key: cord-0035419-6mrfe0ha
authors: Haque, Abida K.; Adegboyega, Patrick A.
title: Pneumocystis jiroveci Pneumonia
date: 2008
journal: Dail and Hammar’s Pulmonary Pathology
DOI: 10.1007/978-0-387-68792-6_13
sha: fb886d7c9ebe135dba5e8b6de3a98e2ef84d0f76
doc_id: 35419
cord_uid: 6mrfe0ha

Pneumocystis pneumonia (PCP) is one of the most common pulmonary infections in persons with impaired cell-mediated immunity, and particularly those infected with human immunodeficiency virus (HIV).1–7 Pneumocystis was first described in the lungs of guinea pigs, during experiments on American trypanosomiasis by Carlos Chagas8 in 1909 and by Antonio Carinii9 in 1910. Both considered the cysts of Pneumocystis as part of the trypanosome’s life cycle. Shortly afterward the Delanoes10 found identical forms in the lungs of rats that had not been infected with trypanosomes and recognized the organism as a separate species. The name Pneumocystis carinii, was given to this organism as a generic name (Greek:pneumon, “lung”; kystis, “cyst”), honoring Carinii.11

Pneumocystis pneumonia (PCP) is one of the most common pulmonary infections in persons with impaired cell-mediated immunity, and particularly those infected with human immunodeficiency virus (HIV).1-7 Pneumocystis was first described in the lungs of guinea pigs, during experiments on American trypanosomiasis by Carlos Chagas 8 in 1909 and by Antonio Carinii 9 in 1910 . Both considered the cysts of pneumocystis as part of the trypanosome's life cycle. Shortly afterward the Delanoes lo found identical forms in the lungs of rats that had not been infected with trypanosomes and recognized the organism as a separate species. The name Pneumocystis carinii, was given to this organism as a generic name (Greek: pneumon, "lung"; kystis, "cyst"), honoring CariniiY

The organism attained medical significance, when van der Meer and Brug l2 in 1942, and later Vanek, Jirovec, and Luke suggested it to be the cause of interstitial plasma cell pneumonia, a disease affecting premature and debilitated infants in central and eastern Europe. 12 ,13 In the 1960s P. carinii was recognized as an important cause of pneumonia in immunocompromised adults on corticosteroids and cancer chemotherapy, in organ transplant recipients, and in children with primary immunodeficiency syndromes. 6 The emergence of acquired immune deficiency syndrome (AIDS) in the 1980s thrust P. carinii to the forefront once again, as a leading cause of morbidity and mortality in immunocompromised individuals, 14 Pneumocystis organisms infecting human beings have recently been named P. jiroveci. 15 The complete identification and classification of pneumocystis has taken many decades. Although initially considered to be a protozoan, it is now generally agreed that pneumocystis is a fungus. The ribosomal RNA is homologous to that found in fungi. 16 ,17 A study of the small subunits of ribosomal RNA (16S-like rRNA) of P. carinii and the fungus Saccharomyces cerevisiae shows close evolutionary linkage between the two. 16 The pneumocystis organisms also stain with methenamine silver stains, further supporting a closer link to fungi rather than pro-tozoa. Recent molecular genetic studies that demonstrate the thymidylate synthase and dihydrofolate reductase genes to be similar to their counterparts in S. cerevisiae support the classification of pneumocystis as a fungus. 17 Furthermore, ultrastructural studies have failed to show the cytoskeletal elements and complex organelle systems characteristic of protozoa. 18 ,19 Pneumocystis organisms are ubiquitous and globally distributed, having been identified in virtually every mammalian species including humans as well as rabbits, dogs, goats, cats, swine, chimpanzees, owl monkeys, and horses,z0-26 The organisms have a wide range of genetic characteristics that are host specific. 27 The pneumocystis that infects humans, P. jiroveci, is different from the one that infects rats, and there is no cross-species infection. 15 ,27 This observation was confirmed when polymerase chain reaction (PCR) applied to the human pneumocystis identified only P. jiroveci. 28 ,29 Epidemiology The epidemiologic features of P. jiroveci are poorly understood. Experimental studies have shown that the infection is acquired by inhalation. 3 O-33 There does not appear to be a natural transmission of P. jiroveci across species. 27 ,34-36 Immunosuppressed rats and nude mice acquire the infection by direct and distant contact with infected animals. In humans, the major predisposing factor is impaired cellular immunity as seen in AIDS, protein-calorie malnutrition, primary immunodeficiency diseases, immunosuppressive therapy with corticosteroids or other agents, and prematurity. 37 The organisms are present in practically every part of the world, including the temperate, tropical, and polar regions. 38 The use of molecular technology through the dihydropteroate synthase (DHPS) locus analysis has facilitated epidemiologic study of the prevalence of P. jiroveci in the human population. Identical genotypes of P. jiroveci were found in the two groups of immunocompetent infants and adults with pneumocystis infection in a study from France, suggesting that the transmission cycles of infection in all individuals parasitized by P jiroveci are linked with a common human reservoir. 39 ,40 It is not clear whether there is an environmental reservoir for P jiroveci, although mammalian lung appears to be a natural home. 35 The isolation of pneumocystis DNA from rural outdoor locations and a seasonal variation in infection in patients suggest that there may be an environmental reservoir. 41 Primary exposure to pneumocystis occurs early in life, so that most children have serum antibody by the age of 2 to 3 years. 42 -45 This is presumed to be an asymptomatic infection. The organisms remain latent within the host, and propagate when the host immune system becomes compromised. Several studies using PCR have found no evidence of pneumocystis DNA in the lungs of immunocompetent individuals, and do not support the latent reactivation theory. 41,46--48 There are also experimental data in rat models that suggest that pneumocystis organisms do not persist in the lungs of immunocompetent individuals. 49 Transmission of pneumocystis has been shown to occur animal to animal when they had a common air supply, in immunosuppressed as well as immunocompetent models, although the latter had a subclinical transient infection. 41 ,5o,51 Clinical studies have suggested the occurrence of human to human transmission of pneumocystis organisms. [52] [53] [54] [55] [56] Acquired immune deficiency syndrome patients have been found to develop immunoglobulin M (IgM) antibodies with recurrent episodes of pneumocystis pneumonia. 57 It has also been shown by immunoblotting that P jiroveci (carinii) antigen recognition patterns in bronchoalveolar lavage (BAL) fluid can change with recurrent episodes of pneumonia. 58 These findings may represent infections with different antigenic strains or antigenic changes in the existing strain of P jiroveci. Recently, mutations in the DHPS gene of P jiroveci were identified and used in epidemiologic studies of P jiroveci. In one study of 139 HIV-infected patients with pneumocystis infection, 19% of patients with prior sulfa treatment had the gene mutation, compared to 4 % of those without treatment. 59 Co-infection with multiple strains of P jiroveci was found in 20% to 30% of cases, suggesting that recurrent infections may be related to reinfection with a new strain rather than reactivation. 60 The DHPS gene-type variation was related to the place of diagnosis and not the place of birth, suggesting the infection to be recently acquired. Additionally, 54 % of P jiroveci strains in newly diagnosed HIV-infected patients demonstrate DHPS gene mutations. Since these patients were not treated with sulfa drugs, such mutations suggest that the infection was acquired from patients who had received prophylactic sulfa. The authors therefore believed that these findings represent evidence for person-to-person AK. Haque and P.A Adegboyega transmission of pneumocystis. Person-to-person spread of P jiroveci is also suggested by outbreaks of infection in malnourished infants in orphanages and in hospitals caring for immunosuppressed patients. 37

The major obstacle to study the life cycle and biology of pneumocystis is the inability to sustain propagation of the organism outside the lung. 28 Our understanding of the life cycle of P jiroveci derives mainly from detailed ultrastructural studies. 61 -65 Four developmental forms are described: trophozoites, cysts, precysts, and sporozoites (also known as intra cystic bodies).66,67 All investigators have consistently identified the trophic (trophozoite) and the cyst stage, and also an intermediate precyst stage. Experiments using P carinii from lungs of infected rats and human lung cell cultures have been partially successful in growing the organisms to study the life cycle. 68 Based on the cell culture studies, it was proposed that several developmental pathways may exist. 68 The environment may play a significant role in determining the predominant method of replication as it does in many yeast or other microorganisms. 69 }O In tissue culture, at least two methods of development were proposed: an asexual cycle and a sexual cycle. Figure 13 .1 shows the two cycles of P jiroveci pneumonia development. The asexual cycle involves mitotic replication of the trophic forms. The sexual cycle involves the continued development of precyst stages to mature cyst forms, and the development of elongated daughter forms within the cyst, followed by excystation and collapse of the cyst.

The vegetative forms of pneumocystis, the trophozoites, are 2 to 811m in diameter, and attach to type I alveolar epithelial cells. Although initially haploid, the trophozoites are believed to attain a diploid chromosomal number by gametic fusion. 37 The trophozoites enlarge and develop into diploid precysts through a process of cell wall thickening. 37 Sporozoites then develop within the precysts following meiosis and mitosis, a process referred to as asporogony.67,69 Mature cysts contain eight haploid sporozoites that become trophozoites, following rupture of the cyst wall, and recapitulate the life cycle.

