Pneumocystis and Other Less Common Fungal Infections

History

In 1909 in Brazil, Chagas first described the morphologic forms of Pneumocystis in the lungs of guinea pigs infected with Trypanosoma cruzi. He believed the forms to represent a sexual stage in the life cycle of the trypanosome and not a different organism. Carini, an Italian working in Brazil, saw the same organism-like cysts in the lungs of rats experimentally infected with Trypanosoma lewisi. His slide material subsequently was reviewed by P. and M. Delanoë and their colleagues at the Pasteur Institute in Paris. They recognized that these alveolar cysts were present in the lungs of local Parisian sewer rats and thereby established that the “organisms” were independent of trypanosomes. They proposed the name Pneumocystis carinii for the new species.

At about this time, Chagas may have unwittingly described the first human case of pneumocystosis when he reported the presence of similar organisms in the lungs of a patient with interstitial pneumonia who had died of American trypanosomiasis. Nevertheless, no definite etiologic connection was made between P. carinii and human pneumonic disease for another 30 years. The reason for this delay was the belief during this period that infantile syphilis was responsible for virtually all instances of interstitial plasma cell pneumonia. In 1938, Benecke and Ammich identified a histologically similar pneumonic illness in nonsyphilitic children that was characterized by a peculiar honeycombed exudate in alveoli. Subsequent scrutiny of photomicrographs in their reports revealed the presence of Pneumocystis organisms, but it was not until 1942 that Van der Meer and Brug, in the Netherlands, unequivocally recognized the organism in lungs from two infants and one adult. The first epidemics of interstitial plasma cell pneumonia were reported shortly thereafter among premature debilitated babies in nurseries and foundling homes in central Europe. In 1952, Vanek and Jirovec in Czechoslovakia provided the most convincing demonstration of the etiologic relationship of Pneumocystis to this disease in an autopsy study of 16 cases.

Pneumocystosis was first brought to the attention of pediatricians in the United States in 1953 by Deamer and Zollinger, who reviewed the pathologic and epidemiologic features of the European disease. In 1957, Gajdusek presented an in-depth perspective on the history of the infection that included an extensive bibliography. This review was particularly timely because the next decade was to see the disturbing emergence of P. carinii pneumonia in the Western world—even while the epidemic disease in central Europe was waning—to the degree that it would become preeminent among the so-called opportunistic pulmonary infections in the immunosuppressed host. In 1988, DNA analysis demonstrated that Pneumocystis was not a protozoan but a fungus. Most recently, a change in nomenclature from P. carinii to P. jirovecii, a name chosen in honor of the parasitologist Otto Jirovec, who now is credited by some with the original description of this organism, has been put forth. The rationale for this change is the unique antigenic, genetic, and restricted infectivity profile of the Pneumocystis organisms associated with each mammalian species.

The Organism

The taxonomic status of P. jirovecii as a fungus has been defined on the basis of molecular analysis. Because the organism cannot be adequately propagated in vitro, efforts to classify it and to elucidate its structure and life cycle have been based exclusively on morphologic observations of infected lungs from animals and humans. The earliest of these investigations was performed by parasitologists; in accordance, the terminology applied to the forms of Pneumocystis seen in diseased tissue has been that reserved for protozoal organisms.

Three developmental forms of this presumably unicellular microbe have been described: a thick-walled cyst, an intracystic sporozoite (intracystic body), and a thin-walled trophozoite (trophic form). The form of Pneumocystis that assists with diagnosis is the cyst, which may contain up to eight sporozoites. Each sporozoite is round to crescent shaped, measures 1 to 2 μm in diameter, and contains an eccentric nucleus. This cystic unit with its intracystic bodies is seen well in Giemsa-stained imprint smears of infected fresh lung. Giemsa stain, however, results in staining of background alveoli and host cell fragments and does not stain empty cysts. Gomori methenamine silver stain (GMS), which highlights only the cyst wall of Pneumocystis , is preferable to Giemsa stain when tissues must be screened for the presence of organisms. The cysts stained with silver have a black cell wall and appear round, crescentic, or disk shaped. Cysts measure 4 to 6 μm in diameter and must be distinguished from erythrocytes. The cysts often occur in clusters within an alveolus.

The typical honeycombed intraalveolar exudate of Pneumocystis pneumonia is a result of negative staining of clumps of cysts held together by proteinaceous debris. The internal structure of the silver-stained cyst is variable. In the lighter-staining round cysts, there are sometimes visible thickenings in the cell wall that are circular or comma shaped ( Fig. 34-1 ). The significance of these cell wall variations is unknown, but they are helpful in confirming the identification of Pneumocystis .

Staining procedures, other than those using Giemsa and methenamine silver, have been used less frequently to delineate the cyst form of the organism. The cyst wall stains red with periodic acid–Schiff (PAS) stain. A modified Gram-Weigert method stains both the cyst wall and the intracystic sporozoites. Gridley fungus stain may identify cyst outlines. More reliable stains for this purpose are the modified toluidine blue stain of Chalvardjian and Grawe and the crystal violet stain, which color the cyst wall purple. Electron microscopy has been an invaluable tool in morphologic studies of P. jirovecii . It has helped to confirm that the structures regarded as Pneumocystis under light microscopy are, in fact, typical microorganisms and not just degradation products of host cells.

The trophozoite is thin walled and measures between 1.5 and 2.0 μm in diameter. It has numerous evaginations or pseudopodia-like projections that appear to interdigitate with those of other organisms in the alveolar space. The projections have been postulated to allow for attachment of Pneumocystis, but the prevailing opinion, however, is that no specialized organelle of attachment exists. Rather, the surfaces of P. jirovecii and alveolar cells (specifically, type I pneumonocytes) are closely opposed, without fusion of cell membranes. This adherence of P. jirovecii to alveolar lining cells may explain why organisms are not commonly found in expectorated mucus or tracheal secretions.

