Vol. 4, No. 2

CLINICAL MICROBIOLOGY REVIEWS, Apr. 1991, p. 137-149

0893-8512/91/020137-13$02.00/0 Copyright © 1991, American Society for Microbiology

Pneumocystis carinii, an Opportunist in Immunocompromised Patients M. S. BARTLETT* AND J. W. SMITH Department of Pathology, Division of Clinical Microbiology, N340 University Hospital, Indiana University School of Medicine, Indianapolis, Indiana 46202-5250

INTRODUCTION ............................ TAXONOMY ............................ EPIDEMIOLOGY ............................ PATHOLOGY AND PATHOGENESIS ............................ CLINICAL MANIFESTATIONS ............................ LABORATORY DIAGNOSIS ............................ TREATMENT ............................ REFERENCES ............................

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will be adopted remains to be seen. It is also not clear whether there are strain differences among Pneumocystis organisms from the same host species. Pulsed-field gel electrophoresis has been used to study Pneumocystis spp. from humans and rats. Two groups of researchers have shown that there are differences in organisms isolated from the same host species (82, 116). Hong et al. presented data that suggest two Pneumocystis strains coinfected one rat. Because most karyotype studies have been done with organisms separated from rat lung and because the organism has been variously reported as having 16 to 20 (197), 17 to 22 (82), and 13 (116) chromosomes, it is possible that contaminating rat lung DNA is being included in the evaluations. In addition to uncertainty as to species, subspecies, and strain differences, the taxonomic position of the organisms has yet to be decided. One investigator even proposed that P. carinii is a mitochondrion (15). Early literature contains arguments supporting the inclusion of Pneumocystis spp. among the fungi (37) or the protozoa (95). Some arguments for including it among the fungi probably resulted from the growth of colonizing yeast cells from samples of lung tissue cultured on fungal medium. Arguments for including Pneumocystis spp. among the protozoa were based on morphologic similarities between Pneumocystis spp. and various protozoa. Vavra and Kucera (182) favored inclusion of Pneumocystis spp. among the fungi because the characteristic organelles of protozoa, such as rhoptries, subpellicular tubules, and conoids, could not be demonstrated in their ultrastructural studies. Recently, there have been several reports of taxonomic assignments based on evaluations of rRNA. Edman et al. (44), evaluating sequences of 16S-like rRNA, concluded that the organism is most closely related to the yeast genus Saccharomyces. Sogin and Edman (172) also found that the 16S-like rRNA contains a group I intron, the first report of such an intron in a fungal nuclear rRNA gene. On the other hand, Watanabe et al. (189), comparing the 5S rRNA of Pneumocystis carinii with that of other organisms, suggested a close relationship with Rhizopoda, Myxomycota, and Zygomycota but not with fungi such as Saccharomyces spp. A study of the 16S-like rRNA of Giardia lamblia showed that Giardia rRNA was more similar to that of bacteria than that of other protozoa (173). As technology provides new methods of assessing relationships among organisms, there may be many changes in

INTRODUCTION Although Pneumocystis carinii was described in 1909 by Chagas (29), who believed it to be a form of Trypanosoma cruzi, it was not appreciated as a pathogen of humans until years later, when there were epidemics of "plasma cell pneumonia" (65) in malnourished infants in European orphanages at the end of World War II. Gajdusek (59) alerted pediatricians in the United States to the threat of Pneumocystis pneumonia in children with immune deficiencies. Sporadic cases or clusters of cases of Pneumocystis pneumonia were reported in patients receiving immunosuppressive therapy for transplantation or malignancy (52, 133). Pneumocystis pneumonia was a frequent cause of severe disease in children being treated for acute lymphocytic leukemia (195) and in transplant patients, but overall, P. carinii was considered an uncommon pathogen. Even so, with the advent of trimethoprim-sulfamethoxazole (TMPSMX) for prophylaxis of patients known to be at risk (89), the incidence of Pneumocystis pneumonia declined until the epidemic of AIDS. The occurrence of Pneumocystis pneumonia in apparently healthy homosexual men (51) was one of the first factors that led to recognition of the epidemic of AIDS. Today P. carinii is a major cause of life-threatening pneumonia in immunocompromised patients, occurring in up to 80% of patients with AIDS. Morphologic detection of P. carinii is required to establish the diagnosis and institute an appropriate therapy. TAXONOMY The genus Pneumocystis has been considered to contain one or several species (65). Because strains from humans, rats, and other animals have not been clearly differentiated, most reports use the species P. carinii for all Pneumocystis organisms. In 1976, Frenkel (53) proposed that Pneumocystis organisms from humans be called Pneumocystis jiroveci; however, that nomenclature has not been used widely. Recently, Hughes and Gigliotti (87) proposed retaining the name P. carinii but following it with the Latin designation for the host genus so that organisms from humans would be called P. carinii Humanus and Pneumocystis from rats would be designated P. carinii Rattus. Whether this system *