The cyst is the largest and most easily recognized developmental stage of P jiroveci. As demonstrated with Gomori's methenamine silver (GMS) stain, the cysts are thick-walled spherules, 5 to 7 11m in diameter, that assume a cup or crescent shape when collapsed. The cyst wall is trilaminar, 70 to 160nm in thickness, and shows a thick electron-dense outer layer, an electron-lucent middle layer, and a thin inner cell membrane. 37 The cyst wall stains well with methenamine silver stain, cresyl echt A Q) > (8 <::J-BQ C)

• FIGURE 13.1. Diagrammatic representation of the developmental cycles of P carinii, in vitro. The asexual cycle: AI, trophic form; Bl, mitotic replication of trophic form; Cl, trophic forms, products of binary fission. The sexual cycle: A,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, migration of nuclei to periphery; I, late precyst, initiation of compartmentalization 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 infected rat lung homogenates, although the actual process of excystment was not observed. (From Cushion et al. 68 Copyright 1988, with permission from Macmillan Publishers Ltd) violet, and toluidine blue. It has been shown that the silver particles are deposited only on the electron-lucent middle layer of the cell wall; this explains the lack of staining of the trophozoites, which lack this layer.?l Many 489 studies have examined the composition of the cell wall. The cyst wall is rich in carbohydrates, lectins, ~-1 ,3-g1ucan, and lipids, including sphingolipid fatty acids and cholesterol. 68 . 72 -?4 Treatment of P. carinii cysts with zymolyase, a ~-1,3-g1ucanase, disrupts the cyst wall and liberates the surface antigens.?5 Ergosterol has not been detected in many studies, which explains the resistance of P. jiroveci to sterol synthesis inhibitors, such as amphotericin Band ketoconazole.? 6 Most cysts contain up to eight spherical daughter forms or intracystic bodies, also known as sporozoites that are 1 to 2 urn in diameter and surrounded by a double unit membrane. The sporozoites have a single nucleus, a mitochondrion, abundant endoplasmic reticulum, ribosomes, as well as microtubules and vacuoles. They are reported to be attached to each other and to the cyst wall by a thread or stalk-like structure?? Many cysts contain single or paired, comma-shaped structures that are localized areas of thickening of the inner layer of the cyst wall. These distinctive argyrophilic structures help to distinguish the cysts of P. jiroveci from nonbudding yeast-like fungi and other pathogens. Although seen best with GMS, the thickened foci can also be stained with toluidine blue 0, but not reliably with the Gram-Weigert stain. None of the cyst wall stains demonstrate the sporozoites or trophozoites, although the membranes of the latter may stain faintly in specimens heavily overstained with GMS .

The sporozoites and trophozoites can be visualized with the appropriate special stains in smears and imprints, mainly because of their nuclear staining. The sporozoites are seen as clusters of small nuclei surrounded by an unstained cyst wall, whereas the trophozoites appear as a meshwork of honeycombed spaces, many of which contain a dot-like nucleus.

The trophozoites may be difficult to distinguish from background fibrin or tissue elements. In lung sections they appear ameboid, with broad pseudopodia and polar aggregates of tubular cytomembranous processes, termed "filopodia." The filopodia increase the surface area of the trophozoites and may help in nutrition and transport of enzymes to the environment; they are not organelles of attachment. 3 ? In unfixed preparations the trophozoites appear as oval bodies resembling a cluster of grapes?? The trophozoites have a cell wall with three layers similar to the cyst; however, the middle electron-lucent layer is thin and poorly developed. Each trophozoite contains a single nucleus, cytoplasmic organelles, and glycogen, although not all of these elements are seen in every trophozoite. Electron micrographs of the trophozoites demonstrate the filopodia and the relationship between the filopodia and the pneumocytes (Figs. 13.2 and 13.3). Occasionally, trophozoites assume shapes that suggest reproduction by either binary fission or endogeny. The trophozoites tend to cluster in large aggregates, where FIGURE 13.2. Electron micrograph of alveolar exudates in AIDS patient with pneumocystis pneumonia. Cyst containing six merozoites is seen between arrows. Next to the cyst is a maturing trophozoite (T) without filopodia. Two collapsed cysts (C) are also present. Note that the upper cyst has discrete areas of capsular thickening corresponding to the darkly staining bodies seen with Gomori's methenamine silver stain. Uranyl acetate, lead citrate. (Courtesy of Dr. Richard Sobonya, University of Arizona.) they mold against each other in sheet-like pieces of a jigsaw puzzle. Each large cluster of trophozoites contains only a few admixed cysts. It is the clustering of the trophozoites that produces the appearance of alveolar "honeycomb exudate" seen on light microscopy. The intermediate stage between the trophozoite and the cyst forms the precyst stage, which demonstrates considerable morphologic variability. The precysts are oval in shape and 4 to 6/-lm in diameter. The cell wall is up to 100 nm in thickness, with an electron-lucent layer that is thicker than that of trophozoites. The cytoplasm contains one or more nuclei, mitochondria, free ribosomes, microtubules, vacuoles, and glycogen granules. Ultrastructural studies have identified three subtypes of the precyst stage, characterized by progressive thickening of the cell wall and an increasing number of nuclei from one nucleus in the early precyst to four or more in the late precyst.38.66.77.7S

The developmental forms of P. jiroveci are not seen clearly in routine histologic sections. A variety of special stains may be used to make the diagnosis. These stains are grouped into two categories: those that stain the cyst wall, and those that stain the nuclei of the trophozoites and sporozoites. 79 The latter group, which can be applied only to imprint smears and cytology specimens, includes a variety of Romanowsky stains (Giemsa, Wright, Diff-Quik), polychrome methylene blue, and Gram's stain. The cyst wall stains can be applied to tissue sections as well, and are preferred for routine diagnostic work. These stains include Gomori's (Grocott) methenamine silver (GMS) and its rapid variants, toluidine blue 0, and Gram-Weigert methods; GMS is preferred by most pathologists. 8o Stains for sporozoitesitrophozoites have been advocated for diagnosis because of their simplicity and rapidity. However, a well-prepared GMS stain is by far the most reliable and sensitive nonimmunologic stain for the diagnosis of P jiroveci. Recently described rapid GMS variants have shortened the time required for specimen staining. 8o -s4 A microwave method, described originally by Brinn,s3 can provide high-contrast slides of optimal electron micrograph in A, taken from the area to left of CAP Filopodia (arrows) are apparent especially in conjunction with type I pneumocyte epithelium. Uranyl acetate, lead citrate. (Courtesy of Dr. Richard Sobonya.)

Pneumonia quality in about 20 minutes and can be applied to any standard specimen. Combined methods that demonstrate both cyst walls and the nuclei of sporozoites and trophozoites, although aesthetically pleasing, offer no advantage over a good methenamine-silver stain.

Molecular studies of rat-derived P carinii show the genome to be between 7 x 10 6 and 1 x 10 7 based on summation of P. carinii chromosomes in pulse-field gel electrophoresis. 85 . 86 This genetic material is arranged as 13 to 19 chromosomes and is between 295 and 710 kilobase (kb) in size. The karyotype of human-derived P. jiroveci appears different from that of rat-derived organisms, but has not been well characterized. 86 Three genes have been cloned from rat-derived P. carinii, including 18S RNA, dihydrofolate reductase, and thymidylate synthase gene. 87 -89 Since then, the DHPS gene and the internal transcribed spacer (ITS) region of P. jiroveci have been studied extensively in human pneumocystis infections. DHPS gene mutations are reported to be associated with sulfa/sulfone resistance in patients who had previously received prophylaxis. 9o ,91 The prevalence of DHPS gene mutations was reported to be markedly increased in the United States since the use of prophylactic sulfa drugs.92 However, another study from Portugal reports no association between DHPS types or therapy and response to anti-Pneumocystis therapy.93 This same study found a subtype of the ITS region of nuclear rRNA, type Ne, to be associated with treatment failure, childhood infection, and early death.

