms cultured for short periods, and by the detection of dihydropteroate synthetase a c t i ~ i t y .These ~ ~ , ~findings ~ correlate well with the sensitivity of the organism to dihydrofolate reductase inhibitors and sulfa drugs. Additional enzyme capabilities of P. carinii have been identified by starch gel electrophoreS ~ SThis . ~method ~ has allowed clear delineation of P. carinii enzymes from those of the host rat enzymes. The presence both of oxi-reductases, such as lactate dehydrogenase, glucose-6-phosphate dehydrogenase , and glutamate dehydrogenase, and the antioxidants, superoxide dismutase and catalase, was confirmed in these studies. P. carinii's malate dehydrogenase was also identified. Abundant glycogen storage granules have been identified within the cytoplasm of the trophic forms. 11~15*33.41 Although the chemical structure and linkages of this glycogen has not been characterized, its presence implies the ability to generate glucose- 1-phosphate and acetyl coenzyme A.

VII. IMMUNOBIOLOGY Antigenic studies of P. carinii have been plagued by the same problems that have faced other areas of research into this organism: the difficulty in obtaining preparations of P. carinii free from host cells and microbes from infected lungs; the marked adherent properties of P. carinii; and the scarcity of human-derived organisms. Early studies were limited to examination of organisms by immunofluorescence or immunoperoxidase staining in infected lung or respiratory secret i o n ~ . ~ .Sources ~ " ~ ~ of antibody have included sera from immunized animals, exposed subjects and animals, as well as monoclonal antibodi e ~ . ~ O -These ~' reagents demonstrate brightly staining cysts with a typical rim pattern of fluorescence and trophic forms visible as clusters.

Immunofluorescent techniques have demonstrated that P. carinii obtained from humans, rats, mice, and other animals appear to have shared and species-specific epitopes. 88 Immunofluorescence is finding increased use in the clinical diagnosis of P. carinii pneumonia using commerqially available monoclonal antibodies. 87 Western blot studies have revealed several major P. carinii antigens (Figure 6).46**93*96-101 The most prominent moiety is a band of about 116 kDa on polyacrylamide gel electrophoresis (PAGE) under reducing conditions. Although different workers have described this immunodominant band as being from 100 to 120 kDa, these findings are thought to represent the same moiety under varying experimental conditions. Under nonreducing conditions, this antigen migrates as an aggregate with a molecular weight of >2 x lo6 m a . Analysis by monoclonal and polyclonal antibodies has revealed cross reactivity between this antigen from P. carinii isolated from humans, rats, mice, rabbits, and ferrets as well as species-specific epitopes. Surface labeling and electron microscopy studies have demonstrated that this 116-kDa antigen resides on the surface of the organism. Purification of the 116-kDa band has been technically difficult using standard chromatographic procedures, but harsh techniques using enzymes, detergents, or denaturation have achieved some success. These biochemical studies have shown that it is an acidic glycoprotein with a pKa of 5.6 to 6.2.w The glycoprotein of rat-derived P. carinii is composed of a 66- to 68kDa protein core and a carbohydrate portion rich in glucosyllmannosyl and N-acetyl glucosamine residues.95~"*98*99~10z-103 A blocked amino terminus has prevented sequencing of the small amounts of core protein thus far obtained. Another major class of antigens found in rodent-derived P. carinii migrates in the 45- to 50kDa range.a*91-93*97Jw This moiety appears as a single broad band or as two distinct bands on immunoblots. The 45- to 50-kDa antigen appears to be a glycoprotein in nature but differs from the 116-kDa band in susceptibility to proteolytic enzymes and lectin binding. The relationship of the 45- to 50-kDa band to the 116-kDa antigen, its location on P. carinii, and its function are all unclear.

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FIGURE 6. Analysis of P. carinii by immunoblotting and lectin blotting. 1: Rat-derived P. carinii reacted with rabbit antiserum to rat-derived P. carinii (lane A) and Concanavalin A (lane B). 2: Human-derived P. carinii reacted with rabbit antiserum to human-derived organisms (lane A) and Con A (lane B). The most prominent immunoreactive bands are 1 16 kDa and 45-50 kDa in rat-derived organisms and 116 kDa and 35-45 kDa in human-derivedP. carinii. The Con A reactivity with the 116 kDa bands of organisms from both sources can be seen. Molecular weight markers are shown on the left. (Adapted from Reference 46.)