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Corresponding author. 137

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traditional taxonomy. For the construction of meaningful

phylogenetic trees, it will be necessary to use sequences from a type species as a standard for comparison with sequences from other organisms and to use standardized techniques of sequence preparation and analysis. Current knowledge of evolutionary relationships may be faulty, and relatedness diagrams may thus be inaccurate. It has been suggested that a better understanding of the relationship of P. carinii to other eucaryotes will be a natural consequence of the in-depth studies now being carried out (54, 84). EPIDEMIOLOGY It is well documented that healthy animals may become infected when housed with infected animals (12, 55, 76). For years, researchers have capitalized on the airborne transmission of Pneumocystis spp. in maintaining infected rat colonies. Although person-to-person transmission has not been documented, clusters or outbreaks of infection suggest that this occurs (161, 168). The reports of hundreds of cases of Pneumocystis pneumonia in children in institutions or other crowded conditions in the years following World War II also

point

to

person-to-person spread. Certainly, epidemics

or

clusters of cases of Pneumocystis pneumonia, beginning with the epidemics in Western Europe, outbreaks in hospitals (31, 49, 159), and outbreaks in organ transplant units (72, 161, 176) suggest person-to-person transmission. A report of increased incidence of Pneumocystis infection in immunosuppressed patients without AIDS in an institution where there was an increased incidence of AIDS is suggestive (74). Whether strains differ in their abilities to be transmitted and infect various hosts is not known. It has been demonstrated that most children in the United States and the Netherlands acquire antibody to the organisms by the age of 4 years (122, 137). Work by Kovacs et al. (101) showed that none of seven infants had antibody specific to P. carinii but that seven of eight normal adults did have specific antibody. It is thus presumed that the organisms are commonly present in the environment. Differences in organisms from rats and humans have been described by various workers (101, 187), suggesting that if infections are acquired by transfer across species lines, the hosts then modify the organisms. It is possible that P. carinji has an as yet undiscovered life stage that does not require a mammalian host or that there are dormant stages that persist in plants and soil. The one condition required for proliferation sufficient to cause pneumonia in any recognized host appears to be suppression of the host immune response. Pneumocystis pneumonia has been seen in most countries of the world, but its prevalence varies. There are specific case reports from Johannesburg (139), Puerto Rico (32), and Scandinavia (109), while in North America, approximately 60 to 80% of patients with AIDS develop Pneumocystis pneumonia at least once. In contrast, it is estimated that in Africa, only 7% of patients with AIDS develop Pneumocystis pneumonia. Although few cases of Pneumocystis pneumonia have been reported from Africa (46, 114), the disease is seen in African patients in Europe (17). Whether this much lower African infection incidence results from differences in the virulence of Pneumocystis strains in Africa or from a low incidence of subclinical (latent) infection in the population or both is not known. Furthermore, although plasma cell pneumonia caused by P. carinii was originally described in children in orphanages of Western Europe, such infection still occurs. For example, there have been recent reports of infection in adopted Vietnamese infants (45, 78, 152).

PATHOLOGY AND PATHOGENESIS

Traditionally, Pneumocystis pneumonia has been diagnosed by pathologists examining sections of lung tissue. The original descriptions of organism-host cell interactions were based on appearances of pulmonary lesions in hematoxylineosin stains. The foamy eosinophilic exudate and "honeycomb" appearance of the lung tissue were described as hallmarks of Pneumocystis pneumonia. Cysts could be revealed in this exudate by using methenamine-silver nitrate stains. With additional techniques for the evaluation of tissue and fluid specimens and with a much larger number of case studies, a great deal more has been learned about the infection processes. P. carinii proliferates as a trophozoite form that appears to preferentially associate with type I pneumocytes (198) by apposition of parasite and host cell membranes. It has been suggested that type II pneumocytes are sometimes affected (113). A damaging "aggressive" parasite attachment (73) to host cells was demonstrated by electron microscopy. The Pneumocystis trophozoites damage pneumocytes and cause desquamation of alveolar lining cells. Proliferation of Pneumocystis trophozoites, formation of cysts, and host response to the damaged tissue lead to filling of the alveoli with the foamy exudate described above. Host immune globulins are associated with the Pneumocystis exudate (21, 26). P. carinii may occasionally elicit other host responses. Granulomatous pulmonary lesions (18, 19, 36, 75) and cavitary lung disease (47, 140) have been described. However, it is important to note that patients with AIDS may have clinical and morphologic evidence of pulmonary damage in which there is no evidence of an etiologic agent (148). The host cell population in bronchoalveolar lavage specimens from patients with P. carinii pneumonia has fewer alveolar macrophages and more neutrophils than specimens from patients with adult respiratory distress syndrome (50). Various pathogenic mechanisms have been postulated (175). The organisms produce foci of necrosis and cellular debris in extrapulmonary sites (146). It has been suggested that fibronectin plays a role in specific binding (143) and that there is cytoskeletal protein binding and damage to host cells (110). The roles of humoral and cell-mediated immunity are still being assessed. Because AIDS patients have deficient T4 cells and development of Pneumocystis pneumonia correlates with depletion of CD4 cells to below 200/mm3 (102), some investigators postulate that cell-mediated immunity is paramount in protection. leki et al. (91) reported that the role of neutrophils is negligible; Furuta et al. (58) showed that both T cells and antibody are important for resistance to infection. The presence of antibody in AIDS patients has been evaluated, and it was found that anti-P. carinii immunoglobulin M was absent (80). Some episodes of Pneumocystis pneumonia have been seen in patients with hypogammaglobulinemia or agammaglobulinemia (149, 164). Probably both humoral and cell-mediated immunity are important in host defense. AIDS patients who do not receive prophylaxis after treatment often develop second episodes of Pneumocystis pneumonia (39) that may be refractory to therapy. These episodes may result from Pneumocystis organisms from the initial infection remaining viable or from reacquisition of P. carinii. The relative importance of these modes of recurrence is not known. A comparison of patients with AIDS and patients without AIDS who developed Pneumocystis pneumonia