The intracystic bodies (sporozoites) measure 1.0 to 1.7 μm across and bear a marked similarity to small trophozoites. In addition, thick-walled cysts rich in glycogen particles but without intracystic bodies (“precysts”), partly empty cysts, and collapsed cystic structures have been identified. The collapsed cysts are crescentic and presumably are the same crescentic forms seen frequently in silver-stained specimens under light microscopy.

Life cycles for P. jirovecii have been proposed. They have been based on the variant forms of the fungus detected by light and electron microscopy. One scheme suggests that the thick-walled round cyst undergoes dissolution or “cracking,” whereupon the intracystic bodies pass through tears in the wall ( Fig. 34-2 ). It is not known whether the bodies escape from the cyst by active motility or whether they are extruded passively as a consequence of cyst collapse. At this stage, the intracystic bodies resemble free thin-walled trophozoites. The small trophozoites evolve to larger forms, their walls thicken, and a precyst develops that is devoid of intracystic bodies. The cyclic process is completed when formation of the mature cyst, containing eight daughter cysts, is achieved.

Classification of Pneumocystis as a protozoan or as a fungus was complicated by the inability to maintain the organism in culture to further characterize the biochemical nature of the organism. Arguments in favor of a protozoan taxonomy were based mainly on the resemblance of its structural features to those of other protozoa. The organism has cystic and trophozoite stages, pseudopodia in cell walls, and pellicles around intracystic sporozoites. In addition, the disease caused by Pneumocystis responds to antiprotozoal medications, such as pentamidine, atovaquone, fansidar, trimethoprim-sulfamethoxazole (TMP-SMX), while not responding to many antifungal drugs, namely, amphotericin, azoles, 5-flucytosine, and Nystatin. On the other hand, like fungi, P. jirovecii contains a paucity of cellular organelles, its nucleus is not visibly prominent, and its cell membrane is layered throughout an entire life cycle. Finally, there is a high degree of homology between “housekeeping” genes of Pneumocystis and other fungi.

The question of host species specificity of Pneumocystis remained similarly unanswered until the development of highly specific monoclonal antibodies provided the tools to demonstrate the uniqueness of Pneumocystis isolated from different mammalian hosts. Subsequent genetic analyses confirmed the host species specificity of Pneumocystis . The initial demonstration that Pneumocystis could not be transmitted between different host species provided biologic confirmation for the observed host-defined phenotypic and genotypic differences among Pneumocystis .

Epidemiology and Transmission

The distribution of human infection is worldwide, and a wide variety of wild and domestic animal species has been demonstrated to be infected by Pneumocystis . The natural habitat of P. jirovecii is unknown, but what is known of the biology of Pneumocystis strongly supports the idea that it is maintained in the population through subclinical or mild infection of immunologically normal persons, especially infants. The prevalence of infection with Pneumocystis remains to be determined because studies to detect latent carriage of the organism in large populations have not been performed. Serologic surveys, however, indicate that infection is widespread and acquired in early life. Meuwissen and colleagues, in the Netherlands, noted that antibodies to P. jirovecii are first detectable in healthy children at 6 months of age, and by age 4 years, nearly all children are seropositive. Pifer and associates, in the United States, found significant titers of antibody to Pneumocystis in healthy 7-month-old infants and in two thirds of normal children by age 4 years. Gerrard and coworkers, in England, detected P. jirovecii antibodies in serum from 48% of 94 young healthy children. Pifer also found that serologic evidence of Pneumocystis infection is present before immunosuppressive therapy with corticosteroids and that Pneumocystis elicits pneumonia in healthy rats. A prospective, longitudinal study of infants bled every 2 months from birth to 2 years demonstrated 85% of the infants seroconverted by 20 months of age. Authors of a number of autopsy reviews have attempted to determine the incidence of Pneumocystis infection, but the results have been divergent, because of the heterogeneity of the populations studied. Those studies conducted in central Europe after World War II or in cancer referral centers in the United States have yielded higher rates of infection.

Few published reports have been devoted exclusively to the descriptive epidemiology of Pneumocystis pneumonia in the United States. In a literature review of the subject, Le Clair accumulated 107 accounts of the disease recorded from 1955 through 1967. The male-to-female ratio of infected persons was in excess of 2:1, but ethnic distribution was even. The disease was reported from diverse geographic locales (21 of the 50 states). The largest number of cases occurred in infants younger than 1 year. Proved or presumptive congenital immunodeficiencies were identifiable in virtually all of the children in this group. In patients 1 to 10 years of age, who constituted the next largest group, only six had a primary immune deficit, whereas most of the other children had an underlying hematologic malignancy. The remaining patients, ranging in age from 10 to 81 years, were persons with assorted malignancies or renal allografts, who almost always had had prior exposure to corticosteroids, radiation, or cytotoxic drugs. The mortality rate for the entire group of patients was 95%.

The Centers for Disease Control and Prevention (CDC) updated Le Clair’s study by investigating the epidemiologic, clinical, and diagnostic aspects of all confirmed cases of pneumocystosis reported to its Parasitic Disease Drug Service between 1967 and 1970. The first of these reports has particular relevance because it focused only on the infectious episodes in infants and young children. A total of 194 documented cases of P. jirovecii pneumonia were analyzed, and 29 occurred in infants younger than 1 year. The attack rate for this group (8.4 per million) was more than five times higher than that for other age groups. Eighty-three percent of these infants had an underlying primary immunodeficiency disease. Moreover, because the inheritance of the primary immunodeficiency state often was sex linked, the preponderance of infection (88%) occurred in males. The mean age at diagnosis in the immunodeficient infants was 7.5 months, whereas the epidemic form of the infection in European and Asian infants was associated with peak morbidity in the third and fourth months of life. Twenty-four percent of the infected children with immunodeficiencies had at least one sibling with an identifiable immune deficiency in whom P. jirovecii pneumonia also developed.