Antigenic studies of pneumocystis using Western blot technique have revealed several major antigens. 75 ,94-96 The most prominent surface antigen of pneumocystis is seen as a band of 116 kDa on polyacrylamide gel electrophoresis. 37 There is cross-reactivity between this antigen from humans and other mammals. Another major class of antigens in rodent-derived P. carinii migrates in the 45-to 50-kDa range. The relationship of this antigen to the 116-kDa antigen is not clear. A third antigen that is the most prominent feature of human-derived pneumocystis is a broad intensely staining band between 35 and 45 kDa. 75 . 9 4-96 This is the most common band found in the lungs and bronchoalveolar fluid of patients with P jiroveci infection, while the 116-kDa band is less prominent in these specimens. 96 Exposure to pneumocystis organisms evokes an immune response in the host. 97 Serologic studies using indirect immunofluorescence (IF) and enzyme-linked immunoabsorbent assay (ELISA) have demonstrated serum antibodies to P. jiroveci in childhood under conditions of natural exposure. 43 ,98 These antibodies are primarily directed against the 35-to 45-kDa band antigen, although antibodies to other bands were also recognized. 57 This study also found that >90% of AIDS patients developed IgM or IgG antibodies to the 35-to 45-kDa band and other antigens with recurrent episodes of pneumocystis infection. The role of these antibodies is unclear; however, there is some evidence that they may function as opsonins. 99 The development of PCP is related to impairment of cell-mediated immunity. The most prominent disease in this category is AIDS, although there has been an increase in non-AIDS patients also. In AIDS and HIV infection, there is a decline in the number and function of circulating CD4+ T-helper cells, the increased incidence of PCP being directly proportional to the fall in CD4+ T lymphocytes. 100 Pneumocystis organisms are host-specific, but the reason for this stringent specificity is not clear.27 P. jiroveci, the only pneumocystis identified in humans, has a unique tropism for the lung, where it exists primarily as an alveolar pathogen. A few cases of invasive infection with dissemination are reported with severe immunosuppression or overwhelming infection of the hOSt. 101 The availability of molecular techniques has identified key molecules in the cell cycle, signal transduction, and metabolic pathways of pneumocystis. The first specific molecule identified was glycoprotein A, a major surface glycoprotein with an integral role in the attachment of pneumocystis to host cells. 45 .102-104 This surface glycoprotein is immunogenic and antigenically distinct in each form of pneumocystis infecting various mammalian hosts. 102,104 The major component of the cyst wall is ~-1,3glucan, and the pneumocystis ~-1,3-g1ucan synthetase gene, GSCl, which mediates the polymerization of uridine 5' -diphosphoglucose into ~-1 ,3-g1ucan. I05 Inhibitors of ~-1,3-g1ucan synthetase are effective in clearing the cyst forms of pneumocystis from lungs of infected mammals. 106 ,107 The cyst wall also contains chitins and other complex polymers, including melanins that provide stability to the cell wall and invoke an inflammatory response in the lungs. 108, 109 Several signal-transduction molecules have been recently identified in pneumocystis. These include cdc2 cyclin-dependent kinase, cdc13 B-type cyclin, cdc25 mitotic phosphatase, and pneumocystis mitogen (PCM)-activated protein kinaseYO-l15 Furthermore, the finding of enhanced activity of PCM in the trophozoites as compared with the cyst forms suggests that the trophozoites may use this pathway for transitions in the life cycle. I I} During infection, the trophozoites adhere tightly to the alveolar epithelium, activating specific signaling pathways in the organism, including the gene encoding PCSTE20 kinase, which signals responses for mating and proliferation in fungal organisms. 114 Other signaling molecules identified in pneumocystis include the pheromone receptors, heterotrimeric G-protein subunits, and transcription factors. 115 · 116 Currently, some of these specific molecules are under intense research studies as potential drug targets. These include dihydrofolate reductase (target of trimethoprim), thymidylate synthase, inosine monophosphate dehydrogenase (target of mycophenolic acid), S-adenosyl-L-methionine:sterol C-24 methyl transferase (involved in biosynthesis of sterol), and lanosterol 14(a)-demethylase (the target enzyme of azole antifungal drugs ). 59, 117, 118 Pathogenesis of Infection Pneumocystis infection is acquired by inhalation. When the organism is deposited in the alveoli, the first critical step in the establishment of infection is attachment to the type I pneumocyte, through interdigitation of their cell membrane to the alveolar cell membrane. l19 This binding is facilitated by the interaction of host proteins, fibronectin and vitronectin, which bind to the surface of the trophozoites and mediate the attachment to integrin receptors of the alveolar cells. 120 The subsequent events depend on the immune status of the host; the organism can remain quiescent for a long period of time, and then proliferate when the host becomes immunocompromised.l19 Other investigators suggest that the organisms remain in the lungs for only a short time, In an immunocompromised host, the organisms start to proliferate, and the alveoli become progressively filled with masses of the pneumocystis organisms. In animal models, P carinii organisms increase from <10 5 to 10 9 or 1010 in 8 to 10 weeks. It is presumed that P jiroveci replicates in a similar manner in humans. The proliferation of P jiroveci results in the accumulation of the typical foamy, honeycomb-like alveolar exudates. Although the type I alveolar cells appear vacuolated and eroded in infected tissues, there is no evident disruption of structure or barrier function in lung epithelial cell cultures. I21 43 There is increasing evidence to support the role of humoral immunity in host defense against pneumocystis. Passive immunization with specific monoclonal antibody is shown to confer partial protection against P carinii in animal models. 129 Pneumocystis infection invokes a cellular response with production of inflammatory cytokines and chemokines, These inflammatory responses are required to control the infection, but an exuberant inflammatory response may result in pulmonary injury. Complex interactions between the inflammatory cells and the soluble mediators produced by these cells facilitate the clearing of infection, but may result also in lung injury. The key inflammatory cells include CD4+ T lymphocytes, alveolar macrophages, and neutrophils. The complex interaction among the inflammatory cells, the soluble mediators, and the immune responses is presented in Figure 13 .4.

The activity of CD4+ T lymphocytes is pivotal in host defense against pneumocystis, in both humans and animals. A reduction in CD4+ T-lymphocyte count to less than 200 to produce cytokines and chemokines that are protective, but also induce lung injury. IFN-y, (interferon-y); IL-8, (interleukin-8); TNF-a, (tumor necrosis factor-a), Ell, cell recruitment and/or activation; *, produced by alveolar macrophages. pneumocystis antigens and generate cytokine mediators, including lymphotactin and interferon-y.l3l Lymphotactin is a chemokine that serves as a potent chemoattractant for lymphocyte recruitment. 132 Interferon-y activates macrophages to produce tumor necrosis factor-a (TNF-a), superoxides, and reactive nitrogen radicals. 133 . 134 Aerosolized interferon-yhas been shown to reduce pneumocystis infection in rats, regardless of CD4+ cell depletion.133 Macrophage-derived TNF-a and interleukin-1 (IL-1) are necessary for initiating pulmonary responses that are mediated by CD4+ T cells. An undesirable effect of T-Iymphocyte activation, however, is pulmonary injury and functional impairment. 132 Pneumocystis infection also results in a marked accumulation of CD8+ T lymphocytes in the lung. 133 Macrophages Alveolar macrophages are the principal cells mediating the uptake and degradation of pneumocystis in the lung, mediated either through the opsonins in the epitheliallining fluid, or through the macrophage mannose receptors that interact with the surface mannoprotein, glycoprotein A, of the pneumocystis. 135 Once phagocytosed, the organisms are incorporated into phagolysosomes and degraded. 136 In response to the phagocytosed pneumocystis organisms, a variety of inflammatory cytokines, chemokines, and eicosanoid metabolites are produced.136 These mediators, however, in addition to eradicating pneumocystis, also produce lung injury. The macrophage function is impaired in patients with AIDS and malignancies, resulting in impaired clearance of pneumocystis. 137

The production of TNF-a by alveolar macrophages is mediated by recognition of the ~-glucan component of the pneumocystis cell wall. 138 Macrophages possess several potential receptors for glucans, including CDllbl CD18 integrin (CR3), dectin-1, and toll-like receptor 2. 139 The activation of macrophages is facilitated by host vitronectin and fibronectin that bind glucan components on pneumocystis cell wall. 138 Tumor necrosis factor-a plays a significant role in the clearance of pneumocystis. It promotes the recruitment of neutrophils, lymphocytes, and monocytes, and induces the production of other cytokines and chemokines, including IL-8 and interferon-y, which stimulate further recruitment and activation of inflammatory cells. 140 ,141 Chemokines such as IL-8, macrophage-inflammatory protein-2, and the interferon-inducible protein of lOkDa are chemoattractants for neutrophils and important inflammatory mediators during pneumocystis infection. Interleukin-8 production is correlated with neutrophil 493 infiltration with release of reactive oxygen radicals, proteases, and cationic proteins, resulting in capillary endothelial and alveolar epithelial cell injury, in turn resulting in impaired gas exchange. Increased levels of IL-8 in BAL fluid are correlated with poor prognosis. 142

There are two major clinical forms of P.jiroveci pneumonia, each with a different epidemiologic pattern: (1) the infantile form, designated plasma cell interstitial pneumonia; and (2) the adult form, or pneumonia in immunocompromised host.

Plasma cell interstitial pneumonia, also called the "epidemic form," historically occurred in institutional settings in underdeveloped countries, mainly affecting premature, malnourished children. 11 • 143 ,144 The infection is characterized clinically by progressive respiratory distress in infants, and histologically by a prominent pulmonary interstitial plasma cell infiltrate. 143 ,I44 This disease has practically disappeared from the Western world, but is still found in developing countries. Clinically, the infants gradually develop respiratory difficulty characterized by tachypnea, cyanosis, cough, and progressive respiratory failure. The chest roentgenogram shows diffuse pulmonary infiltrates and focal or diffuse consolidation.

The adult infection, also called the "sporadic form," is one of the leading causes of fatal opportunistic infection in AIDS patients and other immunocompromised hosts. This infection is more frequent in the United States and other developed countries, and indicates an underlying cellular immune deficiency with impairment of CD4+ (T-helper) lymphocyte function. 145 Diseases that predispose to pneumocystis pneumonia include congenital immunodeficiency diseases, immunodeficiency induced by cytotoxic agents and corticosteroids, and other acquired immunodeficiency.6,146,147 Before the AIDS epidemic, most adult patients with pneumocystis either had acute leukemia or were leukopenic from chemotherapy.6,147 Now these patients are vastly outnumbered by AIDSassociated pneumocystis. The incidence of PCP has undergone a significant reduction with the institution of chemoprophylaxis. 148 At the beginning of the AIDS epidemic, PCP represented the most common AIDS index diagnosis, and the most life-threatening opportunistic infection, with an incidence of 44% to 74%.149 Approximately 80% of AIDS patients developed PCP during the course of their disease. 15o The incidence of PCP in HIVseropositive patients started to decrease in 1987, with only 16% of new index AIDS cases reported in 1993, almost exclusively due to the institution of chemoprophylaxis and antiretroviral therapy.149,151-157 In those individuals not receiving chemoprophylaxis, it still represents a serious pulmonary infection. 151 Rarely, PCP may develop in patients without a recognized predisposing illness. '58 The clinical and radiographic findings in sporadic P. jiroveci infection are not specific, and mimic those of other opportunistic infections. In AIDS patients, the onset of symptoms is more insidious. The fall in the circulating CD4+ T cell count usually correlates with the development of the pneumonia, with the vast majority of infections occurring when the CD4+ lymphocyte count is <200 cells. 100,15) The typical clinical presentation in HIVseropositive patients is an insidious onset, including fever, dyspnea, tachypnea, and nonproductive cough. 6 ,7,l59 Less frequently, weight loss, chest pain, night sweats, chills, fatigue, and malaise have been reported.'6O-'65 This is in contrast to the more acute presentation of HIV-seronegative immunocompromised patients. '66 Physical findings are often mild compared to the degree of symptomatic respiratory impairment. There is, however, a subset of HIV-seropositive patients that presents with an acute, fulminant onset, progressing to respiratory failure within 1 to 2 weeks.'60.l62,'67 The disease in non-AIDS patients may vary from an acute illness with an abrupt onset to a more indolent disease. Symptoms often start after corticosteroids are tapered or discontinued, and are present for a short time, 1 to 2 weeks before a diagnosis of pneumocystis pneumonia is established.