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showed that those with AIDS had many more organisms but that those without AIDS had greater lung inflammation, as evidenced by more neutrophils. The greater inflammation was associated with lower arterial oxygen tension and poorer survival (111). CLINICAL MANIFESTATIONS Because P. carinii may be present with no apparent deleterious effect on the host, it is assumed that most clinical manifestations result from activation of latent infections. Organisms proliferate when there is some compromise of the immune system. In patients receiving treatment for acute lymphocytic leukemia, the time of onset of clinical Pneumocystis pneumonia varied from 30 to 100 days after the start of chemotherapy, suggesting that in this situation, a minimum of one month is required for the P. carinii organisms to reach sufficient numbers to cause disease (159). Progression of disease, types of host cell infiltration, and response to anti-Pneumocystis therapy all vary according to the type and severity of immunosuppression. Probably most infections remain subclinical. Differences in rapidity of development of pneumonia, degree of lung involvement and hypoxemia, and response to therapeutic regimens have been described for patients immunocompromised by malignancy and chemotherapy and for those immunocompromised by human immunodeficiency virus (201). Children immunocompromised by protein calorie malnutrition have a slowly developing disease, while individuals given chemotherapy for malignancy or organ transplantation frequently have rapidly developing pulmonary disease. Patients with AIDS most often have slowly developing disease (83, 177). Malnutrition probably contributes to immunosuppression in those with AIDS just as it did in children with plasma cell pneumonia. Radiographic patterns may be variable. Although it is usually reported that P. carinii causes bilateral fluffy infiltrates, atypical (42), one-sided, nodular, and cavitary lesions have also been reported (10, 56). Patients receiving aerosolized pentamidine for prophylaxis may show peripheral apical pneumonia because these areas are more difficult to reach with aerosol. Chest radiographs often appear normal when blood gas levels and clinical appearances suggest severe pneumonia (67). Gallium-67 scans have been reported to be more sensitive than chest radiographs (11, 196); however, it appears that the uptake of gallium is not specific for P. carinii but indicates an inflammatory process (79, 104). Prior to the AIDS epidemic, few cases of disseminated disease were reported (6, 9, 147). In fact, workers tried to determine why P. carinii had such a clear predilection for lung tissue and seemed to be confined to it. Now reports have shown infection in almost every organ; manifestations include thymoma (27), choroiditis (117, 150), pneumocystoma (60), necrotizing vasculitis (112), and infections of the small intestine (28), liver (142), spleen (181), bone marrow (151, 158, 159), ear (165, 170), and skin (35). The use of aerosol pentamidine for prophylaxis has contributed to this variety by suppressing lung infection while organisms disseminate to other organs. The classic symptoms of patients with Pneumocystis pneumonia are a dry, nonproductive cough, fever, weight loss, and shortness of breath. Often, the P02 is lower than would be expected from the clinical signs and symptoms and the chest radiograph.

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LABORATORY DIAGNOSIS

Although some have suggested that empiric therapy is justified for patients with symptoms compatible with Pneumocystis pneumonia (115), this is not prudent because antiPneumocystis treatment would be inappropriate for other opportunistic infections. A variety of other common opportunistic infections have been reported (200), including infections with Histoplasma capsulatum (191), Cryptococcus neoformans (193), Coccidioides immitis (171), Mycobacterium avium complex (120), Nocardia spp. (132), Toxoplasma gondii (71), cytomegalovirus, and bacteria such as Streptococcus pneumoniae. Some patients may have coinfections with P. carinii and one or more of these infectious agents. In our experience at Indiana University Medical Center, histoplasmosis frequently has been the diagnosis when history and clinical presentation suggested Pneumocystis infection. The treatment used for Pneumocystis pneumonia is not effective for histoplasmosis, just as antifungal agents are ineffective against Pneumocystis pneumonia. In addition, the infiltrates demonstrated on chest radiographs and altered lung function with a lowered PO2 and diminished diffusion capabilities may result from noninfectious processes. Ruling out infection prevents these patients from being treated with potentially toxic antimicrobial agents.