After this analysis of cases indigenous to the United States was complete, it became evident that infantile pneumocystosis could be introduced into the United States from epidemics abroad. The first such case was reported in 1966, when a 3-month-old Korean infant died of Pneumocystis infection after being brought to the United States from an orphanage in Korea. The potential for imported pneumocystosis received renewed publicity with the cessation of the war in Vietnam. Surveillance for Pneumocystis infection in American-adopted Vietnamese orphans was urged when it was recognized that large numbers of infants exposed to the hardships of war and malnutrition in Indochina had experienced fulminant Pneumocystis pneumonia. In quick succession, multiple cases of Pneumocystis infection among these refugee Vietnamese were reported. Most of the affected infants were approximately 3 months of age; this was exactly the age at which pneumocystosis had emerged in the marasmic children infected during the earlier nursery epidemics in central Europe and Asia.

The epidemiology of P. jirovecii infection has changed as cases of human immunodeficiency virus (HIV) infection have occurred in infants. As is true in adults with acquired AIDS, infants with AIDS are at high risk for this opportunistic infection. Among children with perinatally acquired HIV infection, P. jirovecii pneumonitis occurs most often among infants 3 to 6 months of age. Another important change in the epidemiology of Pneumocystis pneumonia relates to the use of more potent immunosuppressive therapies, which places new patient groups at risk for Pneumocystis pneumonia. For example, it is now suggested that patients with inflammatory bowel disease who are receiving anti–tumor necrosis factor (TNF) treatment should be considered for Pneumocystis pneumonia prophylaxis because of the recognized risk of Pneumocystis infection in these patients.

It has been suggested that P. jirovecii may be an important cause of pneumonitis in immunologically intact infants. As noted above, primary infection with Pneumocystis occurs in most healthy infants by 2 years of age. However, seroconversion occurred for the most part without a recognizeable clinical illness. More cases need to be confirmed histologically before it is established that Pneumocystis infection produces morbidity in previously healthy infants.

A role for Pneumocystis in the pathogenesis of the sudden infant death syndrome (SIDS) has been postulated. The cause of SIDS, the unexplained death of previously healthy infants, remains a mystery. In 1999, Vargas and colleagues, in Santiago, Chile, reported finding Pneumocystis in the lungs of many infants whose death was diagnosed as SIDS. Shortly thereafter, this observation was confirmed in SIDS cases occurring in the Northeast United States. Interpreting these observations presented two problems: defining appropriate case controls for the presence of Pneumocystis in infants without SIDS and explaining the mechanism of death, given the light infections and lack of demonstrable pathology in these infants. After a series of investigations, which included control infants, Vargas and colleagues clearly established the presence of Pneumocystis in infants dying of SIDS but came to the conclusion that Pneumocystis was not likely the cause of SIDS. More recently, Vargas and colleagues reported finding near-universal focal Pneumocystis that was associated with upregulation of mucus expression in the lung of SIDS cases. Whether and how this observation relates to the role of Pneumocystis in the pathogenesis of SIDS is yet to be determined.

The mode of transmission of P. jirovecii is difficult to prove, but animal studies provide unequivocal evidence for animal-to-animal transmission via the airborne route. Epidemiologic studies support a similar mode of transmission among humans. For example, Pneumocystis infections among transplant patients have been shown to increase as their exposure to patients with or at risk for Pneumocystis pneumonia increases. Furthermore, using molecular probes, it is possible to demonstrate that clusters of Pneumocystis pneumonia appear to be caused by the same organism. Occurrence of pneumocystosis among family members also has been reported with pneumonia developing in three family members in a strikingly related time sequence. More commonly, cases within a family emerge over a period of several years, and affected members almost always are infant siblings with either proven or suspected underlying immunodeficiencies. In at least three family studies, no fewer than three siblings succumbed to the infection. It is unlikely, however, that direct patient-to-patient transfer of the organism occurred in any of these settings because, in almost all instances, development of disease in the sibling occurred months or years later. Contagion could still be implicated in the family milieu if a reservoir of asymptomatic infection with P. jirovecii existed among healthy family members. Supporting evidence suggests that infants may serve in the role of reservoir. Maternal transfer of Pneumocystis to infants from colostrum or from the genital tract at parturition also might maintain Pneumocystis within a family, but it is difficult to test this hypothesis. The paucity of documented cases of overt Pneumocystis pneumonia in stillborn infants or in the early neonatal period, however, argues against frequent intrauterine passage of the organism. Congenital infection has been suggested based on very few case reports, but the data presented is inconclusive.

The possibility that Pneumocystis pneumonia is a zoonotic disease and that infestation of rodents or even domesticated pets could provide a sizable reservoir for human infection has been postulated based on the frequent finding of Pneumocystis in animals. For example, abundant infection of rodents with Pneumocystis was discovered in patients’ homes in many of the index cases in ward epidemics in Czechoslovakia. However, with only microscopy, it is not possible to distinguish animal from human Pneumocystis. The development of reagents that could distinguish Pneumocystis from one host species from that infecting a different host allowed for definitive experiments to address host species specificity. Controlled experiments demonstrated that Pneumocystis from ferrets could not infect severe combined immunodeficient (SCID) mice. These results were quickly confirmed using Pneumocystis from different hosts, including P. jirovecii from humans. Thus the concept of Pneumocystis pneumonia as a zoonosis is not tenable.

Pathology

The gross and microscopic pathologic features of P. jirovecii pneumonia have been elucidated in a number of excellent reviews. At autopsy in typically advanced infection, both lungs are heavy and diffusely affected. The most extensive involvement often is seen in posterior or dependent areas. At the lung margins anteriorly, a few remaining air-filled alveoli may constitute the only portion of functioning lung at the time of death. Subpleural air blebs not infrequently are seen in these anterior marginal areas. On occasion, prominent mediastinal emphysema or frank pneumothorax can be noted. The color of the lungs is variously described as dark bluish purple, yellow-pink, or pale gray-brown. The pleural surfaces are smooth and glistening, with little inflammatory reaction. Hilar adenopathy is uncommon. Necrosis of tissue is not a feature of the disease.