In general, patients with AIDS-associated PCP have significantly more pneumocystis organisms in their lungs, with fewer neutrophils, than those patients without AIDS. '66 The smaller number of inflammatory cells in AIDS-related pneumocystosis correlates with better oxygenation and survival compared with non-AIDS-related infection. The mortality rate in patients with AIDS is 10% to 20% during the initial infection, but the rate increases substantially with the need for mechanical ventilation. 168 Patients with non-AIDS-related pneumocystosis present with an abrupt onset of respiratory insufficiency that may correlate with a tapering or increased dose of immunosuppressive therapy. These patients have more neutrophils and fewer pneumocystis organisms in the lungs than do patients with AIDS-associated pneumocystis pneumonia. 166 The mortality rate in patients with non-AIDS-related pneumocystis pneumonia is 30% to 60%, with a greater risk of death among patients with cancer than among patients with transplants or other diseases such as connective tissue disorders. 4 , '69 Extrapulmonary Infection Extrapulmonary infection is being recognized with increasing frequency, and appears to correlate with the use of inhaled prophylactic pentamidine therapy.17o,171 A review of autopsy cases with AIDS shows an incidence of extrapulmonary pneumocystosis of 2.5 % to 2.8 % A.K. Haque and P.A. Adegboyega between 1980 and 1994. 172 -175 Concomitant pneumocystis pneumonia was present in 45% of patients. The most frequent spread of infection is to the hilar lymph node, followed by bone marrow, liver, spleen, and adrenal glands, followed by gastrointestinal tract, thyroid, genitourinary tract, heart, pancreas, eyes, and ears.174 Less frequently, skin, cerebral cortex, pituitary gland, and parathyroid glands are involved.I72,l75 Most patients who have disseminated extrapulmonary pneumocystosis have no specific symptoms. Some patients, however, may experience local pain or a mass; rarely, there is clinical and laboratory evidence of organ (e.g., renal, otic, or bone marrow) failure.172,l75 A case of digital vasospasm and necrosis secondary to microemboli containing pneumocystis organisms has been reported. 176 P.jiroveci probably spreads from the lungs by hematogenous or lymphatic routes to these sites.

Clinical manifestations of extra pulmonary pneumocystosis are variable, and may range from asymptomatic infection to end-organ failure.172,174 Computed tomograms may show low-attenuation lesions that progressively calcify in a rim-like or punctuate manner. 177 .l 78 Although chemoprophylaxis is considered a risk factor in extrapulmonary infection, only 50% of the patients had received the therapy in one series. 174 The detection, using PCR amplification, of P.jiroveci (carinii) DNA in the blood of patients with PCP provides supporting information in cases with extrapulmonary infection. 179,180 Pathologic findings in the extrapulmonary sites consist of the typical eosinophilic foamy material with a variable inflammatory infiltrate. The organisms can be demonstrated with the usual silver stains or immunoperoxidase stain. 37

Typical radiographic findings in PCP include bilateral diffuse interstitial and alveolar infiltrates, initially predominating in parahilar regions and lower lung fields, which may progress to extensive areas of air-space consolidation involving much of both lungs. The radiographic picture presents a combination of air-space consolidation with variable interstitial infiltrate. Atypical manifestations include unilateral and upper lobe infiltrates, nodular lesions, lobar consolidation, cavitation within a mass, localized nodule or consolidation, pneumatocele, pneumothorax, pneumomediastinum, subsegmental atelectasis, bronchiectasis,hilar and mediastinal lymphadenopathy, and pleural effusion. 18 l,l82 Classically, ground-glass perihilar interstitial infiltrates are seen in the early stages, which progress to involve all lung fields in untreated patients. The infiltrates may develop a coarse pattern with homogeneous consolidation and air bronchograms.183.l84 The lung apices are relatively spared, but the use of prophylactic inhaled pentamidine has resulted in involvement of the apices, presumably due to the relatively poor distribution of aerosol pentamidine to these regions. The radiographic features of PCP in those with and without HIV infection and with and without chemoprophylaxis are summarized elsewhere.185.186 As many as 10% to 20% of AIDS patients with PCP present initially with no detectable radiographic abnormalities. 186 In these cases, highresolution computed tomography may reveal extensive ground-glass attenuation or cystic lesions.

Recently, the phenomenon of immune reconstitution syndrome was described in a small group of patients who developed a pneumonic illness after treatment for pneumocystis pneumonia and subsequent highly active antiretroviral therapy (HAART) initiation. 187 During the second illness, none of these patients had an identifiable pathogen on bronchoscopy. The mean BAL CD4/CD8 ratio was 0.54, which was much higher than the ratio of 0.07 seen in a cohort of HIV-infected patients undergoing bronchoscopy for a variety of respiratory complaints. The authors suggested that this influx of CD4 cells might represent immune reconstitution. Another similar case was reported in which the BAL and tissue samples taken at the time of recurrent respiratory illness were negative on routine stains, but PCR confirmed the presence of pneumocystis. 1RR These reports suggest that subclinical infection with P. jiroveci can be present after clinical response to therapy and may trigger an inflammatory response upon reconstitution of the immune system.

The pathologic features of PCP have been described in patients with the epidemic or infantile infection, and more recently in the sporadic or adult infection. [189] [190] [191] [192] [193] Epidemic Pneumocystis Pneumonia (Infantile PCP) Interstitial plasma cell pneumonia, also known as the epidemic or infantile form of non-AIDS-related PCp, is rarely encountered in the United States. It has an intense plasmacytic and lymphocytic interstitial inflammation. 143 .l44 This morphologically nonspecific interstitial pneumonia resembles pneumonia associated with other opportunistic (especially viral) infections that are also prone to occur in premature, debilitated, or malnourished infants. Often, the correct diagnosis is suggested by the "honeycomb exudate" in alveolar spaces, but definitive 495 diagnosis requires demonstration of the pathogen with an appropriate special stain or immunohistochemical reagent (see Fig. 7 .42 in Chapter 7).143

Pathologic features of the sporadic P. jiroveci pneumonia, almost exclusively seen in immunocompromised adults, can be categorized in two major histopathologic groups: typical and atypical (Table 13 .1).

The typical or classical features are seen in the lungs of patients who are not on prophylactic treatment (HAART). Grossly, the lungs are heavy with a pale gray or tan, granular, firm, consolidated cut surface as seen in Figure  13 .5. Nodules and cavities are occasionally encountered. Microscopically, a variety of patterns have been described in patients with and without AIDS.

The typical histopathologic pattern in AIDS-associated PCP is characterized by a mild interstitial chronic inflammation and type II pneumocyte hyperplasia, associated with eosinophilic, foamy intra alveolar exudates that expand the alveolar spaces ( Fig. 13.6 ). At higher magnification, the exudate is punctuated with round basophilic dots that correspond to the nuclei of the sporozoites and trophozoites, and are best seen in tissue preserved with a good nuclear fixative, such as BS solution. 189 Recognition of the basophilic nuclear "dots" is helpful in the differential diagnosis of pneumocystis from alveolar edema and alveolar proteinosis, both with eosinophilic alveolar exudates, without any demonstrable nuclei. The alveolar exudate is composed predominantly of abundant P. jiroveci trophozoites admixed with cysts, membranotubular extensions, surfactant, and cellular debris enmeshed in fibrin. The alveolar exudate may be focal or diffuse, depending on the severity of infection. In formalin-fixed tissue, the exudate is often separated from the adjacent alveolar septa by an artifactual clear space due to retraction. The histologic appearance is so characteristic that a presumptive diagnosis of pneumocystis pneumonia can be made on hematoxylin and eosin (H&E)-stained slides, in the appropriate clinical setting. 19 The eosinophilic alveolar exudate stains with periodic acid-Schiff (PAS) stain. 194 Cysts are round to oval and focally curved disk-shaped or boat-shaped, and stain well with GMS or toluidine blue 0 (Fig. 13.7) . 194.195 Trophozoites are not seen on H&E stain, but easily can be seen in smears with Giemsa, Wright-Giemsa, or Diff-Quick stains (Table 13 .2). 196 In a progressive and chronic infection, interstitial and intraluminal fibrosis may be seen. One study reported interstitial and intraluminal fibrosis in 63% and 36% of lung biopsies, respectively.194.1 95 In acute progressive infection with respiratory failure, diffuse alveolar damage, A.K. Haque and P.A. Adegboyega hyaline membranes, and reactive epithelial cell proliferation may be seen. 189 ,196 Because P jiroveci cannot be cultivated on cell-free media in the clinical laboratory, the diagnosis of PCP depends on demonstration of the organisms in biopsy or cytology specimens,

Several studies have suggested that the histologic spectrum of pneumocystis pneumonia is much broader than the typical pattern described above. The first study that documented a broad spectrum of patterns was conducted before the onset of the AIDS epidemic; only about one third of the lung biopsy specimens showed the typical pattern of PCp ' 197 Notably, the foamy alveolar material was not found in approximately 50% of the specimens. Atypical findings included dense interstitial lymphocytic infiltrates, interstitial fibrosis, epithelioid granulomas, multinucleated giant cells, and focal calcifications (Table 13 .1). This spectrum of changes in PCP has been confirmed in subsequent studies. 192 . 197 In a study from the National Institutes of Health (NIH), the atypical manifestations reported were interstitial chronic inflammation (57%), fibrosis (50%), numerous alveolar macrophages with desquamative interstitial pneumonia-like pattern (9%) , granulomatous inflammation (5%), hyaline membranes (4%), interstitial pneumonitis (3%), cystic or cavitary lesions (2%), microcalcifications (2 %), and vascular infiltration and vasculitis (1 %). In 19% of the patients, alveolar exudates were absent, and in 2% there was a minimal histologic reaction. 194.195 Interstitial Chronic Inflammation