Currently, the only proven method for establishing a diagnosis of infection with P. carinii is morphologic demonstration of organisms. Clinical presentations, radiographs, and pulmonary function tests may be suggestive but are not specific for P. carinii because they can only indicate lung damage (33). Reports on elevated serum lactate dehydrogenase levels (61, 97, 167) and angiotensin converting enzyme (169) suggest that these enzymes may be useful in assessing patient response to therapy and prognosis. Increased levels of these enzymes indicate inflammation, alveolar damage, or macrophage dysfunction. Although organisms from rat or mouse lung have been grown to a limited extent in culture (13, 38, 106, 129, 178), organisms from human lung have not been cultured successfully. Culture, the gold standard of microbiology, is not useful for diagnosis at present. Tests for antibody to P. carinii are not helpful for diagnosis because most healthy individuals have antibody that can be detected by methods currently in use, while severely immunocompromised individuals with proven Pneumocystis pneumonia may not have antibody. Detection of P. carinii antigen in serum has been described (136, 138) but has not been specific and sensitive enough for establishing a diagno-

sis and thus is not recommended. Studies of patient groups with positive antigen tests have not always included sufficient controls to differentiate false-positive tests from those of subclinically infected individuals (85). A DNA probe has been described (183) which has been used successfully to detect P. carinii in bronchoalveolar lavage samples and appears promising as a diagnostic test but it is not commercially available (184). At present, diagnosis of Pneumocystis pneumonia requires demonstration of organisms in tissue or secretions from the lung (or from some extrapulmonary site). Tissue obtained by open-lung biopsy or transbronchial biopsy samples may be used. Transthoracic cutting-needle biopsy is rarely used because of the high rate of complications associated with this technique (8). Open-lung biopsy, historically the method of choice (7, 93), is seldom performed today except for some pediatric patients or patients with unusual

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diagnostic problems. Transbronchial biopsies are useful (22), but they must be performed by a skilled bronchoscopist and multiple biopsy samples must be obtained (94). Percutaneous needle aspiration has been used to obtain specimens, especially from children, and is a relatively safe procedure (8).

Most pulmonary specimens are collected by bronchoscopy and, in addition to transbronchial biopsy (see above), may include bronchoalveolar lavage fluid, bronchial brushings, and bronchial washings. Bronchoalveolar lavage fluids are superior to bronchial washings because they contain cells and organisms from the alveoli, whereas bronchial washings may contain material only from the tracheobronchial tree. Bronchial washings may not show organisms in some patients with Pneumocystis pneumonia. A variety of bronchoscopic techniques with and without fluoroscopic guidance have been described (123), and their yields have been compared. The usefulness of bronchial washings (155); endobronchial brushings (162); and minilavages, samples obtained from instillation of 20 to 50 ml of saline (199), have been compared, and their diagnostic efficacy varies from institution to institution. There are many additional reports on these procedures and various modifications. In our experience, bronchoalveolar lavage and transbronchial biopsy have been most useful. Sputum induction is a less expensive method than bronchoscopy to procure suitable specimens from both adults and children (16, 103, 135). Although at some institutions the diagnostic yields from sputum samples have been excellent, at others the yields have not been satisfactory (40, 154). Induced sputum specimens have been less useful for diagnosing P. carinii pneumonia in non-AIDS than AIDS patients. Patients with AIDS usually have very large numbers of organisms present, so that induced sputum specimens are likely to allow diagnosis. However, sputum specimens cannot be used to detect certain other organisms, whereas bronchoalveolar lavage specimens have been reported to be useful in the detection of infectious agents such as fungi (171). In one report on the successful use of induced sputum specimens for diagnosing P. carinii pneumonia, it was emphasized that training for those performing the induction process, the presence of a special team to work with patients, and careful attention to all details of the protocol are required (107). One report suggests that nasotracheal suctioned specimens are superior to induced sputum for diagnosis of P. carinii pneumonia (105). Unless induced sputum examination has been demonstrated to be both sensitive and specific for diagnosis in a particular institution, a negative sputum sample should be followed by a bronchoalveolar lavage to ensure optimal detection of organisms. Examination of induced sputum samples for diagnosis of Pneumocystis pneumonia may not be cost-effective in institutions in which only a few patients with opportunistic infections are seen. Individuals with AIDS often have severe infections, with numerous clumps of Pneumocystis organisms present in specimens, making diagnosis easy. However, bronchoalveolar lavage specimens from patients who have been given aerosolized pentamidine do not always contain organisms, and diagnosis may require a transbronchial biopsy (96). In non-AIDS patients, Pneumocystis organisms may be sparse, making their demonstration more difficult (111). Procuring specimens and processing them for optimal detection of P. carinii and other opportunists requires skilled individuals, including bronchoscopists or respiratory therapists and microbiologists or pathologists. Selection of appro-