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The most prominent feature of human-derived P. curinii on immunoblot is a broad intensely staining band of between 35 and 45 kDa.46*92.93.95 This band appears to be a glycoprotein with properties similar to the 45- to 50kDa band of rat-derived P. curinii. Antigen bands can be much more readily detected in clinical specimens from AIDS patients with P. curinii pneumonia than in other immunocompromised hosts, perhaps reflecting the greater organism burden. The 35 to 45 kDa band is the most common one found in the lungs and bronchoalveolar fluid of humans with P.curinii pneumonia, while the 116-kDa band is less prominent in these specimens. 93 A number of other P. curinii antigens of various sizes (e.g., 92 and 66 kDa) have been detected, but their location on P. carinii and their function are currently uncertain. Some cross reactivity has also been demonstrated with P. curinii antigens and cardiolipin, but the functional significance of this remains to be determined.Io5Further detailed study of the function and structure of the antigens of P. carinii will be partially dependent on efforts to clone these antigens molecularly and on improvements in organism cultivation.

A. Animal Models Excellent models for P . curinii pneumonia have been established in many small experimental animals. These systems have contributed much to our knowledge of the pathogenesis and hostparasite interaction in P. carinii pneumonia because they present a histological picture that is virtually identical to that found in the human pneumocystosis. 106*107A unique feature of P. curinii infection is that pneumonia can be provoked in the animals without the need for inoculation of the organisms. Thus, the naturally acquired infection is utilized and manipulated under experimental conditions. The essential requirement for the provocation of P . curinii pneumonia is a prolonged period of immunosuppression or a state of immunodeficiency resulting in activation of latent infection.

The most widely studied model is that of corticosteroid-treated rats, although corticosteroid-suppressed ferrets, rabbits, and mice have all been ~ s e d . ~ ~ * ' ~With * ' ~ immunosuppres&''~ sion, P . carinii organisms replicate slowly and gradually fill the alveoli with organisms and honeycombed frothy rnate~ial."~ A low (8%) protein diet enhances the development of infection and antibiotics are required to decrease bacteria infections during the profound immunosuppres~ i 0 n . The l ~ ~trend in recent years among commercial vendors to develop higher grade animals for research has led to the development of virusfree rat colonies that are also apparently free of latent P . curinii.'I6 One approach has been to intratracheally inoculate virus-free rats with P. curinii-infected lung homogenates from other animals. lo8 This technique has been successful in some laboratories, although is not in widespread use. Congenitally immunodeficient mice offer an alternative model for pneumocystosis without the confounding issues of steroid immunosuppression. 104* 117~118Outbreaks of P. curinii pneumonia have been reported in several colonies of athymic (nulnu) and scidlscid mice. That these outbreaks could be controlled by the administration of agents known to be active against human P . curinii pneumonia suggests that this model might be useful in drug development. Currently, athymic mice are being used as an animal model by workers in Japan, while scid mice are the subject of ongoing investigations in some centers in the U.S.104,117-122 Scid mice, which lack both B- and T-cell function, offer the hope that infection with strains of human-derived P . curinii can be established and then tested for host immune responses and response to drug therapy. Another model undergoing development is the prolonged administration of anti-CD4 antibodies to render animals immunodeficient and susceptible to P. curinii infection.