Although these gross features of widespread infection are strikingly characteristic, focal or subclinical pneumocystosis presents a less recognizable picture. In this condition, the lung has tiny 3- to 5-mm reddish brown retracted areas contained within peribronchial and subpleural lobules, where hypostasis is greatest. Even these features, however, may be absent because of variable involvement of adjacent lung tissue by concomitant pathologic processes.

The microscopic appearance of both the contents and the septal walls of pulmonary alveoli in Pneumocystis pneumonia are virtually pathognomonic of the infection. The outstanding histologic finding with hematoxylin and eosin stain is an intensely eosinophilic, foamy, or honeycomb-like material uniformly filling the alveolar sacs ( Fig. 34-3 ). This intraalveolar material is composed largely of Pneumocystis , host cells, immunoglobulin, and proteinaceous debris. Typical cyst forms of the organism within alveoli are visible only after application of special stains such as methenamine silver.

The type and degree of cellular inflammatory responses provoked by the intraalveolar cluster of Pneumocystis organisms vary based on host response (see “Pathogenesis” ). The descriptive histologic term for pneumocystosis—interstitial plasma cell pneumonia—is derived from the pronounced plasma cellular infiltration of the interalveolar septa observed almost exclusively in newborns in European nursery epidemics. Distention of alveolar walls to 5 to 10 times the normal thickness, with resultant compression of alveolar spaces and capillary lumens, typically is noted in this form of the disease. Hyaline membranes develop occasionally. Septal cell hyperplasia, a nonspecific reaction of lung tissue to injury induced by infections of diverse etiology, is also seen.

Hughes and colleagues studied the histologic progression of typical Pneumocystis pneumonia based on the number and location of organisms and the cellular response in pulmonary tissue. The lung samples were from children with underlying malignancy who had received intensive chemotherapy. The authors categorized three sequential stages in the course of the disease. In the first stage, no septal inflammatory or cellular response is seen, and only a few free cyst forms are present in the alveolar lumen; the remainder are isolated on the alveolar septal wall. The second stage is characterized by an increase in the number of organisms within macrophages fixed to the alveolar wall and desquamation of these cells into the alveolar space; again, only minimal septal inflammatory response is seen at this time. Finally, a third stage is identified, in which extensive reactive and desquamative alveolitis can be seen. Such diffuse alveolar damage may be the major pathologic feature in certain cases. Variable numbers of cysts of the organism, presumably undergoing dissolution, are present within the alveolar macrophages. These findings underscore the thought that the so-called foamy exudate within alveoli is neither foamy edema fluid nor the product of an exudative inflammatory reaction but largely a collection of coalesced alveolar cells and macrophages that contain sizable digestive vacuoles and remnant organisms.

The mechanism of spread of Pneumocystis throughout pulmonary tissue is not completely understood. Direct invasion by the organism through septal walls into the interstitium or the lymphatic or blood vascular spaces of the lung is considered unlikely, except in rare instances when systemic dissemination of the organism occurs.

Interstitial fibrosis is a distinct but infrequently reported complication of Pneumocystis pneumonia in older children and adults but has been reported in infants only rarely. Nowak, in Europe, first emphasized that fibrosis was not unusual in the lungs of infants at autopsy who had especially protracted infection with P. jirovecii . Pneumocystis -infected lungs sometimes demonstrate, in addition to fibrosis, other pathologic features compatible with a more chronic destructive inflammatory process. Multinucleate alveolar giant cells occasionally accompany alveolar cell proliferation. Whether presence of these cells is more often a response to undetected concomitant viral infection is unknown. Typical granulomatous reactions with organisms visible in the granulomas also have been described. Extensive calcification of Pneumocystis exudate and adjacent lung tissue may ultimately develop.

Pathogenesis

The clinical conditions that predispose patients to the development of Pneumocystis pneumonia are associated with impaired immune responses, leading to the presumption that Pneumocystis causes disease, not because it is intrinsically virulent but because the host’s immune mechanisms fail to contain it. The severity of P. jirovecii pneumonia in infants with AIDS illustrates this phenomenon dramatically. The primary role of immunocompromise also would explain, in part, why Pneumocystis pneumonia did not emerge as a serious health problem until more than 30 years after the disease was first recognized. European epidemics of Pneumocystis arose out of the devastation of World War II and widespread use of antibacterial drugs. Each of these two seemingly unrelated events served ultimately to disrupt the normal host-organism immunologic interaction in favor of the organism. The war resulted in institutionalization of inordinate numbers of orphans under conditions of overcrowding and malnutrition. At the same time, antibacterial therapy dramatically enhanced survival rates of these institutionalized infants, who would otherwise have succumbed to bacterial sepsis during the first days or weeks of life. In addition, it was realized that Pneumocystis infection appeared in these marasmic children at an age when their immunoglobulin G (IgG) levels reached a physiologic nadir. By 1960, the orphanage epidemics had abated in Europe as environmental conditions improved, but they persisted in Asia, where poverty and overcrowding continued. Subsidence of the epidemic disease and more widespread antibacterial drug therapy, as well as sophisticated immunosuppressive drug treatment, contributed thereafter to awareness in Europe and North America of isolated instances of Pneumocystis infection among children suffering from a variety of identifiable immunodeficiencies.

Weller, in Europe, was among the first to experimentally induce Pneumocystis pneumonia in animals. His crucial observation relative to pathogenesis of the infection was that in rats pretreated with cortisone (and penicillin) and exposed to suspensions of Pneumocystis -containing lung tissue, Pneumocystis pneumonia develops with the same frequency and severity as in corticoid-treated animals that were not subsequently inoculated with organisms. The intensity of such artificially induced animal infection also was noted to be less marked than that in spontaneous human pneumocystosis of the epidemic variety. Comparable observations in the rabbit model were made by Sheldon in the United States. He showed that cortisone and antimicrobial agents were sufficient to induce Pneumocystis infection without direct exposure of animals to an exogenous source of organisms. The inescapable conclusion of these carefully designed studies was that Pneumocystis infection is latent in rats and rabbits and becomes clinically manifest only when host resistance is altered or that Pneumocystis is ubiquitous in the environment and capable of infecting a susceptible host.