CLIN. MICROBIOL. REV.

priate procedures should be based on the numbers and types of patients seen at an institution as well as the availability of appropriately skilled personnel. For these reasons, diagnostic yields from various specimens will vary among institutions. If a bronchoscopy is performed, there may be one or more specimens, including lavages, brushings, washings, and tissue. Tissue may be submitted for surgical pathology examinations and/or used to make impression smears for staining and examination in the microbiology or cytopathology laboratory. For impression smears, the tissue is blotted on a sterile surface to remove excess liquid and then pressed against glass slides to make impressions. Usually, several slides are prepared. Brushes may be pressed against slides for impression smears, or brushings may be vortexed in saline and centrifuged to pellet the cells before smears are made of the pellet. Lavage and wash samples are centrifuged, and slides are prepared from the pellets. A bloody specimen may require treatment of a portion of the specimen with an agent such as saponin, Zaptoglobin, or Lyse to lyse the erythrocytes. An erythrocyte lysis product used for automated leukocyte counts can usually be obtained from the hematology laboratory. By lysing erythrocytes, it is possible to concentrate the specimen and yet not destroy the appearance of the organisms. Wash samples may contain quantities of mucus that require treatment with a mucolytic agent such as Sputolysin. Specimen is added to an equal volume of the mucolytic agent and then centrifuged to pellet the cells, after which the sediment is used to prepare slides. Induced sputum samples should be treated with mucolytic agents as described for wash samples. Protocols for handling induced sputum or bronchoalveolar lavage specimens may include processing by cytocentrifugation (119), which is widely used to evaluate cell populations. Mucolytic agents must be used and the number of cells present must be evaluated to determine the amount of material to cytocentrifuge. How slides are fixed depends on the staining procedures to be performed. For most histochemical stains, methanol fixation is satisfactory. For some immunologic stains, acetone fixation is required. Opinions differ as to the most effective stain or combination of stains. The three kinds of stains are (i) organism stains, such as Giemsa and various rapid Giemsa stains, which stain trophozoites or intracystic bodies; (ii) cyst wall stains, such as the traditional methenamine-silver nitrate, which stain cyst walls and do not stain trophozoites or intracystic organisms; and (iii) immunospecific stains. Some laboratories use only a cyst wall stain, and others use only an organism stain; we recommend that both an organism and cyst wall stain be used. For patients with numerous clumps of organisms, staining lavage fluid with a rapid Giemsa stain such as Diff Quik or Giemsa Plus may allow diagnosis in 1 or 2 min. Traditional Giemsa staining takes approximately 30 min, but it provides better morphologic detail and may reveal rare clusters of trophozoites more clearly than the rapid modifications. A comparison of stain methods emphasizes the usefulness of Giemsa stain for demonstrating trophozoites (81). Wright stain (41) is similar to Giemsa stain but results in less color contrast. In Giemsa stains, trophozoites and intracystic bodies have a dotlike red to violet nucleus with a blue cytoplasm surrounding it (Fig. 1). Trophozoites are from 2 to 5 ,um in diameter and often occur in groups. Individual trophozoites may be difficult to distinguish from cell fragments. When mature, cysts have intracystic bodies which vary from round