B. Host-Parasite Interaction Exposure to P . carinii stimulates an immune response in the host. Serologic studies, which

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employed indirect fluorescent antibody and enzyme-linked immunoabsorbent assay techniques, have shown that healthy humans develop serum antibodies in childhood to P . carinii under conditions of natural e x p o ~ u r e .A~ ~recent . ~ study focusing on the serologic response to specific P . carinii antigens by Western blot techniques has shown that these antibodies are directed primarily against the 35- to 45-kDa band, although other bands were also recognized. 58 Surprisingly, this study also found that >90% of A I D S patients followed sequentially develop IgM and/or IgG antibody responses to the 35- to 45-kDa band and other P . carinii antigens with recurrent episodes of pneumocystosis. This is in contrast to the serologic response of these patients to other opportunistic infections where responses have been markedly impaired. The role of these antibody responses, as well as the finding of antibodies to P . carinii in bronchoalveolar lavage fluid of humans and laboratory animals after exposure to the organism, is unclear. Support for the role of antibodies can be found in the occurrence of P . carinii pneumonia in some patients with B-cell defects and that passive immunization with monoclonal antibodies directed against the 116-kDa antigen decreased the severity of infection in laboratory animal^.^-^^^ Available evidence suggests that these antibodies may function as opsonins.125 Diseases predisposing to the development of P . carinii pneumonia exert their major effects on cell-mediated immune function. Of these conditions, AIDS is numerically the most important, although an increase in P . carinii pneumonia has been noted in non-AIDS patients as well. The principal immunologic deficit in HIV infection is a decline in the number and function of circulating CD4 T-helper cells. The incidence of P . carinii pneumonia in HIV patients has been shown to rise in direct proportion to the fall in CD4 cells. Laboratory studies on the cellular immune response have been hampered by the lack of available purified antigen preparations. This has been overcome to some extent by the use of ratderived organisms propagated in short-term culture combined with stringent controls. Two studies have used this type of preparation to measure the blastogenic responses of peripheral blood

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mononuclear cells.76.‘27 The responses in healthy adults were found to be specific for P . carinii; in contrast, newborn infants, who had no known exposure, exhibited no responses. HIV patients exhibited a decline in responses which paralleled progression of the infection, and AIDS patients demonstrated no response even following recovery from a bout of P . carinii pneumonia. All subjects in this study had serum antibodies to the rat-derived P . carinii preparation by indirect immunofluorescence; however, there was no correlation between antibody titer and blastogenic response. These results raise the possibility that humoral and cellular responses are directed against different epitopes. The role of specific effector cells in host defenses against P . carinii is poorly understood. Attachment of P . carinii to alveolar macrophage occurs by a fibronectin and calcium-dependent mechanism, but does not trigger a phagocytic response. 128*129Ingestion and killing of P . carinii by macrophage is enhanced by the presence of specific antibodies. 1 2 5 ~ 1 3 0A recent study demonstrated that the mannose receptor also plays a role in ingestion of P . carinii by macrophages.”’ Several studies have suggested that neutrophils, alveolar type I1 epithelial cells, and cytokines such as y-interferon and tumor necrosis factor (TNF) may participate in the host effector mechanisms against P . carinii.8 1 ~ 1 3 2 - 1 3 5These studies have been hampered by the confounding effects of corticosteroids in the animal models. A more recent study using rat-derived P . carinii separated from either alveolar macrophage or type I1 epithelial cells by a semi-permeable membrane demonstrated that type I1 pneumocytes alone, but not normal alveolar macrophage, significantlyreduced P . carinii viability; similar reduction of viability could be produced by TNF in the absence of cells or by endotoxin or y-interferon in the presence of cells.136These data require some caution in their interpretation because assays of P . carinii viability have not been standardized.

VIII. PATHOGENESIS AND PATHOLOGIC FINDINGS Pathogenic mechanisms have been most clearly defined in the corticosteroid-treated ro-


