Keywords

Case Presentation

A 57-year-old man with a past medical history of bilateral lung transplant presented with increasing shortness of breath to his outpatient pulmonologist. Chest computed tomography (CT) showed diffuse patchy infiltrates, most prominent in the para-hilar region, with associated lymphadenopathy (Fig. 53.1). The patient underwent bronchoscopy and transbronchial biopsy, which showed granulomatous inflammation with few associated organisms, thought to be consistent with P. jirovecii. Prolonged treatment for pneumocystis pneumonia was not effective, and the patient eventually underwent wedge biopsy, which showed necrotizing granulomas and small, narrow-based budding yeasts, morphologically most consistent with H. capsulatum (Fig. 53.2). Fungal cultures obtained from fresh tissue confirmed the diagnosis.

Fig. 53.1
A C T of the chest illustrates the diffuse patchy white spots, in the para hilar region with visible swelling of lymph nodes.

Chest CT showing diffuse patchy infiltrates, most prominent in the para-hilar region, with associated lymphadenopathy

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Fig. 53.2
The histological images of histoplasmosis illustrate the necrotizing granulomas and small, narrow-based budding yeasts,

Typical morphology of histoplasmosis. The organisms are small, ovoid yeast forms with narrow-based budding, as contrasted with the folded, umbilicated forms with an argyrophilic dot as seen in P. jirovecii

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Final Diagnosis: Small Yeasts with Narrow-Based Budding

Comment: These findings are suggestive of Histoplasma capsulatum but may be confused with other small yeasts such as Candida glabrata, Pneumocystis jirovecii, and others. Correlation with microbiology studies is necessary to identify the infectious organism.

Clinical Considerations

Histoplasma capsulatum is an invasive endemic pulmonary fungal pathogen that infects immunocompetent hosts and is seen as narrow-based budding yeast on histologic examination. In North America, Histoplasma is most frequently found in the distribution of the Ohio and Mississippi rivers [1], but it is globally distributed and prevalent in sub-Saharan Africa [2, 3] The clinical presentation of histoplasmosis has a wide range of clinical severity: most frequently it is asymptomatic and self-limiting [4] but can produce an acute presentation, a chronic infection akin to mycobacterial disease, or disseminated disease.

P. jirovecii, previously characterized as a protozoan, is now classed as a fungus. It is present worldwide, and although it is overwhelmingly a disease of the immunocompromised, at least one study has found a carrier rate of up to 20% in healthy, immunocompetent adults [5]. Pneumocystis pneumonia is one of the most commonly encountered lung infections in people with impaired cell-mediated immunity, such as those with advanced HIV [6], organ transplants, or hematological malignancy. Unlike Histoplasma spp. and other fungi, P. jirovecii cannot be grown by microbiologic culture [7].

Radiologic Features

Typical radiographic findings in Pneumocystis pneumonia are bilateral, diffuse alveolar, and interstitial infiltrates that are typically either lower lobe or hilar predominant but may diffusely involve the lungs. However, multiple atypical radiographic patterns have been reported and include nodular lesions, lobar consolidation, unilateral involvement, cystic spaces, and hilar lymphadenopathy [8]. Up to 20% of patients with AIDS may present with no radiographic abnormalities on chest radiographs, although they will likely have ground-glass opacities on subsequent CT [9]. The key message is that Pneumocystis pneumonia is compatible with a wide array of radiographic findings.

In acute histoplasmosis, radiographs most often show patchy consolidation in one or more lobes, frequently with prominent hilar or mediastinal lymph nodes [10]. Severe disease can show diffuse reticulonodular infiltrates, and resolved cases may have calcified nodules. However, a substantial portion of cases may have normal chest radiographs [4, 10]. The variety of radiographic findings reflects the diversity of pathologic manifestations: histoplasmosis can produce an acute presentation, chronic infection akin to mycobacterial disease, disseminated disease, mediastinitis, or a circumscribed nodule of largely resolved infection.