The presence of interstitial inflammation is the most common finding in PCP. The lymphocytic and plasma cell infiltrates within the alveolar septa are associated with mild expansion of the septa with type II pneumocyte hyperplasia. In one large series of biopsies of HIVpositive patients, an interstitial infiltrate was graded only as mild or moderate in nearly 90% of the patients. 195 Figure 13 .8 shows the classical features of interstitial chronic inflammation. If severe, the chronic infiltrate may be misinterpreted as lymphocytic interstitial pneumonia (LIP) or nonspecific interstitial pneumonia (NSIP), particularly if the typical foamy exudate is minimal. Intraalveolar cellular infiltrates are not a usual feature of active PCP, and when present, consist of macrophages that may contain intracytoplasmic pneumocystis cysts by silver stains. 189 The presence of numerous intraalveolar macrophages may simulate desquamative interstitial pneumonia (DIP) .197 Cysts have been described in the alveolar pneumocyte cytoplasm in the early phase of PCP. FIGURE 13.6. A. Low-magnification photomicrograph shows thickened alveolar septa with mild interstitial inflammation and fibrosis, lined by hyperplastic type II pneumocytes. The alveoli are filled with the typical foamy, eosinophilic exudate of Pneumocystis. B. Higher magnification shows an alveolus with the foamy/bubbly eosinophilic exudates and a few mononuclear cells. Basophilic dots are visible within the exudate. The alveolar septa have a few inflammatory cells and collagen, and are lined by hyperplastic type II pneumocytes. C. Immunostain demonstrates the cyst forms of Pneumocystis organisms, surrounded by inflammatory cells. The trophozoites also stain with the immunostain; however, these are not as clearly visualized as the cyst forms. This pattern of P. jiroveci pneumonia may complicate long-standing, treated, or recurrent PCp ' 190 ,192 Interstitial fibrosis may be seen in >50% of patients with chronic PCp ' 195 This usually presents as a mild thickening and fibrosis of the alveolar septa, with deposition of loose, edematous fibrous tissue, and without architectural distortion of the lung parenchyma ( Fig. 13.9A ). In some cases, alveolar intraluminal fibrosis consistent with organizing pneumonia may be seen (Fig. 13.10) . Many, if not most, of these cases are consistent with the organizing or proliferative phase of diffuse alveolar damage (DAD), which may also be the result of oxygen toxicity, concurrent viral infection, shock, or other recognized causes of DAD in HIV-positive patients, rather than injury by P. jiroveci (Fig. 13.9 ).

Diffuse alveolar damage may be seen both in AIDS patients and in other immunodeficient patients. [198] [199] [200] Respiratory failure as a terminal event is one of the leading causes of death in AIDS patients. In a study of 196 autopsied AIDS patients, 34% died of acute respiratory distress syndrome (ARDS), expressed histologically as DAD. P. jiroveci was the most common organism, identified in 55% of these patients. 201 Often, there is very little foamy material in the alveolar spaces, and the changes, therefore, may be misinterpreted as due to viral infection, oxygen toxicity, or a drug reaction unless an appropriate stain is used to demonstrate the cysts. The cysts may be quite sparse, and are usually found within the hyaline membranes lining the walls of alveolar ducts and alveoli (Fig. 13.llA-C) . Figure 13 .5 shows gross findings in the A.K. Haque and P.A. Adegboyega counterstain for pneumocytes and brown stain for Pneumocystis highlights the chronic interstitial inflammation. One of the alveolar walls is focally devoid of pneumocytes.

lung from a patient with DAD. Figure 13 .11 demonstrates the features of DAD in the lung shown in Figure 13 .5. The pneumocystis organisms are plastered along the alveolar walls within the hyaline membranes, and also present in the alveolar exudate (Fig. 13.llB) . Dual immunostain for pneumocystis and alveolar pneumocytes (cytokeratin AEl/3) shows invasion of the alveolar interstitium and destruction of type II pneumocytes by the pneumocystis organisms (Fig. 13 .SB). This finding may not be appreciated by the routine H&E or the usual single immunostain for pneumocystis.

Necrotizing PCP, seen mainly in patients with AIDS, is characterized by confluent parenchymal necrosis with lysis of alveolar septa. Grossly, parenchymal nodules and cavities ranging from 1 to 6 cm in diameter may be seen ( Fig. 13.12) . 189, 198 Histologically parenchyma is replaced by confluent eosinophilic foamy material in which there are abundant cysts of P. jiroveci (Fig. 13.13 ). Pneumocystis exudate infiltrates alveolar septa, a pattern referred to as "septal lysis," resulting in lung necrosis and cavity formation (Fig. 13.13 ).189 Rarely, parenchymal necrosis is associated with pneumocystis vasculitis, characterized by massive infiltration of blood vessel walls by characteristic foamy material accompanied by mononuclear inflammatory cells and mural necrosis (Fig. 13.14) . 202 The histogenesis of the cavitary lesions is not clear; however, pulmonary infarction due to vascular invasion by pneumocystis trophozoites is considered a possible mechanism. 202 Interestingly, about half of the patients with necrotizing pneumocystis pneumonia also have extra pulmonary pneumocystosis; vascular invasion is a possible mechanism of spread of infection. C. Immunostain for pneumocystis may also be used to demonstrate the cysts and trophozoites in the hyaline membranes. D. Photomicrograph of lung from a patient with long-standing pneumocystis infection shows enlarged and atypical type II pneumocytes with high nuclear cytoplasmic ratio, as seen in atypical alveolar hyperplasia. Other lesions associated with necrotizing PCP include poorly formed granulomas (Fig. 13.15 ) and microcalcifications ( Fig. 13.16 ), described in more detail below.

Thin-walled, pneumatocele-like cysts may be seen in PCP, either in the parenchyma or in the subpleural areas ( Fig. 13.17) . Initially the walls of the cysts may be formed by alveoli filled with pneumocystis exudates in necrotizing PCP. Chronic or healed cavities have a thin fibrous wall with focal chronic inflammation ( Fig. 13.17B,C) . Focal micro calcifications may be present in the cyst wall. The large subpleural cysts presumably develop from either rupture or confluence of smaller parenchymal cavities. 195 The cysts are usually empty, or contain a few macrophages and lymphocytes. Occasionally cysts may be colonized by organisms other than pneumocystis such as aspergillus or mycobacteria. Rupture of cavities leads to spontaneous pneumothorax. 203 

Microcalcifications are an important atypical feature of PCP, and may be seen with or without the foamy exudates. 204 Several patterns of calcification may be seen, including a "bubbly" pattern with vacuolations, a platelike pattern, an elongated pattern, and a conchoidal pattern; these patterns were described in a study of 13 patients with microcalcifications. 204 The authors postulate that the initial lesion is manifested by a form of long, thin calcifications that are always associated with active PCP. The "bubbly" plate-like calcifications are usually associated with focal interstitial fibrosis and are a later phase of calcification. All calcifications contained P. jiroveci cysts on GMS stain, and thus represent dystrophic calcification of degenerated P. jiroveci organisms. 204 The margins of cavitary lesions, as well as subpleural areas, may also develop microcalcifications. Figure 13 .16 shows plate-like and conchoidal calcifications in the lung of a patient with AIDS.

Vascular permeation is not a common feature of PCP; however, when present, it may be a marker of extrapulmonary dissemination of P. jiroveci. The vascular wall shows an eccentric expansion of the intima by the characteristic eosinophilic foamy exudate, which may also infiltrate the muscular layer of the vessel wall. Chronic inflammation may be associated with the mural exudate ( Fig. 13.14) . and necrotic exudate. C. Photomicrograph of cystic lesion shows no discernible lining. The cyst wall is composed of collapsed and fibrotic lung parenchyma. 

Granulomatous inflammation in response to pneumocystis infection has been described both in AIDS patients and in immunodeficient patients without AIDS. In some patients, it has been associated with a nodular or cavitary pattern on chest radiograph. 190.196.205.206 Frequently seen in necrotizing PCP, the granulomas are predominantly in alveolar spaces, and are usually loose and poorly formed, with or without necrosis (Fig. 13.15 ); see also ( Fig. 13.18 ). 205.206.207 An even less frequent pattern of granulomatous PCP is that of a solitary nodule or a few discrete fibrocaseous lesions, similar radiographically and histologically, to tuberculomas or histoplasmomas. 210 . 211 Cyst forms may be sparse in the center of these well-formed necrotizing granulomas. An immunohistochemical stain for pneumocystis is helpful in locating sparse cyst forms and distinguishing them from histoplasma organisms (Fig. 13.18D) .

Granulomatous PCP has been particularly associated with dissemination of pneumocystis to extra thoracic viscera. 21o • 211 It is speculated that granulomatous PCP occurs in patients who are marginally, rather than severely, immunosuppressed.192.l98 Because granulomatous reactions are infrequent in pneumocystis infection, every effort should be made to exclude other pathogens such as fungi and mycobacteria using special stains. FIGURE 13.19 . Photomicrograph shows Pneumocystis in the alveolar spaces and cytomegalovirus inclusions in the surrounding type II pneumocytes.

This is an infrequent manifestation of PCP. In some patients, lymphadenopathy is caused by underlying disease (e.g., lymphoma or leukemia) or reactive hyperplasia. In one patient, an intravenous drug abuser, enlarged lymph nodes contained typical foamy masses of P jiroveci similar to those found in the alveolar spaces. 212 This patient also had disseminated pneumocystosis.