dent model and in limited in vitro studies. Development of pneumocystosis in the rat begins with attachment of the organism to the type I pneumocyte. 34.L37-141Ultrastructural analysis has demonstrated that the organisms adhere tightly to these cells without fusion of cell membranes. In v i m studies have suggested that this attachment is mediated by fibronectin and the 116-kDa antigen. 128.129*142 These studies, performed in an assay utilizing A549 cells, a human alveolar epithelial cell line with features of type II and type I pneumocytes, also suggest adherence interferes with the function of the host lung cells. Following attachment of the organism, electron microscopy demonstrates a decrease in the surface glycocalyx of the type I cells and increased alveolar-capillary permeability. 137~139.143~1MThis has been demonstrated by the leakage of the marker, horseradish peroxidase, from the vascular space into the alveolar space. These changes result in subepithelial bleb formation and denudation of the basement membrane, manifestations of degenerative changes in alveolar type I cells. Thereafter, hypertrophy of the type 11 cells is noted which is a nonspecific reparative response to diffuse alveolar damage. Functional abnormalities have been noted concurrent with the structural changes. Studies of bronchalveolar lavage fluid of rats and small numbers of patients with P. carinii pneumonia have shown a fall in surfactant phospholipids. 145-147 This is accompanied by alterations in compliance and lung mechanic^.'^^,'^^ The mechanisms of decreased surfactant are not fully understood, although it has been shown that P. carinii can bind surfactant to its surface inhibit surfactant production and/or secretion. 150~151 When animals are permitted to recover from pneumocystosis (by removal of their immunosuppression), they mount a vigorous inflammatory and immune response. This is characterized by phagocytosis of P. carinii by alveolar rnacrophage, a prominent mononuclear cell infiltrate, a return in circulating lymphocytes and T-cell subsets to normal, a rise in serum antibodies to P. carinii, and the gradual development of pulmonary fibrosis. Pathologic changes noted in human pneumocystosis depend on whether the disease is diffuse, focal, or nodular.152In diffuse disease, all

lobes of the lung are involved in the disease process. The lungs are densely consolidated with a rubbery consistency. Severely affected areas demonstrate diffuse alveolar damage similar to that seen with a wide variety of etiologies. In less severely affected areas, foci of consolidation, atelectasis, and hyperinflation are seen. The focal form differs only in the extent of involvement, while rarely, single or multiple nodules and cavitation can be seen. Histopathologically, P. carinii pneumonia in humans shows both alveolar interstitial thickening and frothy honeycombed material in the alveolar lumen. The interstitial thickening is attributed to hyperplasia and hypertrophy of type 11 pneumocytes, interstitial edema, cellular infiltrates composed of lymphocytes with few rnacrophage or plasma cells, and mild fibrosis. The alveolar lumens filled with frothy honeycombed material contain numerous P . carinii cysts. Characteristically, organisms are much more numerous in AIDS patients than in others with pneumocystosis . Ultrastructural features have been discussed above with reference to the pathogenesis of the disease.

IX. EPIDEMIOLOGY The epidemiologic features of P. carinii are poorly understood. Experimental studies have shown that infection is acquired by inhalation of an airborne particle, but the nature of this particle remains In immunosuppressed rats and nude mice, it has been demonstrated that pneumonia could be produced by distant or direct contact of uninfected animals with infected animals. The fungal nature of P. carinii raises the possibility of as yet unidentified stages (e.g., spores) or environmental sources of P. carinii which may play an important role in the infection. P. carinii causes pneumonia almost exclusively in the immunocompromised host. The major predisposing conditions are characterized by impaired ceIlular immunity: AIDS, protein calorie malnutrition, prematurity, primary immunodeficiency diseases, and the use of corticosteroids and other immunosuppressive agents for the treatment of cancer and other disorders. Review

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of worldwide reports of P. carinii pneumonia reveal that few countries are free of the organism.4oCountries in temperate, tropical, and polar regions have all reported infection. The few countries, such as China and certain South and Central American states, where pneumocystosis has not been reported probably reflect a failure of disease recognition or reporting. Primary exposure to P. carinii among humans occurs early in life, so that by age 3 most children have developed serum antibodies to the organism. No clinical manifestation of the primary infection has been documented and it is presumed to be an asymptomatic infection. In infants with AIDS, the poor prognosis associated with clinical pneumocystosis may reflect an overwhelming primary infection in a severely immunocompromised host. It is generally thought that, following this original infection, organisms remain latent within the host and begin to propagate as the host’s immune function becomes compromised. Alternatively, pneumocystosis in immunocompromised hosts may arise from organisms acquired more recently from the environment. Whether the organisms that are transmitted can establish a new infection or can complicate a preexisting latent infection is unclear. AIDS patients have been found to develop IgM antibody responses to specific P . carinii antibodies with recurrent episodes of P . carinii pneumonia.58It has also been demonstrated by immunoblotting that P. carinii antigen recognition patterns in bronchoalveolar lavage fluid specimens from these patients can change with recurrent episodes of pneumonia. 153 These findings may represent infections with different antigenic strains or antigenic changes in the existing strain of P . carinii. Attempts are currently being made to answer these questions with studies utilizing exposure to strains with demonstrably different karyotype patterns on pulse field gel electrophoresis. Person-to-person spread of P . carinii has been suggested by outbreaks of pneumocystosis in malnourished infants in orphanages and in hospitals caring for immunosuppressed patients. A recent outbreak was reported in a group of elderly people with apparently mild defects in immune function.154This outbreak was noteworthy for the fact that none were admitted with an illness highly ’