Histologic Features

The diagnosis of Pneumocystis pneumonia can be accomplished by recognition of characteristic histomorphologic features of the organism and intra-alveolar exudates (Fig 53.3a). Four developmental forms of P. jirovecii are described, in keeping with its former protozoan classification: trophozoites, precysts, cysts, and sporozoites (also known as intracystic bodies). For the purpose of pathologic identification, cysts and trophozoites are the most relevant. The cyst is the largest, most easily recognized stage, and is easily demonstrated with GMS stain. The vegetative forms of Pneumocystis, trophozoites, are 1–5 μm in diameter and characteristically attach to type I pneumocytes. The cyst is 5–7 μm in diameter and most contain up to eight intracystic bodies (sporozoites), each 1–2 μm in diameter. These sporozoites have a single nucleus. Some cysts will contain internal comma-shaped structures, either singular or in pairs, that are localized thickenings of the internal layer of the cyst wall. Ruptured cysts that have released their sporozoites will have a characteristic cup or crescent shape when collapsed. Intracystic sporozoites are not visible on histopathologic stains. The cysts are round to oval, focally curved, with crushed “boat” forms present (Fig. 53.3c). These structures are useful in identifying P. jirovecii as compared to budding yeast pathogens. When found separately from the cysts, sporozoites and trophozoites are difficult to identify and distinguish from background debris or tissue, but they can have an amoeboid appearance in tissue sections.

Fig. 53.3
The histological images illustrate the alveolar filling pattern in pneumonia. It indicates the alveolar filling pattern with eosinophilic exudate typical and silver stain for P. jirovecii .

Typical morphology and alveolar filling pattern of P. jirovecii pneumonia. Low (100×) magnification sections of a postmortem lung stained with H&E (a) and GMS (b) demonstrate an alveolar filling pattern with eosinophilic, proteinaceous exudate typical for P. jirovecii pneumonia. The opportunistic pathogens are abundant within intra-alveolar exudates and easily seen with a silver stain (b, c). Note the central umbilication and slightly folded appearance of the organisms at high (400×) magnification (c)

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Grossly, in patients with P. jirovecii pneumonia, the lungs are typically heavy with a gray or tan consolidated cut surface. The most typical histologic features are that of mild interstitial mononuclear inflammation, type II pneumocyte hyperplasia, and eosinophilic foamy intra-alveolar exudates that expand the alveolar spaces (Fig. 53.3a). On higher magnification, the exudates can be punctuated by round basophilic dots that correspond to sporozoite and trophozoite nuclei. These help to distinguish the findings from pulmonary edema or alveolar proteinosis. In tissue sections, there is often characteristic retraction of the exudate from the adjacent alveolar wall.

Although the above features are the most common histologic pattern, P. jirovecii can provoke essentially any lung injury reaction pattern. In chronic infection, interstitial fibrosis may be seen. In an acute and progressive infection, diffuse alveolar damage, hyaline membranes, and reactive epithelial proliferation can be seen. There is often robust lymphoplasmacytic inflammation and type II pneumocyte hyperplasia that can be misinterpreted as lymphocytic interstitial pneumonia (LIP) or nonspecific interstitial pneumonia (NSIP). In one study, 19% of patients lacked the characteristic alveolar exudates, and many showed atypical features such as interstitial inflammation (63%), fibrosis (50%), numerous alveolar macrophages resembling desquamative interstitial pneumonia (9%), granulomatous inflammation (5%), hyaline membranes (4%), and interstitial pneumonitis (3%) [11]. Due to this multitude of possible histologic patterns, the differential diagnosis of opportunistic lung infections frequently includes both Pneumocystis pneumonia and endemic mycoses such as H. capsulatum. In such cases, diagnosis may be delayed without definitive histologic or microbiologic evidence of organisms.

Several infectious etiologies could be confused for P. jirovecii. These include Histoplasma capsulatum, Candida glabrata, and Cryptococcus spp. Each of these entities has characteristic findings to suggest it. H. capsulatum has small, ovoid yeast forms (2–5 μm) that show narrow-based budding, which is absent in Pneumocystis. Yeast forms of H. capsulatum typically stain poorly in routine preparations but stain well with GMS (Fig. 53.1). In addition, H. capsulatum is primarily an intracellular pathogen, whereas P. jirovecii is primarily an extracellular organism within alveolar spaces. Cryptococcus yeasts (5–15 μm) are larger than either H. capsulatum or P. jirovecii and typically have a capsule. Pseudohyphae production by a small yeast, like C. glabrata, would argue strongly against the above pathogens.