VOL. 4, 1991

P. CARINII IN IMMUNOCOMPROMISED PATIENTS

to spindle shaped. The cyst wall does not stain and is seen as a clear halo around the intracystic organisms. In patients with severe infections, especially patients with AIDS, there may be very large clumps of P. carinii which can be easily overlooked because they are so dense and deeply stained that individual organisms are not distinguishable. On close examination, cysts and trophozoites can often be recognized in the thinner areas of these clumps. P. carinii is generally extracellular, but organisms may occasionally be detected in macrophages. Cyst wall stains include methenamine-silver nitrate and modifications of this stain for rapid processing (25, 77, 118, 130, 141), cresyl echt violet (23, 62), toluidine blue 0 (30, 69), Gram-Weigert (156), and calcofluor (14). Problems with toluidine blue 0 sulfation reagents (166) and differences in dye lots (194) have been reported. Because cyst walls of P. carinii contain chitin (185), whitening agents such as calcofluor may be used (14) to demonstrate P. carinii by fluorescence microscopy. In cyst wall stains, cysts frequently have dark areas that are often described as "parentheses" or dots (Fig. 1). Cyst walls may have folds, and cysts may be cup shaped. The dot and parenthesislike shapes indicate differences in cell wall thickness, a correlation demonstrated by the excellent histochemical electron micrograph studies of Watts and Chandler (190). There may be some variation in the intensity of staining of cysts. If only occasional silverstained organisms are seen, it may not be possible to differentiate Pneumocystis cysts from yeast cells or other fungal elements. This may make evaluations of specimens from patients with AIDS more difficult, as these patients frequently have oropharyngeal candidiasis. Both Gram (48) and Papanicolaou (63) stains have detected cysts but are not optimal stains and should not be relied upon for detection of P. carinii. Since these methods demonstrate cyst walls of P. carinii and walls of fungi, Pneumocystis-positive, Pneumocystisnegative, and yeast-positive samples should be included as controls. Experience is required to ensure that yeasts are not mistakenly identified as Pneumocystis cells. Control slides are not needed for Giemsa stain and its modifications because host cells in the specimen serve as internal controls. Immunologic staining with monoclonal antibodies specific for P. carinii from human lung (64) may allow more accurate diagnosis than histochemical staining. Commercial products are available, and some have been evaluated (20, 134). Immune-specific staining with commercial reagents has not been used widely enough to predict its usefulness, and difficulties have been reported (99). An evaluation of one commercial immune-specific stain method suggests that the indirect fluorescent-antibody stain (IFA) is more sensitive than conventional histochemical stains (134). Of 182 specimens examined, 17 were positive by IFA but none of the other stains; thus, the sensitivity of IFA was 30% higher than that of histochemical stains. However, of these 17 IFApositive specimens, only 3 could be determined to be true positives. If the 14 that were positive only by IFA were excluded, there would be no significant difference in sensitivity between IFA and histochemical staining. In this study, a change in IFA evaluation criteria to minimize false positives would have changed the detection rate for knownpositive specimens (positive by other stains) to 51%. When the laboratory examines specimens from many patients with Pneumocystis pneumonia or from AIDS patients who have numerous organisms, diagnosis can be made in minutes with a rapid Giemsa stain; therefore, the cost of and time for immune-specific staining are not justified (20). Another dis-

advantage of immunologic stains is that they do not allow detection of other organisms, such as Histoplasma and Cryptococcus spp., bacteria, and Toxoplasma gondii. Immunologic staining might be reserved for specimens that are negative by rapid histochemical stains. With kits for immunologic stains, the package directions must be followed closely. Positive controls must be included with all cyst wall and immunologic stain procedures for P. carinii. If those examining slides are not highly skilled in differentiating P. carinii cysts from yeast cells, a slide of material containing yeast cells should be included as a negative control. The morphology of P. carinii is quite different in cyst wall stains and in organism stains. We recommend that a laboratory perform one cyst wall stain and one organism stain to ensure accurate diagnosis of Pneumocystis pneumonia and to allow detection of other opportunistic infections. If one or two P. carinii cysts are found in both organism and cyst wall stains, most likely the organisms represent infection. Determining whether the infection is the cause of clinical symptoms requires interpretation by the attending physician.

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TREATMENT

Pentamidine isethionate was used to treat children with immune deficiencies or patients receiving transplants who developed Pneumocystis pneumonia until Hughes demonstrated the usefulness of TMP-SMX. He first tested it in animal models (90) and then used it to treat children with Pneumocystis pneumonia (86) and for prophylaxis (89). Since 1975, TMP-SMX has been considered the drug of choice for the treatment of Pneumocystis pneumonia and has been widely used for prophylaxis in patients receiving transplants or antineoplastic therapy. Although, as with most therapeutic agents, there were side effects in non-AIDS patients, these effects were not common and were less common and less severe than those caused by pentamidine. In AIDS patients, the proven-effective regimen of TMPSMX caused severe adverse reactions (68, 92), including skin rashes, granulocytopenia, and liver function changes in up to two-thirds of patients. Because of these adverse reactions, desensitization strategies (192) and changes in dosage (121) have been tried. Some clinicians prefer to continue TMP-SMX despite rash development because rashes may clear. However, because of the high frequency of adverse reactions, TMP-SMX has not been widely used for prophylaxis in AIDS patients. Parenteral pentamidine has been reported to cause hypo-

glycemia (174, 188), hyperglycemia (131), neutropenia (5), abscess formation at injection sites (5), and fatal acute pancreatitis (202). Intravenous administration is the usual route when treating Pneumocystis pneumonia. Most of the side effects of pentamidine can be avoided by changing the

route of administration from intravenous or intramuscular injection to aerosol inhalation. When pentamidine is aero-

solized, it is deposited in the lungs, where organisms are concentrated. Levels of the drug in serum and nonpulmo-