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suggestive of pneumocystosis, and the diagnosis was made after 7 to 32 days of hospitalization, raising the question of nosocomial infection. Unfortunately, the limited available epidemiological data on these outbreaks provide few definitive answers. X. CLINICAL FEATURES

P . carinii causes two clinical pictures of pulmonary infection: (1) interstitial plasma cell pneumonia, and (2) pneumonia in the irnmunocompromised host. Interstitial plasma cell pneumonia, known as the “epidemic” form of pneumocystosis, is a cause of progressive respiratory distress in infants characterized histologically by a heavy interstitial plasma cell infiltrate.4.sIn immunocompromised hosts, there are significant differences in the disease picture in AIDS and non-AIDS patients.’55P . carinii also causes extrapulmonary infection in severely compromised hosts. The infantile form of the disease, interstitial plasma cell pneumonia, was described in orphanages or related institutions that house chronically debilitated infants under crowded conditions. This form of the disease has largely disappeared from the Western world, but continues to be found in other areas. The disease occurs throughout the first year of life and presents with nonspecific clinical manifestations. Respiratory difficulties develop gradually and are characterized by tachypnea, cyanosis, and cough and progressive respiratory distress. Physical findings are unusual but may include focal or diffuse signs of consolidation. Chest X-ray shows diffuse pulmonary infiltrates. P . carinii pneumonia is a leading cause of fatal opportunistic infection in AIDS patients and other immunocompromised hosts. The disease in non-AIDS patients may vary from an acute illness with an abrupt onset to a more indolent disease course seen more often in AIDS patients. In patients with cancer and other diseases requiring immunosuppressive therapy, symptoms are present for 1 to 2 weeks before a diagnosis of P. carinii is established. The symptoms frequently begin after corticosteroids have been tapered off or discontinued. In AIDS patients, the onset of


symptoms is often more insidious. The reduction in circulating CD4 cells has been found to correlate with the risk for the development of P . carinii pneumonia, with the vast majority of cases occumng with CD4 cells

Editorial: Pneumocystis carinii pneumonitis.

Pneumocystis carinii karyotypes.

Pneumocystis carinii pneumonia.

Pneumocystis carinii in a pig.

Repeated DNA in Pneumocystis carinii.

Pneumocystis carinii--new clinical spectrum?

The risk of Pneumocystis carinii pneumonia.

Pneumocystis carinii pneumonia in children.

Treatment of pneumocystis carinii pneumonia in children.

Seasonal incidence of Pneumocystis carinii pneumonia.

Propagation of Pneumocystis carinii in vitro.

Prevention and treatment of Pneumocystis carinii pneumonia.

Detection of Pneumocystis carinii with DNA amplification.

Diagnosis and treatment of Pneumocystis carinii pneumonia.

False negative biopsy in Pneumocystis carinii pneumonia.

Pneumocystis carinii pneumonia complicating multiple myeloma.

The value of Pneumocystis carinii antibody and antigen detection for diagnosis of Pneumocystis carinii pneumonia after marrow transplantation.

Immunocytochemical detection of chitin in Pneumocystis carinii.

Letter: PNEUMOCYSTIS CARINII IN A Vietnamese foundling.

Why have Pneumocystis carinii trophozoites been ignored?

Quantification of the detection of Pneumocystis carinii by DNA amplification.

Pneumocystis carinii, an opportunist in immunocompromised patients.

[Ultrasonic nebulised pentamidine for Pneumocystis carinii pneumonia].

Pneumocystis carinii pneumonitis: a serological study.