The issue of latency is an important one in defining the pathogenesis of Pneumocystis pneumonia. To determine whether Pneumocystis establishes latency after infection, SCID mice, which resolve naturally acquired Pneumocystis carinii pneumonia (PCP) after reconstitution with immunocompetent spleen cells, were observed for evidence of a latent Pneumocystis infection. Neither P. carinii nor amplified P. carinii DNA was detected in the lungs of SCID mice killed 21 days after spleen cell reconstitution. Furthermore, SCID mice that recovered from P. carinii infection failed to reactivate the infection after they were either depleted of CD4 ⁺ cells for up to 84 days or depleted of CD4 ⁺ cells and treated with corticosteroid for 35 days. These results indicate that an immune response to P. carinii can completely clear the organism from the host. This supports the hypothesis that P. carinii pneumonia that develops in immunocompromised patients is due to exposure to an exogenous source of P. carinii rather than reactivation of latent infection. Experiments using the steroid-treated rat model of Pneumocystis infection demonstrated similar findings.

In 1966, Frenkel and colleagues published a hallmark study of rat pneumocystosis. They showed that clinical and histopathologically significant involvement with Pneumocystis is regularly inducible in rats by “conditioning” them with parenteral cortisone over a period of 1 to 2 months. Premature death from complicating bacterial infection was prevented by simultaneous administration of antibacterial agents. Of interest is their finding that regression of established interstitial pneumonitis occurs if cortisone conditioning is stopped early enough; on the other hand, rats continuing to receive cortisone die of coalescent alveolar Pneumocystis infiltration, and the infiltrate is almost devoid of inflammatory cells. These histologic changes are, in fact, an exact replica of those observed in sporadic cases of human Pneumocystis infection developing in congenitally immunodeficient and exogenously immunosuppressed patients. Attempts to precipitate clinical pneumocystosis with a variety of immunosuppressants other than cortisone were also explored. Of eight cytotoxic agents and antimetabolites tested, only cyclophosphamide was shown to make the rats susceptible to infection by Pneumocystis . Total-body irradiation and lymphoid tissue ablation (splenectomy, thymectomy) by themselves were incapable of inducing overt Pneumocystis pneumonia.

The clinical association between pneumocystosis and protein-calorie malnutrition also has been reproduced in a rat model. Healthy rats given either a regular or a low-protein diet gain weight and exhibit little to no evidence of pneumocystosis postmortem. By contrast, in rats fed a protein-free diet, which produces weight loss and hypoalbuminemia, fatal infection regularly developed; administration of corticosteroid only foreshortened their median survival time.

None of the experimental models described thus far permit a precise appraisal of the relative importance of the cellular and humoral components of host defense against Pneumocystis . Although corticosteroids, cytotoxic drugs, and starvation interfere primarily with cell-mediated immunity, they do not always induce purely functional cellular defects. Rather, the immunosuppressive effects of chemotherapeutic agents or of malnutrition are far more complex, and ultimately both cellular and humoral arms of the immune system may be impaired by them.

The production of pneumocystosis in the nude mouse without the use of exogenous immunosuppressants implies that susceptibility to the infection relates most to a defect in thymic-dependent lymphocytes. The role of antibody deficiency is less clear. A role for antibody in control of infection with the organism has been shown in vitro by demonstrating that Pneumocystis organisms adherent to rat alveolar macrophages become interiorized only after anti- Pneumocystis serum is added to the culture system.

That primary humoral immune deficits could predispose to sporadic pneumocystosis was first reported, unwittingly, by Hutchison in England in 1955. He described male siblings with congenital agammaglobulinemia who died of pneumonia of “similar and unusual” histology. P. jirovecii was implicated as the etiologic agent of these fatal infections only when the pathologic sections were reviewed by Baar. Burke and colleagues stressed what was to be regarded as a typical histologic finding in Pneumocystis -infected agammaglobulinemic children, namely, the absence or gross deficiency of plasma cells in pulmonary lesions (and in hematopoietic tissues). This deficiency contrasted sharply with the extensive plasmacytosis seen in epidemic infections.

Pneumocystis been reported in association with a pure T-cell deficiency, namely, in DiGeorge syndrome. That the integrity of the cellular immune system is critical for resistance to Pneumocystis may be inferred from the steroid-induced and congenitally athymic animal models of pneumocystosis described earlier. Most Pneumocystis infections occurred in infants with SCID, a state characterized by profound depression of both cellular and humoral immunity.

For many years it had not been possible to study in vitro the cellular immune response to P. jirovecii because of the impurity of available antigens. Preliminary experiments with an antigen derived from a cell culture suggested that specific cell-mediated immunity may be depressed in children with active Pneumocystis pneumonia. Lymphocytes from two such children failed to transform in the presence of the antigen, whereas lymphocytes from healthy, seropositive adults were, in most cases, stimulated specifically to undergo blastogenesis.