nary tissue are much lower than when it is given intramuscularly or intravenously, and therefore toxicity is decreased. Aerosolized pentamidine has been used both for prophylaxis and for treatment (34, 125-127), although it is most widely used for prophylaxis. Since the adoption of pentamidine for prophylaxis, many reports of disseminated Pneumocystis infection in patients receiving aerosolized pentamidine prophylaxis suggest that it only suppresses development of

pulmonary infection (142, 151, 170). One report of extrapul-

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c FIG. 1. P. carinii in stained smears. Magnification, x970. (a) Rat lung impression smear, Giemsa stain. Cysts containing up to eight intracystic organisms are evident. Cyst walls are unstained and appear as clear spaces around the intracystic organisms. The small dotlike nuclei of trophozoites are present in other areas. Trophozoites are as large as or larger than the individual intracystic organisms. The large dark masses are host cells. (b) Human bronchoalveolar lavage sediment smear, Giesma stain. This large clump of Pneumocystis organisms could be easily overlooked or misinterpreted as an artifact. Note the numerous dotlike nuclei of Pneumocystis organisms and the circular outlines of some cysts in the mass of organisms. (c) Human bronchoalveolar lavage sediment smear, Giesma stain. This small clump of Pneumocystis trophozoites shows dark nuclei with irregular shapes. Pneumocystis organisms generally are found in clumps of various sizes. 142

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(d) Human bronchoalveolar lavage sediment smear, rapid methenamine-silver nitrate stain. This clump of Pneumoc-ystis organisms is similar panel b. The number of cysts is much smaller than the number of trophozoites, and some clumps do not contain cysts (panel c). Note that cysts often have thickened areas in the wall that stain more darkly and may appear to be a single dot or a set of parentheses. Some cysts show linear folds. (e) Human bronchoalveolar lavage sediment smear, rapid methenamine-silver nitrate stain. These cysts are similar to those in panel d but stain somewhat darker. Folds are prominent in some cysts. (f) Human bronchoalveolar lavage sediment smear, modified toluidine blue 0 stain. These cysts are similar in appearance to those stained with rapid methenamine-silver.

to that shown in

143

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CLIN. MICROBIOL. REV.

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infection in 2.5% of AIDS patients at autopsy suggests that aerosol pentamidine prophylaxis may not be monary

optimal (179). In addition, recurrences of pulmonary Pneumocystis infection may occur in some patients while they are receiving prophylaxis (1, 24). Successful Pneumocystis treatment and prophylaxis with TMP-SMX suggested that other antifolate inhibitors might be effective. The effects of drugs on dihydrofolate reductase extracted from Pneumocystis organisms were studied (3), and the drugs were tested in animal models. Two potent dihydrofolate reductase inhibitors, trimetrexate and piritrexim, showed anti-Pneumocystis activity in animal models (100, 144) and clinical trials (2, 163). Trimetrexate was effective for therapy, but relapse after treatment was frequent. Patients treated with trimetrexate required concurrent leucovorin rescue, making the regimen costly. Dapsone has been used both alone and in combination with SMX or TMP (43, 108). The dapsone combinations are effective, but some problems with adverse reactions remain (153). Supplemental steroid therapy in addition to the use of anti-Pneumocystis drugs has been successful with certain groups of patients (4, 128, 186), but was usually reserved for those with severe pneumonia who were failing on other treatments. A report that using corticosteroids early in treatment of patients with moderately severe P. carinii infection improves survival has led to wider use of corticosteroids (124). Other drugs that have been used in limited trials include difluoromethylornithine (66) and fansidar, which is a combination of pyrimethamine and sulfadiazine (70). Data on the usefulness of difluoromethylornithine are difficult to interpret because the drug has been used primarily in salvage protocols and the residual effects of previously used drugs make responses difficult to interpret. Recurrent infection after treatment with fansidar has been reported (57), and this drug may cause serious adverse reactions, such as Stevens-Johnson syndrome, which limit its usefulness. Drugs directed to targets other than folate metabolism may avoid the adverse reactions associated with these compounds. The combination of clindamycin and primaquine was shown to be effective in animals (145) and has been used successfully for the treatment of Pneumocystis pneumonia (98, 160, 180). Other 8-aminoquinolines have been evaluated for treatment and prophylaxis in animal models and appear to be very effective (12a, 12b). The compounds are being considered for clinical trials. A new compound, hydroxynaphthoquinone (566C80), has been reported to be effective in animals (88), and early clinical trials are in progress. A drug with little toxicity which can be taken orally for long-term prophylaxis is urgently needed because patients with AIDS have recurring or multiple infections. Patients at high risk for opportunistic infections (CD4 counts of less than 200/mm3) and patients who have recovered from Pneumocystis pneumonia require prophylaxis. As improved antiviral therapies, such as zidovudine and dideoxyinosine, increase the lifespans of AIDS patients, opportunistic infections become more important. Prophylactic regimens which simultaneously prevent infections with P. carinii plus T. gondii or Cryptosporidium spp. would be especially appealing.