A PCP infection in patients with AIDS is often associated with other infections, cytomegalovirus infection being one of the commonest. Figure 13 .19 shows both infections in the same alveolus in a patient who died with AIDS.

The effects of therapy on pathologic features of pneumocystis pneumonia were studied in patients who died of fulminant infection following therapy.213 Patients who died within the first week of therapy showed compact alveolar exudates that lacked the foamy appearance of typical infection, with fewer stainable organisms as compared to pretreatment biopsy. A second group of patients who died from 8 days to 8 weeks following therapy, had features of organizing DAD; however, all patients were treated with high concentration of oxygen. A third group of patients who responded to therapy and were considered to be cured of infection showed no residual pneumocystis organisms when autopsied 6 months to 2 years later. This study suggests that chronic interstitial lung disease does not develop as a result of pneumocystis infection in long-term survivors.

A.K. Haque and P.A. Adegboyega P jiroveci can persist in the lungs of AIDS patients following therapy, even when there is a good clinical response. Pneumocystis cysts can persist in the lungs for up to 5 weeks or more following treatment with sulfa drugs. The viability of the trophozoites is not known, but if viable they may cause recurrent infection. Since cyst forms of P jiroveci are known to persist for several weeks, the interpretation of repeat biopsy or cytology during this period should be cautious. There is also a case report that indicates preferential elimination of the cysts and persistence of trophozoites on treatment. In such cases, silver stain can be falsely negative, and immunohistochemical stains or electron microscopy should be used for diagnosis, if clinical suspicion of recurrence is high.214

Extrapulmonary disease is seen primarily in patients with overwhelming pulmonary infection, those with severe underlying immunodeficiency, and those (particularly HIV-infected patients) who received aerosolized pentamidine for prophylaxis against PCp'lR9. 215 Since 1982 there have been increasing reports of extra pulmonary dissemination of pneumocystis in AIDS patients receiving aerosol pentamidine. 176 ,215 Sites of extrapulmonary dissemination include lymph nodes, skin, ears, eyes, bone marrow, spleen, liver, kidneys, thyroid gland, gastrointestinal tract, and a variety of other organs and tissues. 189 The lesions of extrapulmonary pneumocystosis are gray-white or yellow nodules 0.5 to 4.9 mm or larger, and they resemble micro abscesses. Histologically, the lesions are often angiocentric with eosinophilic foamy exudates similar to the ones seen in the lungs, and show abundant cyst forms on GMS stain. An inflammatory response is minimal or none, and when present it consists of histiocytes, lymphocytes, plasma cells, neutrophils, giant cells, and fibroblasts. In most cases the lesions gradually enlarge and contain all the developmental forms of pneumocystis, suggesting replication in anaerobic extrapulmonary sites. The characteristic morphology of the cysts on GMS stain and immunohistochemical stain is sufficient for making a diagnosis. Microscopically, the lesions consist of sheets of eosinophilic, foamy material identical to that described previously, composed of cysts and trophozoites, with little or no inflammatory response. l96 Smaller lesions and satellite nodules may have an angiocentric distribution.

The ultrastructural pathology of PCP is mainly of pathogenetic interest. 65 . 189 Ultrastructural studies have shown that the single or paired argyrophilic "intracystic bodies" are plaquelike thickenings of the cyst wall rather than sporozoites or other organelles. 189 Such studies also have confirmed that P. jiroveci is strictly an extracellular pathogen. Trophozoites attach to the surface of type 1 pneumocytes by specialized interdigitations, without membrane fusion or communication between cytoplasmic compartments. 216 . 217 Trophozoite attachment may eventually cause epithelial and endothelial damage, resulting in acute diffuse alveolar damage.218

The cytologic features of PCP are well described and illustrated in the literature. 219 ,22o Cytologic examination of induced sputum and BAL specimens has become the primary diagnostic modality for PCP, particularly in an HIV-infected patient population in whom the sensitivity of cytodiagnosis has been shown to be reasonably high.220 In Papanicolaou-stained smears, P. jiroveci is seen as aggregates of cysts and trophozoites with a granular, foamy, or honeycomb appearance (Fig. 13.20) . These aggregates are usually spherical, with considerable depth of focus, and approximately the size of a normal alveolar sac. 219 Sometimes referred to as "alveolar casts," these aggregates can be recognized at screening magnification if present in sufficient quantity. However, the diagnosis should be confirmed with GMS stain. The honeycomb aggregates in Papanicolaou-stained smears are fluorescent when viewed with ultraviolet light and a fluorescence microscope 221 ; however, this method is no more sensitive or specific than GMS stain. 221 ,222 Also, the fluorescent method is not readily available in all laboratories, especially in those in the developing world.

Pneumocystis jiroveci can be demonstrated by special histochemical, fluorescent, and immunohistochemical stains. The staining features of P. jiroveci are presented in Table  13 .2. The easiest and most reliable stain is GMS, which stains the cysts in tissue sections, smears, and cytologic preparations. The cysts are round to oval, 4 to 7!lm in maximum dimension, and often have collapsed crescent and helmet forms, as seen in Figures 13.6 and 13.7. An intracystic density "central dot" or "paired comma structures" due to a plaque-like thickening of the cyst wall is an important diagnostic feature of pneumocystis. 223 The alveolar foamy exudate is composed of trophozoites and sporozoites, which appear as basophilic dots on H&Estained tissue sections, and can be seen in smears (but not tissue sections) by Romanowsky stains (Giemsa, Wright, Diff-Quick), and Gram and methylene blue stains.

Chemofluorescent techniques such as Calcofluor white highlight the cysts, but offer no advantage over GMS stain. 221 ,224 Pneumocystis jiroveci pneumonia is not autofluorescent, unlike other fungi.221 Fluorescent antibody techniques, although specific, are time-consuming, require a fluorescent microscope, and do not allow visualization of morphologic details. 225 A variety of monoclonal antibodies are available commercially to detect and confirm the identity of P. jiroveci in routinely processed biopsy and cytology specimens. 226 These antibodies, which are specific and sensitive, identify both the cyst and trophozoite forms of P. jiroveci. For this reason, immunohistochemistry is considered to be more sensitive than GMS for the detection of P. jiroveci in clinical specimens. 227 ,22s The immunostain is particularly useful for the examination of induced sputum and BAL specimens, which may contain abundant argyrophilic mucus and debris that can obscure the organisms, and for specimens from treated patients. 226 ,229 An immunohistochemical stain provides a definitive diagnosis, and is more sensitive than GMS, especially in treated and extra pulmonary pneumocystis infection.23o.231 

Although a large number of pathogenic microbes are stained with GMS, it is the yeast-like fungi, particularly Histoplasma capsulatum, Candida glabrata, and Cryptococcus neoformans, that are most difficult to distinguish from the cysts of P. jiroveci. 189 The comparative features of these organisms are presented in Table 13 .3. The recognition of the single or paired, comma-shaped argyrophilic foci in the walls of P. jiroveci cysts is most important, since structures with this configuration are never seen in the walls of yeast. Production of blastoconidia by the process of external budding is a characteristic feature of yeast, but the cysts of P. jiroveci do not reproduce by budding. Cryptococcus neoformans has a mucopolysaccharide capsule that is mucicarmine positive and Fontana-Masson (melanin) stain positive (even in the capsule deficient organisms), which distinguishes it from cysts of P. jiroveci. The yeast forms of Candida spp. are usually accompanied by mycelial elements (pseudohyphae and true hyphae), which are not a feature of pneumocystis.

The intracytoplasmic inclusions of cytomegalovirus (CMV) are argyrophilic in GMS-stained sections.232 However, they can be distinguished easily from pneumocystis cysts, because the viral inclusions are always located inside cells, and they "keep company" with typical Cowdry type A intranuclear inclusions, seen best in replicate sections stained with H&E. The cyst wall of Toxoplasma gondii, like that of P. jiroveci, is argyrophilic with GMS.

The cysts of 1: gondii, however, are many times larger than those of P. jiroveci and are clearly visible in H&Estained sections, unlike the cysts of P. jiroveci. Finally, tissue elements such as leukocytes, erythrocytes, and mucous vacuoles are weakly argyrophilic and may complicate both screening and diagnosis in slides that are overstained with GMS.189 For this reason, a positive + +, including capsule deficient forms +, usually (gram positive) control slide known to contain cyst forms of P. jiroveci must be stained in parallel with unknown histologic sections and cytology smears, to gauge the intensity of silver staining.

Amplification of P. jiroveci DNA by PCR recently has been used for the diagnosis of P. jiroveci in induced sputum and BAL specimens, and reported to have greater sensitivity over standard methods (special stains and immunohistochemistry) for the detection of P. jiroveci. m - 235 In a study of 29 patients with suspected PCp, PCR of P. jiroveci DNA in induced sputum was evaluated, and it was significantly more sensitive than cytology (54.5% positive versus 4.5% positive).236 A quantitative real-time PCR assay for detection and quantitation of P. jiroveci in 53 BAL specimens based on the probe targeting the gene encoding ~-tubulin compared to immunofluorescence microscopic examination and Giemsa stain showed that all PCR-negative samples were negative by microscopy. Among the PCR-positive BAL specimens, only 35% were positive by microscopy.237 Polymerase chain reaction for detection of P. jiroveci appears to be a useful and noninvasive method that has high sensitivity and specificity for early diagnosis of PCp' 238

Pneumocystis jiroveci can be sustained in cell culture on a limited number of celllines. 239 ,24o To date, no artificial media support the growth of P. jiroveci. Serologic methods to detect antibodies to P. jiroveci are regarded as insensitive, mainly because many patients with PCP are unable to mount a detectable humoral response. 241 ,242

Therefore, demonstration of cysts or trophozoites with an appropriate special stain or by immunohistochemistry remains the only dependable way to establish the diagnosis of pneumocystosis.