Although PCP was clearly defined as an opportunistic pathogen causing clinical disease in patients suffering from primary or, more commonly, secondary immunodeficiencies, for many years the pathogenic mechanisms by which Pneumocystis caused lung injury and respiratory impairment remained mostly undefined. Histologic observations of infected lung tissue demonstrated that Pneumocystis attaches to alveolar epithelial cells in the distal lung and that these cells are preferentially damaged during severe PCP. However, the nature of the injury was unclear, and progress in understanding this organism and identifying potential toxins or virulence factors was hampered by the lack of an axenic culture system. Clinical observations offered some insight into the nature of PCP-associated lung injury. A positive correlation between the degree of pulmonary inflammation and the severity of PCP was noted and suggested that the host response to infection affects the clinical manifestation of PCP. Before the AIDS epidemic, the majority of PCP cases occurred in patients with hematologic malignancies, and often it was noted that the onset of PCP coincided with cessation of corticosteroid treatment. Similarly, the onset of PCP in bone marrow transplant recipients often coincided with engraftment. In both examples, Pneumocystis infection likely occurred during the period of immunosuppression, but the clinical manifestation was not evident until a degree of immune function was restored, suggesting that the host-driven pulmonary immune response contributed to the disease process. The onset of the AIDS epidemic offered a distinct subset of PCP patients who suffered from profound immunosuppression. Comparison of patients with AIDS-related PCP to patients with non-AIDS PCP revealed that AIDS patients had a more subtle onset of symptoms with better pulmonary function and better prognosis, despite harboring higher lung fungal burdens. Furthermore, the severity and prognosis for PCP patients was found to correlate with the degree of pulmonary inflammation but not with organism burden. Together, these findings suggest that the host response to Pneumocystis infection contributes to lung injury and respiratory impairment during PCP.

The development of animal models of Pneumocystis infection has proved invaluable for elucidating the immunopathogenesis of PCP. SCID mice lack functional lymphocytes and are highly susceptible to Pneumocystis infection. An early study found that when Pneumocystis -infected (SCID) mice were immune reconstituted by the adoptive transfer of functional lymphocytes, the mice suffered from rapid deterioration and high mortality rates. Subsequent studies found that immune reconstitution of Pneumocystis -infected SCID mice induced a rapid increase in the pulmonary expression of proinflammatory cytokines and chemokines and the recruitment of cellular infiltrates into the lungs. Although immune reconstitution provided the benefit of restoring an effective CD4 ⁺ T-cell–dependent immune response against Pneumocystis infection, it also had profound effects on physiology, including severe weight loss, tachypnea, hypoxia, and decreased lung compliance. Of importance, nonreconstituted SCID mice with similar fungal burdens appeared physiologically normal and showed little evidence of PCP-related disease, suggesting that Pneumocystis itself is not the direct cause of pulmonary damage, at least early in the evolution of the disease process. The immune-reconstituted SCID mouse model of PCP is similar to the clinical syndrome termed immune reconstitution inflammatory syndrome (IRIS), which has been reported in AIDS patients after institution of combined antiretroviral therapy. The rapid recovery of CD4 ⁺ T lymphocytes causes an intense pathologic pulmonary immune response to preexisting pulmonary infections, including Pneumocystis . Patients with PCP-related IRIS suffer severe pulmonary decompensation and have poorer survival rates than patients with a classic AIDS-related presentation of PCP. These observations highlight the contribution of the immune response to PCP pathogenesis.

Classic AIDS-related PCP has also been effectively modeled in mice. Continual administration of anti-CD4 monoclonal antibody maintains mice in a chronic CD4 ⁺ T-cell–depleted state. CD4-depleted mice are susceptible to Pneumocystis infection and develop a clinical syndrome very similar to AIDS-related PCP in humans. Several studies have demonstrated that in the absence of CD4 ⁺ lymphocytes, large numbers of CD8 ⁺ T cells are recruited to the lung in response to Pneumocystis infection. These lymphocytes are unable to control Pneumocystis infection but directly contribute to lung injury and PCP-related pathogenesis. Mice depleted of both CD4 ⁺ and CD8 ⁺ lymphocytes are much healthier compared with mice depleted of only CD4 ⁺ lymphocytes. Together, these findings support the immunopathogenic features of PCP. Although infection is necessary to cause pneumonia, certain aspects of the immune response cause disease symptoms. Differences in the degree of immunosuppression among PCP patients likely affect their ability to produce an immunopathogenic response to infection and may account for variability in the severity of PCP between different patient groups.

The mechanisms of PCP-related immunopathogenesis are not clearly defined. Although both CD4 ⁺ and CD8 ⁺ lymphocyte-driven events have been implicated in immunopathogenesis, the specific mechanisms have not been elucidated. The appearance of neutrophils and neutrophil chemotactic factors in the lung correlate with a poorer prognosis for PCP patients. However, these cells do not appear to directly contribute to either host defense against Pneumocystis infection or to PCP-related immunopathogenesis. The proinflammatory cytokine TNF appears to be required for successful host defense against Pneumocystis infection, and signaling through TNF receptors is also a major contributor to immunopathogenesis. A possible physiologic consequence of PCP-driven inflammation is pulmonary surfactant dysfunction. Surfactant is critical to normal gas exchange and proper lung function. Pulmonary inflammation elicited during Pneumocystis infection was found to directly disrupt surfactant function, contributing to PCP-related respiratory impairment. Although the specific mechanisms of immunopathogenesis are not defined, the contribution of inflammation and the immune response to this disease process is recognized, and standard treatment of moderate-to-severe PCP typically includes corticosteroids as adjunctive therapy to dampen inflammation.

Clinical Manifestations

Diagnosis

The diagnosis of Pneumocystis pneumonia remains difficult. The organism must be visualized in the respiratory tract of ill persons, and often this can be accomplished only by bronchoalveolar lavage (BAL) or, in infants, a lung biopsy. Recently, polymerase chain reaction (PCR) assay has been used for diagnosis in fluid specimens obtained by BAL. Nevertheless, this technique is still not generally available for routine clinical use. Attempts to isolate Pneumocystis from clinical specimens on synthetic media or in tissue culture have not been successful, and serologic techniques to detect active infection have been too insensitive.

Treatment

Prognosis

Prevention

The first successful attempts to prevent pneumocystosis with drugs were reported in infants with the epidemic form of the infection. In a controlled trial conducted in an Iranian orphanage where the infection was endemic (attack rate of 28%), the biweekly administration of a pyrimethamine and sulfadoxine combination to marasmic infants before the second month of life entirely eradicated Pneumocystis pneumonia from the institution. In a children’s hospital in Budapest, Hungary, pentamidine given every other day for a total of seven doses to premature infants from the second week of life provided equally effective prophylaxis. During the 6 years of the study, Pneumocystis infection did not develop among 536 premature babies who received this treatment, whereas 62 fatal cases were recorded elsewhere in the city.