The role of immune modulators is not clear. As yet, there of their effective use for treatment or prevention of Pneumocystis infection. Good nutrition probably is very important because malnutrition is itself immunosuppressive. Until a niche in the environment has been described for P. are no reports

carinii and until more is known about the spread of the organism among people and the role, if any, of reservoir hosts, ways to avoid acquiring the organisms cannot be determined with certainty. To prevent disease from developing, we must prevent severe immunosuppression or provide drug prophylaxis to prevent proliferation of organisms. Basic studies on host cell-parasite interactions and description of the adherence mechanism may allow specific intervention in the process and prevent initial attachment and establishment of infection. In the future, some of the genes cloned and DNA or RNA sequences specific to P. carinii being developed by workers today may be made available commercially in kits that are convenient and effective for detection of P. carinii. Such methods would rapidly and accurately diagnose Pneumocystis pneumonia and replace the morphologic diagnostic method, which requires skilled personnel and is time consuming. REFERENCES 1. Abd, A. G., D. M. Nierman, J. S. Ilowite, R. N. Pierson, Jr., and A. L. L. Bell, Jr. 1988. Bilateral upper lobe Pneumocystis carinii pneumonia in a patient receiving inhaled pentamidine prophylaxis. Chest 94:329-331. 2. Allegra, C. J., B. A. Chabner, C. U. Tuazon, D. Ogata-Arakaki, B. Baird, J. C. Drake, and H. Masur. 1988. Treatment of Pneumocystis carinii pneumonia with trimetrexate in acquired immunodeficiency syndrome (AIDS). Semin. Oncol. 15:46-49. 3. Allegra, C. J., J. A. Kovac, J. C. Drake, J. C. Swan, B. A. Chabner, and H. Masur. 1987. Activity of antifolates against Pneumocystis carinii dihydrofolate reductase and identification of a potent new agent. J. Exp. Med. 165:926-931. 4. Amandson, D. E., K. M. Murray, S. Brodine, and E. C. Oldfield. 1989. High-dose corticosteroid therapy of Pneumocystis carinii in patients with acquired immunodeficiency syndrome. South. Med. J. 82:711-714. 5. Andersen, R., M. Boedicker, M. Ma, and E. J. C. Goldstein. 1986. Adverse reactions associated with pentamidine isethionate in AIDS patients: recommendations for monitoring therapy. Drug Intell. Clin. Pharm. 20:862-868. 6. Awen, C. F., and M. A. Baltzan. 1971. Systemic dissemination of Pneumocystiis carinii pneumonia. Can. Med. Assoc. J. 104:809-812. 7. Balantine, T. V. N., J. L. Grosfeld, R. M. Knapek, and R. L. Baehner. 1977. Interstitial pneumonitis in the immunologically suppressed child: an urgent surgical condition. J. Pediatr. Surg. 12:501-508. 8. Bandt, P. D., N. Blank, and R. A. Castellino. 1972. Needle diagnosis of pneumonitis: value in high-risk patients. J. Am. Med. Assoc. 220:1578-1580. 9. Barnett, R. N., J. G. Hull, V. Vortel, and J. Schwarz. 1969. Pneumocystis carinii in lymph nodes and spleen. Arch. Pathol. 88:175-180. 10. Barrio, J. L., M. Suarez, J. L. Rodriguez, M. J. Saldana, and A. E. Pitchenik. 1986. Pneumocystis carinii pneumonia presenting as cavitating and noncavitating solitary pulmonary nodules in patients with the acquired immunodeficiency syndrome. Am. Rev. Respir. Dis. 134:1094-1096. 11. Barron, T. F., N. S. Birnbaum, L. B. Shane, S. J. Goldsmith, and M. J. Rosen. 1985. Pneumocystis carinii pneumonia studied by gallium-67 scanning. Radiology 154:791-793. 12. Bartlett, M. S., M. M. Durkin, M. A. Jay, S. F. Queener, and J. W. Smith. 1987. Sources of rats free of latent Pneumocystis carinii. J. Clin. Microbiol. 25:1794-1795. 12a.Bartlett, M. S., S. F. Queener, M. M. Durkin, M. M. Shaw, and J. W. Smith. 1989. Program Abstr. 29th Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. 1023. 12b.Bartlett, M. S., S. F. Queener, M. A. Jay, M. M. Dorkin, and J. W. Smith. 1988. Program Abstr. 28th Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. 1023. 13. Bartlett, M. S., P. A. Verbanca, and J. W. Smith. 1979.

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Pneumocystis carinii, an opportunist in immunocompromised patients.

Pneumocystis carinii has been recognized as a cause of pneumonia in immunocompromised patients for over 40 years. Until the 1980s, Pneumocystis pneumo...
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