The prevention and treatment of pneumocystosis are rapidly evolving controversial subjects that are currently the focus of considerable investigational interest. Two standard drugs used to treat most patients with PCP are trimethoprim-sulfamethoxazole (TMP-SMX) and pentamidine isothionate. 243 They appear to be equally effective and are associated with approximately the same incidence of adverse reactions, which may affect as many as 60% of patients with AIDS.243 Early adjunctive therapy with corticosteroids can reduce the morbidity and mortality associated with moderate or severe PCp '243-246 Patients at high risk for PCp, specifically HIV-infected patients with CD4+ T-Iymphocyte counts of less than 200 cells/mm\ and all AIDS patients who have already had one or more episodes of PCP received prophylaxis with TMP-SMX and aerosolized pentamidine. 247 • 248 The latter regimen, however, may predispose to extrapulmonary pneumocystosis. 248 The recent introduction of combination antiretroviral therapy, including HAART, have contributed to the reduced incidence of PCP. An investigational drug, 566CBO, used in mild to moderately severe episodes of PCP in AIDS patients has shown promising results. 249 Other drugs under development include echinocandins and pneumocandins, which inhibit ~-glucan synthesis, or sordarins, which inhibit fungal protein synthesis.

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Transmission of Pneumocystis carinii from AIDS patients to other immunosuppressed patients: a cluster of Pneumocystis carinii pneumonia in renal transplant patients

An outbreak of Pneumocystis carinii pneumonia at a pediatric hospital

An outbreak of Pneumocystis pneumonia in children with acute lymphocytic leukemia

Factors in an outbreak of Pneumocystis carinii in a transplant unit

Pneumocystis carinii pneumonia: a cluster of eleven cases

Serologic responses to specific Pneumocystis carinii antigens in health and disease

Characterization of Pneumocystis carinii antigen in bronchoalveolar lavage fluid by Western blot

Development of a yeast assay for rapid screening of inhibitors of human-derived Pneumocystis carinii dihydrofolate reductase

Multiple genes encode the major surface glycoprotein of Pneumocystis carinii

Further observations on the ultrastructure of Pneumocystis

Pneumocystis carinii in lungs of rats treated with cortisone acetate: ultrastructural observations relating to the life cycle

Ultrastructure of Pneumocystis in human lung: life cycle in human pneumocystosis

The biology of Pneumocystis carinii

Ultrastructure of Pneumocystis carinii: a review of internal and surface characteristics

Sporogony in Pneumocystis carinii: synaptonemal complexes and meiotic nuclear divisions observed in precysts

Pneumocystis carinii pneumonia: a light microscopical and ultrastructural study

Analysis of the developmental stages of Pneumocystis carinii, in vitro

Atlas of cell morphology

Studies of Blastocystis hominis

Freeze-fracture studies on Pneumocystis carinii. 1. Structural alterations of the pellicle during the development trophozoite to cyst

Detection of surface carbohydrates on Pneumocystis carinii by f1uorescein-conjugated lectins

Analyses of Pneumocystis fatty acids

Yeast glucan in the cyst wall of Pneumocystis carinii

Properties of the major rat and human Pneumocystis carinii antigens

Ultrastructural detection of carbohydrates in the pellicle of Pneumocystis carinii

Ultrastructural study of Pneumocystis carinii in explant cultures of rabbit lung and in cultures with and without feeder cells

Developmental biology of Pneumocystis carinii, an alternative view on the life cycle of the parasite

Pneumocystis carinii pneumonitis: the nature and diagnostic significance of the methenamine silver-positive "intracystic bodies

Rapid methenamine silver stain for Pneumocystis and fungi

Modified Grocott's methenamine silver nitrate method for quick staining of Pneumocystis carinii

Ten-minute silver stain for Pneumocystis carinii and fungi in tissue sections

Rapid metallic histological staining using the microwave oven

A simple and reliable rapid methenamine silver stain for Pneumocystis carinii and fungi

Identification of Pneumocystis carinii chromosomes and mapping of five genes

Pneumocystis carinii karyotypes

Pneumocystis carinii: sequence from ribosomal RNA implies a close relationship with fungi

Isolation and expression of Pneumocystis carinii thymidylate synthase gene

Santi DV Isolation and expression of the Pneumocystis carinii dihydrofolate reductase gene

Pneumocystis jeroveci dihydropteroate synthase genotypes in French patients with pneumocystosis: a 1998-2001 prospective study

Severity and outcome of HIV-associated pneumocystis pneumonia containing Pneumocystis jiroveci dihydropteroate synthase gene mutations

Increase in prevalence of Pneumocystis carinii mutations in patients with AIDS and P. carinii pneumonia, in the United States and China

Pneumocystis jiroveci in Portuguese immunocompromised patients: association of specific ITS genotypes with treatment failure, bad clinical outcome and childhood

Development and characterization of monoclonal antibodies to Pneumocystis carinii

Identification of antigens and antibodies specific for Pneumocystis carinii

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

Monoclonal antibodies to Pneumocystis carinii: identification of specific antigens and characterization of antigenic differences between rat and human isolates

Parasitologic and serologic observations of infection with Pneumocystis in humans

The interaction in vitro of Pneumocystis carinii with macrophages and L-cells

The risk of Pneumocystis carinii pneumonia among men infected with human immunodeficiency virus type I

Pulmonary complications of HIV infection: autopsy findings

Development of murine monoclonal antibodies to Pneumocystis carinii

Carbohydrate recognition domain of surfactant protein D mediates interactions with Pneumocystis carinii glycoprotein A

Pneumocystis carinii glycoprotein A binds macrophage mannose receptors

Cell wall assembly by Pneumocystis carinii: evidence for a unique gsc-1 subunit mediating beta-1,3-glucan deposition

Treatment of Pneumocystis carinii pneumonia with 1,3-beta-glucan synthesis inhibitors

Efficacy of MK-991 (L-743,872), a semisynthetic pneumocandin, in murine models of Pneumocystis carinii

Isolated Pneumocystis carinii cell wall glucan provokes lower respiratory tract inflammatory responses

Pneumocystis carinii cell wall beta-glucan induces release of macrophage inflammatory protein-2 from alveolar epithelial cells via a lactosylceramidemediated mechanism

Pneumocystis carinii contains a functional cell-division-cycle Cdc2 homologue

Differential regulation of growth and checkpoint control mediated by a Cdc25 mitotic phosphatase from Pneumocystis carinii

Pneumocystis carinii uses a functional cdc13 B-type cyclin complex during its life cycle

Complementation and characterization of the Pneumocystis carinii MAPK

Lung epithelial cells and extracellular matrix components induce expression of Pneumocystis carinii STE20, a gene complementing the mating and pseudohyphal growth defects of STE20 mutant yeast

Pneumocystis carinii STEll, an HMG-box protein, is phosphorylated by the mitogen activated protein kinase PCM

The ste3 pheromone receptor gene of Pneumocystis carinii is surrounded by a cluster of signal transduction genes

Characterization of a lanosterol 14 alpha-demethylase from Pneumocystis carinii

The Pneumocystis carinii drug target S-adenosyl-Lmetionine:sterol C-24 methyl transferase has a unique substrate preference

Attachment of microbes to host cells: relevance of Pneumocystis carinii

Vitronectin binds to Pneumocystis carinii and mediates organism attachment to cultured lung epithelial cells

Interaction of rat Pneumocystis carinii and rat alveolar epithelial cells in vitro

Pneumocystis carinii inhibits cyclin-dependent kinase activity in lung epithelial cells

Surfactant protein D interacts with Pneumocystis carinii and mediates organism adherence to alveolar macrophages

Inhibition of lung surfactant protein B expression during Pneumocystis carinii pneumonia in mice

Human surfactant protein A enhances attachment of Pneumocystis carinii to rat alveolar macrophages

Surfactant protein-A reduces binding and phagocytosis of Pneumocystis carinii by human alveolar macrophages in vitro

Surfactant protein D-mediated aggregation of Pneumocystis carinii impairs phagocytosis by alveolar macrophages

Pulmonary inflammation disrupts surfactant function during Pneumocystis carinii pneumonia

Passive immunoprophylaxis with specific monoclonal antibody confers partial protection against Pneumocystis carinii pneumonitis in animal models

A new model of Pneumocystis carinii infection in mice selectively depleted of helper T lymphocytes

Lymphocytes in host defense against Pneumocystis carinii

Chemokine gene expression during Pneumocystis carinii-driven pulmonary inflammation

Reduction in intensity of Pneumocystis carinii pneumonia in mice by aerosol administration of gamma interferon

Pneumocystis jiroveci Pneumonia

Gamma interferon stimulates rat alveolar macrophages to kill Pneumocystis carinii by L-arginine-and tumor necrosis factor-dependent mechanisms

Uptake of Pneumocystis carinii mediated by the macrophage mannose receptor

The role of macrophages in Pneumocystis carinii degradation and clearance from the lung

Reduced binding and phagocytosis of Pneumocystis carinii by alveolar macrophages from persons infected with HIV-l correlates with mannose receptor down regulation

Vitronectin and fibronectin function as glucan binding proteins augmenting macrophage responses to Pneumocystis carinii

Immune recognition: a new receptor for beta-glucans

Pneumocystis carinii stimulates tumor necrosis factor-alpha release from alveolar macrophages through a beta-glucan-mediated mechanism

Importance of endogenous tumor necrosis factor alpha and gamma interferon in host resistance against Pneumocystis carinii infection

Prognostic value of interleukin-8 in AIDS-associated Pneumocystis carinii pneumonia

Endemic infantile Pneumocystic carinii infection: the Shiraz study

Pneumocystis carinii infection and interstitial plasma cell pneumonia: what does history teach?