On the basis of promising results in a rat model of infection, TMP-SMX was evaluated in a randomized, double-blind, controlled trial in children with cancer who were at extremely high risk for Pneumocystis pneumonitis. The daily dosage for prophylaxis was 5 mg of TMP plus 20 mg of SMX/kg body weight, administered orally in two divided doses. Seventeen (21%) of 80 children receiving placebo acquired pneumocystosis, whereas the infection developed in none of 80 patients given TMP-SMX. No adverse effects of TMP-SMX administration were observed, although oral candidiasis was more prevalent among the patients in the treatment group than among the control patients. In a subsequent uncontrolled trial, the prophylactic efficacy of TMP-SMX was confirmed; cases of infection developed only in those children in whom the TMP-SMX was discontinued while they were still receiving anticancer chemotherapy. More recently, a regimen of TMP-SMX prophylaxis given 3 days per week was shown to be as effective as daily administration.

The gratifying success of TMP-SMX prophylaxis in prevention of Pneumocystis infection has been duplicated in other medical centers caring for children with underlying malignancy. Administration of the drug for the duration of antineoplastic therapy has become standard practice. Congenitally immunodeficient children and infants with AIDS who have had a prior episode of Pneumocystis pneumonia would appear to be prime candidates for preventive therapy. The CDC issues a set of guidelines for chemoprophylaxis against P. jirovecii pneumonia in children with HIV infection. These guidelines recommend promptly identifying infants and children born to HIV-infected women, initiating prophylaxis at 4 to 6 weeks of age for all of these children, and continuing prophylaxis through 12 months of age for HIV-infected children and offer new algorithms based on clinical and immunologic status to continue prophylaxis beyond 12 months of age. Although no chemoprophylactic regimens for P. jirovecii pneumonia among HIV-infected children have been approved as labeling indications by the U.S. Food and Drug Administration (FDA), TMP-SMX currently is recommended as the drug of choice in children with HIV infection. This recommendation is based on the known safety profile of TMP-SMX and its efficacy in adults with HIV infection and in children with malignancies. Alternative regimens recommended for HIV-infected children who cannot tolerate TMP-SMX include aerosolized pentamidine in children older than 5 years of age, oral dapsone, and oral atovaquone. One study suggests that TMP-SMX use is associated with a decreased incidence of P. jirovecii pneumonitis and an increased incidence of HIV encephalopathy, both as initial AIDS-defining conditions in infants and children. Most likely this was related to an “unmasking” of progressive encephalopathy among infants who otherwise would have died earlier because of P. jirovecii pneumonia.

The Organism

The genus Aspergillus is within the family Moniliaceae and is classified as an ascomycetous, saprophytic mold. The species that cause disease most commonly are Aspergillus fumigatus, which causes at least 90% of all disease, followed by Aspergillus flavus, Aspergillus niger, and Aspergillus terreus . However, as the number of people living with profound immunosuppression has increased, the number of Aspergillus spp. that have been reported to cause invasive disease has increased; the list contains at least 20 different species. Almost all cases of neonatal aspergillosis have been caused by A. fumigatus. A distinguishing feature of pathogenic species is their ability to grow at human body temperature (37° C); A. fumigatus, for example, can grow at temperatures as high as 50° C, and this characteristic can be used to identify this specific species. Microscopically, Aspergillus is a hyaline, septate, monomorphic mold that shows dichotomous branching. The species are differentiated using a variety of distinguishing morphologic, mycologic, and biochemical characteristics. It is important to note that it can be difficult to differentiate Aspergillus spp. from other types of molds during the initial evaluation of clinical samples.

Epidemiology and Transmission

Aspergillosis is a rare infection in neonates and infants, and the literature of such infections is limited to case reports and case series. Consequently, it is not possible to estimate an incidence rate. In general, the two primary modes of transmission are inhalation of airborne conidia or through direct, localized inoculation of damaged or compromised tissues. Person-to-person transmission has not been documented, whereas the clustering of multiple cases has been linked to exposure to a common environmental source, such as humidifying units. Thus isolating affected patients is unlikely to prevent additional cases within a closed unit. The identification and control of possible sources of environmental contamination is more likely to lead to the prevention of additional cases.

For preterm infants in the NICU setting, PCA is the most common manifestation of aspergillosis, as illustrated by the large case series reported by Groll and associates. As discussed in depth by Walsh, it is likely that the well-characterized fragility of the skin of preterm infants is the primary reason for the disproportionate rate of PCA relative to pulmonary aspergillosis. A large multicenter retrospective analysis of 139 cases of invasive aspergillosis in children of all ages found that cutaneous aspergillosis was more common in children than adults; however, this study did not correlate age with incidence of PCA. In other populations, a primary risk factor acquiring invasive aspergillosis is decreased levels or function of peripheral neutrophils. Although neutropenia is not generally a complication of prematurity, there are numerous studies indicating that premature infants have defects in specific neutrophil functions, including migration, phagocytosis, and microbial killing. No specific studies have examined the ability of neutrophils from premature infants to respond to Aspergillus , but it is certainly possible that an inability of premature neutrophils to make an effective response to microbial pathogens contributes to their risk for aspergillosis. The study of Groll and associates underscores this possibility in that it found that a significant portion of term infants who developed pulmonary aspergillosis also had chronic granulomatous disease. Chronic granulomatous disease is genetic disease caused by mutation of the phox gene, leading to decreased respiratory burst in phagocytes, and is characterized by increased susceptibility to aspergillosis as well as other pathogens. The findings of Groll suggest that infants who develop pulmonary aspergillosis be evaluated for defects in neutrophil function, including chronic granulomatous disease.