Special Stains

Special stains for Pneumocystis can be grouped into two general categories: those that highlight the cyst wall and its internal structures and those that stain the nuclei of trophozoites and sporozoites. Only cytologic specimens are usable in the case of the latter, and multiple stains, including Giemsa, Wright, and Diff-Quik, can highlight the vegetative state nuclei. More commonly used in clinical practice are stains that highlight the cyst wall. Of these, GMS is the most reliable and sensitive and will highlight the walls of round to oval 5–7 μm cysts and any “crescent” or “boat” forms (Fig. 53.3b) within the exudates. Some cysts have a unique argyrophilic peripheral dot-like structure and are helpful to distinguish cysts from budding yeast forms. This feature is also present as a basophilic dot in cyst forms when seen on hematoxylin and eosin (H&E) preparations. Taken together (Fig. 53.3a), these findings are highly suggestive of Pneumocystis pneumonia. In cases where Cryptococcus is in the differential diagnosis, a mucicarmine stain is a useful adjunct to visualize capsule; however, capsule-deficient forms exist.

Molecular and Microbiologic Features

While H. capsulatum grows reliably in culture, P. jirovecii cannot be grown on cell-free media. The inability to culture P. jirovecii underlines the importance of clinical suspicion, awareness of key morphologic features, and importance of ancillary detection methods when P. jirovecii pneumonia is suspected. Both H. capsulatum and P. jirovecii can be detected and distinguished from each other by broad-range fungal PCR with sequence-based identification or species-specific PCR. Species-specific assays employ primer sets optimized to bind the target organism’s DNA, such as the Pneumocystis jirovecii gene cdc2, a conserved species-specific cell division cycle2 [12, 13]. Optimized primer binding is thought to have higher analytical sensitivity than broad-range PCR assays. Clinical sensitivity is influenced by pretest probability of infection, tissue volume [14], organism burden, and target gene copy number.

Some microbiology laboratories continue to offer direct fluorescent antibody (DFA) staining of respiratory fluids for Pneumocystis detection, although a lack of control material threatens the continued use of these assays (personal communication). The clinical sensitivity of DFA staining is also subject to organism burden and sample volume but performs well in high-prevalence populations, with sensitivity ~55% in HIV-infected patients with suspected PJP [15]. DFA is therefore an excellent assay when PJP is suspected; however, the addition of a species-specific PCR increases sensitivity [12, 13] and should be considered when clinical suspicion is high but DFA is negative. PCR is of particular value when cytopathology or histopathology specimens are the only available diagnostic material.

Key Points for Differentiating P. jirovecii and H. capsulatum

Both Pneumocystis jirovecii and Histoplasma capsulatum Cause Pulmonary Infections in Overlapping Patient Populations. What Histopathologic Features Help Distinguish Between P. jirovecii and H. capsulatum?

Two important differences are very helpful in distinguishing these two infections: tissue distribution and tissue reaction pattern. P. jirovecii is predominantly found in the alveolar spaces and is often accompanied by a “frothy” eosinophilic exudate that fills the alveolar space. H. capsulatum, on the other hand, germinates from inhaled conidia inside alveolar macrophages and is primarily intracellular with lymphatic dissemination. Eosinophilic exudates in the alveoli are not a typical feature of H. capsulatum. Confusion in diagnosis can arise when the typical alveolar exudate of P. jirovecii is absent, when atypical tissue reaction patterns—such as granulomatous inflammation or interstitial fibrosis—are present, or when the organisms cannot be well-visualized.

What Morphologic Features Help Distinguish These Two Organisms from Each Other?

These two organisms have similar morphology but can often be distinguished from each other using several morphologic features. First, P. jirovecii cysts are slightly larger (5–7 μm) than H. capsulatum (2–5 μm) and have a characteristic boat- or cup-like forms and perinuclear dot-like structures indicative of sporozoite release. These morphologic features are important to note since the sizes of the organisms are similar. Second, P. jirovecii lack the narrow-based budding typical of H. capsulatum dividing yeasts.

What Laboratory Tests Are Available to Confirm the Diagnosis?

Culture is always important in the evaluation of infectious diseases, but only H. capsulatum will grow in microbiologic culture. Two other types of assays in the clinical microbiology laboratory are both sensitive and specific: PCR (both organisms) and DFA (P. jirovecii only).