Pneumocystis carinii pneumonitis

Pneumocystis carinii infection

Pneumocystis carinii pneumonia among patients without AIDS at a cancer hospital

Effect of aerosolized pentamidine prophylaxis on the diagnosis of Pneumocystis carinii pneumonia by induced sputum examination in patients infected with the human immunodeficiency virus

Pneumocystis carinii infection in the HIV-seropositive patient

Pneumocystis carinii pneumonia: diagnosis

Clinical manifestations of AIDS in the era of Pneumocystis prophylaxis

Changes in survival after acquired immunodeficiency syndrome (AIDS): 1984-1991

Natural mode of acquisition for de novo infection with Pneumocystis carinii

The changing spectrum of AIDS index diseases in Canada

Trends in the incidence of outcomes defining acquired immunodeficiency syndrome (AIDS) in the multicenter AIDS cohort study: 1985-1991

Danish AIDS patients 1988-1993: a recent decline in Pneumocystis carinii pneumonia as AIDS-defining disease related to the period of known HIV positivity

Changing incidence of Pneumocystis carinii pneumonia as initial AIDS defining disease in the United Kingdom

A cluster of Pneumocystis carinii pneumonia in adults without predisposing illnesses

Pneumocystis carinii pneumonia in the patient with AIDS

Clinical features of Pneumocystis pneumonia in the acquired immune deficiency syndrome

Pulmonary disease in the acquired immune deficiency syndrome

Early predictors of in-hospital mortality for Pneumocystis carinii pneumonia in the acquired immunodeficiency syndrome

The diagnosis of Pneumocystis carinii pneumonia in patients with the acquired immunodeficiency syndrome using sub segmental bronchoalveolar lavage

Comparison of biopsy-proven Pneumocystis carinii pneumonia in acquired immune deficiency syndrome patients and renal allograft recipients

Pulmonary manifestations of the acquired immunodeficiency syndrome (AIDS)

Pneumocystis carinii pneumonia. Differences in lung parasite number and inflammation in patients with and without AIDS

Clinical spectrum of infections in patients with HTLV-Ill-associated diseases

Non-invasive tests in the diagnostic evaluation of Pneumocystis carinii pneumonia in patients with or suspected of having AIDS

Use of adjunctive corticosteroids in severe adult non-HIV Pneumocystis carinii pneumonia

Aerosolized pentamidine for prophylaxis against Pneumocystis carinii pneumonia. The San Francisco community Prophylaxis Trial

Widespread dissemination of Pneumocystis carinii infection in a patient with acquired immunodeficiency syndrome receiving long-term treatment with aerosolized pentamidine

Disseminated Pneumocystis carinii infection causing extrapulmonary organ failure: clinical, pathologic, and immunohistochemical analysis

Extrapulmonary pneumocystosis: the first 50 cases

Extrapulmonary infection and other unusual manifestations of Pneumocystis carinii

Extrapulmonary Pneumocystis carinii infections

Digital necrosis and disseminated Pneumocystis carinii infection after aerosolized pentamidine prophylaxis

Widespread visceral calcifications in disseminated Pneumocystis carinii infection: CT characteristics

Extrapulmonary Pneumocystis carinii infections in AIDS: CT findings

Improved diagnosis of Pneumocystis carinii infection in AIDS: CT findings

Application of DNA amplification to Pneumocystosis: presence of serum Pneumocystic carinii DNA during human and experimentally induced Pneumocystis carinii pneumonia

Pneumocystis carinii pneumonia: spectrum of parenchymal CT findings

Radiology of community-acquired pneumonia

Pulmonary complications of AIDS: Radiologic features

Radiographic features in patients with pulmonary manifestations of the acquired immune deficiency syndrome

Aerosolized pentamidine: effect on diagnosis and presentation of Pneumocystis carinii pneumonia

Atypical roentgenographic manifestations of Pneumocystis carinii pneumonia

Immune reconstitution pneumonitis following Pneumocystis carlllll pneumonia in HIV-infected subjects

Immune reconstitution syndrome after successful treatment of Pneumocystis carinii pneumonia in a man with human immunodeficiency virus type 1 infection

Evolving concepts of infection by Pneumocystis carinii

Spectrum of pathologic manifestations of Pneumocystis carinii pneumonia in patients with neoplastic diseases

Histopathology of Pneumocystis carinii infestation and infection in malignant disease in childhood

Pneumocystis carinii infection in the acquired immunodeficiency syndrome

Pneumocystis carinii pneumonitis: the nature and diagnostic significance of methenamine silver-positive "intracystic bodies

Pneumocystis carinii pneumonia

Atypical pathologic manifestations of Pneumocystis carinii pneumonia in the acquired immune deficiency syndrome. Review of 123 lung biopsies from 76 patients with emphasis on cysts, vascular invasion, vasculitis, and granulomas

Pulmonary pathology in AIDS: atypical Pneumocystis carinii infection and lymphoid interstitial pneumonia

Lung biopsy in Pneumocystis carinii pneumonia: a histopathologic study of typical and atypical features

Cavitation and other atypical manifestations of Pneumocystis carinii pneumonia

Pneumocystis infection masquerading as diffuse alveolar damage: a potential source of diagnostic error

Pathologic features of the lung in the acquired immune deficiency syndrome (AIDS): an autopsy study of seventeen homosexual males

A clinicopathologic study of adult respiratory distress syndrome in human immunodeficiency virus-infected patients

Necrotizing Pneumocystis carinii vasculitis associated with lung necrosis and cavitation in a patient with acquired immunodeficiency syndrome

Thin-walled cavities, cysts and pneumothorax in Pneumocystis carinii 13. Pneumocystis jiroveci Pneumonia pneumonia: further observations with histopathologic correlation

Pulmonary calcification caused by Pneumocystis carinii pneumonia. A clinicopathological study of 13 cases in acquired immune deficiency syndrome patients

Granulomatous Pneumocystis carinii pneumonia in three patients with the acquired immune deficiency syndrome

Granulomatous pulmonary lesions in patients with the acquired immunodeficiency syndrome (AIDS) and Pneumocystis carinii infection

Granulomatous Pneumocystis carinii pneumonia in patients with malignancy

Granulomatous Pneumocystis carinii pneumonia complicating hematopoietic cell transplantation

Granulomatous Pneumocystis carinii pneumonia mimicking tuberculosis

Granulomatous pneumocystosis presenting as a solitary pulmonary nodule

Leonidas 1. Pneumocystis carinii pneumonia presenting as a single pulmonary nodule

Pneumocystis carinii pneumonia complicated by lymphadenopathy and pneumothorax

The pathology of treated Pneumocystis carinii infection and lymphoid interstitial pneumonia

Persistence of trophozoites after successful treatment of Pneumocystis carinii pneumonia

Fatal, disseminated pneumocystosis in a patient with acquired immunodeficiency syndrome receiving prophylactic aerosolized pentamidine

Attachment of Pneumocystis carinii to rat pneumocytes

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

Mechanism of pulmonary alveolar injury in experimental Pneumocystis carinii pneumonia in the rat

The recognition of Pneumocystis carinii in Papanicolaou-stained smears

Rapid cytologic diagnosis of Pneumocystis: a comparison of effective techniques

Skolom 1. Fluorescence of Pneumocystis carinii in Papanicolaou smears

Evaluation of calcofluor white stain for detection of Pneumocystis carinii

Pathology of infectious diseases

Calcofluor white stain for the detection of Pneumocystis carinii in transbronchial lung biopsy specimens: a study of 68 cases

The use of an indirect fluorescent antibody test for detecting Pneumocystis carinii

Immunohistochemistry of Pneumocystis carinii

Monoclonal immunofluorescence compared with silver stain for investigating Pneumocystis carinii pneumonia

Monoclonal antibody to Pneumocystis carinii: comparison with silver stain in bronchial lavage specimens

Detection of Pneumocystis carinii: comparative study of monoclonal antibody and silver staining

Detecting Pneumocystis carinii by immunohistochemistry

Immunohistochemistry of Pneumocystis carinii infection

Staining qualities of cytomegalovirus inclusions in the lungs of patients with the acquired immunodeficiency syndrome

Detection of Pneumocystis carinii with DNA amplification

DNA amplification on induced sputum samples for diagnosis of Pneumocystis carinii pneumonia

Improved diagnosis of Pneumocystis carinii infection by polymerase chain reaction on induced sputum and blood

Detection of Pneumocystis carinii by DNA amplification in patients with connective tissue diseases: re-evaluation of clinical features of P carinii pneumonia in rheumatic diseases

Quantitative TaqMan PCR for detection of Pneumocystis jiroveci

Correlation of morphologic diagnosis of Pneumocystis carinii with the presence of pneumocystis DNA amplified by the polymerase chain reaction

Propagation of Pneumocystis carinii in vitro

Growth and serial passage of Pneumocystis carinii in the A549 cell line

Evaluation of serologic tests for Pneumocystis carinii antibody and antigenemia in patients with acquired immunodeficiency syndrome

Diagnosis of Pneumocystis carinii pneumonia

Ruskin 1. Trimethoprimsulfamethoxazole compared with pentamidine for treatment of Pneumocystis carinii pneumonia in the acquired immunodeficiency syndrome

Pneumocystis carinii pneumonia: therapy and prophylaxis

New developments in the treatment of Pneumocystis carinii pneumonia

Consensus statement on the use of adjunctive therapy for pneumocystis pneumonia in the acquired immunodeficiency syndrome

Recommendations for prophylaxis against Pneumocystis carinii pneumonia for adults and adolescents infected with human immunodeficiency virus

Disseminated pneumocystosis without pulmonary involvement during prophylactic aerosolized pentamidine therapy in a patient with the acquired immunodeficiency syndrome

A preliminary evaluation of 566CBO for the treatment of pneumocystis pneumonia in patients with the acquired immunodeficiency syndrome