Pathogenesis

Consistent with their opportunistic nature, Aspergillus spp. do not display virulence factors in the traditional microbiologic sense (e.g., cholera toxin). To replicate and cause disease, the fungus must be able to withstand the host environment, and the thermotolerant nature of Aspergillus spp. that cause disease is an important trait required for virulence. For example, mutants of specific genes (e.g., crgA ) that lead to decreased growth at 37° C are attenuated for virulence in animal models of aspergillosis. Other genes that have been associated with decreased virulence in animal models include amino acid metabolism and iron acquisition, as well as others. Once the fungus has breached the pulmonary epithelium or epidermis, it is well known to invade tissue and blood vessels. Consistent with its saprophytic nature, it is well adapted to growth along environmental gradients, which is thought to contribute to its characteristic angiotropism. Invasion of blood vessels has two consequences. First, it leads to additional tissue damage and necrosis by destroying the blood vessel and depriving the local tissue of oxygen and nutrients. Second, angioinvasion facilitates dissemination to other organ systems within the host. The latter characteristic is particularly relevant to neonatal aspergillosis because many cases of PCA ultimately lead to disseminated disease accompanied by sepsis and multiorgan system involvement. A commonly encountered target organ of Aspergillus is the central nervous system (CNS), and its angiotropism appears to also allow it to readily penetrate the blood-brain barrier. As one might expect, CNS involvement is a complication of aspergillosis that is associated with a very poor prognosis.

Pathology

Because PCA is one of the primary manifestations of aspergillosis in neonates, the histopathologic analysis of biopsy specimens of infected tissue and skin is an important mode of diagnosis. Aspergillus is readily demonstrated in tissue using standard GMS or PAS stains. The hyphae are septate and hyaline and, classically, display dichotomous branches that emerge at an acute angle to the primary filament (<90 degrees). Although these features can allow one to distinguish Aspergillus from Zygomycetes (aseptate, right-angle branching), the histopathologic characteristics of Aspergillus are similar to a wide range of other pathogenic molds, including Fusarium and Scedosporium . However, because these molds are exceedingly rare in infants and are generally treated with the same agents, this ambiguity rarely has clinical significance. Because of its angiotropic nature and destructive effect on blood vessels, hemorrhagic necrosis is typical of infected tissue and is consistent with the necrotic skin lesions that are part and parcel of PCA in neonates.

Clinical Manifestations

The most distinctive manifestation of aspergillosis in neonates and infants is the high percentage of PCA. The characteristic clinical feature of PCA is the appearance of skin lesions. Initially, these lesions can be nonspecific raised erythematous plaques and pustules. However, they generally progress to macerated, ulcerated lesions with a punched out appearance. In almost all cases, the lesions form necrotic eschars. Although these lesions are almost characteristic of PCA, it is important to note that their appearance is not pathognomonic of PCA, and they cannot be distinguished from skin lesions resulting from the systemic dissemination of other microbial pathogens such as Pseudomonas aeruginosa or Staphylococcus aureus. Similarly, as a number of authors have noted, pulmonary aspergillosis does not have distinctive clinical characteristics that allow it to be readily identified.

Regardless of the initial site of infection, many neonates develop disseminated disease with a sepsis-like syndrome and multiorgan system involvement. Symptoms accompanying dissemination include hypotension, coagulopathy, and hepatosplenomegaly. Although almost any organ can be involved after dissemination, CNS disease is a frequent complication. Because aspergillosis is difficult to diagnose, it is not surprising that CNS symptoms or findings may be the initial clue to the diagnosis, as for the patient described by Fuchs and coworkers. In the series of children younger than 3 months reported by Groll and coworkers, four patients with CNS disease as the only manifestation were identified.

Diagnosis/Differential Diagnosis

The diagnosis of aspergillosis in the neonate and infant is extremely difficult for a wide variety of reasons, including its rarity and the fact that its clinical manifestations overlap with other, much more commonly encountered pathogens. The most important method of diagnosing invasive infections in any patient population is the blood culture. Unfortunately, blood cultures are almost invariably negative in the setting of invasive aspergillosis, a fact that is somewhat counterintuitive, given its angiotropism. In older patients, radiographic characteristics of pulmonary aspergillosis, such as the halo sign, can be suggestive of the diagnosis in an at-risk patient population. However, no such characteristics have been identified in neonates or infants. The skin lesions characteristic of PCA provide the most direct opportunity for diagnosis through biopsy, stains, and culture. In contrast to blood cultures, tissue culture yields reasonable results; however, histologic identification of hyphal elements in a biopsy specimen in the absence of culture results should prompt immediate treatment and is certainly sufficient for a presumptive diagnosis. It is also important to consider other potential diagnoses that are associated with a critically ill infant displaying ulcerative or necrotic skin lesions; these include other fungal infections such as invasive candidiasis, bacterial infections such as P. aeruginosa, and disseminated viral infections such as congenital herpes or enterovirus.

Non–culture-based methods for the diagnosis of aspergillosis have been the subject of intensive interest. The most widely used and best characterized of these tests is the serum galactomannan assay. The assay uses an enzyme-linked immunosorbent assay (ELISA)–based technology to detect the presence of galactomannan in serum and, less commonly, other fluids such as BAL samples. Galactomannan is a component of the Aspergillus cell wall but is absent from Candida spp. The assay has been widely adopted to monitor for and diagnose aspergillosis in adult patients. Initial, small studies of the assay in children found that neonates had a significantly higher rate of false-positive tests. Subsequent studies indicate that this high false-positive rate may be due to the high levels of Bifidobacterium in the gut of neonates ; the lipoteichoic acid from this organism cross-reacts with the galactomannan ELISA. The galactomannan assay has been studied in older children and seems to perform similarly to adult patients. However, no prospective studies have evaluated its use in neonates and infants, and thus its value for this patient population remains unknown and may be complicated by false-positive results. Other tests that have been developed for aspergillosis, including serum 1,3-β-glucan and PCR-based methods, remain experimental or have not been sufficiently studied in this population to warrant routine use.