January 2013

A 48 year old female with shortness of breath, cough, and fever

Zoe Kinkade M.D. and Rocco LaSala M.D.


A 48 year old Caucasian female presented to clinic on Friday afternoon with shortness of breath, cough, and fever of several weeks duration. Past medical history was significant for HIV infection for almost 20 years with prior IV drug abuse, COPD, hepatitis C, and hepatitis B. The patient reported not taking anti-retroviral therapy for over one year due to gastrointestinal symptoms and for social reasons.

The patient exhibited pancytopenia (WBC 1800/µL, hemoglobin 8.6 g/dL, and platelets 120,000/µL) with moderately elevated liver enzymes. Her absolute CD4 count was 3/µL and HIV plasma viral load was >500,000 IU/mL. Radiographic imaging showed a focal left pneumonic infiltrate and bilateral ground glass opacities. The patient was admitted to general medicine service, and a diagnostic bronchoalveolar lavage (BAL) was performed that evening. Lavage fluid was submitted for bacterial, mycobacterial and fungal cultures/stains, cell count and differential, viral molecular testing and cytology.

Gross Description


Representative images of the BAL demonstrated the etiology of the pulmonary process(Figures 1 & 2). At that time, all microbiology cultures were pending. Gram stain demonstrated moderate Gram positive cocci in chains and few squamous epithelial cells.

Figure 1 : Wright-stained BAL fluid (400X).
Figure 2 : Wright-stained BAL fluid (1000X).


What is the most likely etiology of this patient’s pulmonary disease ?


Please select an answer above.


Pneumocystis jiroveci is an unusual, opportunistic fungus that infects humans and is among the leading causes of fatal opportunistic infections in HIV patients [1]. There are several species of Pneumocystis including P. carinii, which comprises multiple subspecies and primarily infects rats, and P. jiroveci (formerly P. carinii), which is the causal agent in human infections [2]. The life cycle of P. jiroveci is quite atypical for a fungus, which is probably why the organism was originally classified as a protozoan. Three morphologic forms exist: cysts, sporozoites, and trophozoites. Cysts are 5-8 µm, round or cup-shaped and may resemble dented ping pong balls. Within the cysts are up to eight sporozoites, which are 1-2 µm. Trophozoites are 2-5 microns, extracellular, and usually form clusters [2]. All morphologic forms are found in the lung.

Pneumocystis infections occur mainly in immunocompromised hosts, particularly those with HIV, patients receiving chemotherapy or long-term corticosteroids, patients with hematologic malignancies, and transplant recipients. The clinical presentation includes a dry cough, fever, and shortness of breath. Pneumocystis pneumonia (PCP) may be diffuse or patchy and characteristically produces a “ground glass” infiltrate on radiography. Extrapulmonary infection may occur rarely and has been described involving the brain, liver, kidneys, and spleen [2]. HIV patients are at highest risk when CD4 counts fall below 200/µL. The clinical differential diagnosis includes adult respiratory distress syndrome (ARDS) and other types of infectious pneumonia (bacterial, viral, mycobacterial).

The primary method of diagnosis for PCP is microscopic visualization of the organism. Unlike most other fungal pathogens, P. jiroveci cannot be cultured on standard laboratory media. In most cases, detection of a characteristic foamy, proteinaceous alveolar cast material in histologic lung sections (Figure 7) or respiratory cytology preps (Figures 1, 2, 8) is an initial a clue to the presence of the organism. Host inflammatory response is typically sparse, though granulomatous reactions have been described rarely [3]. The organism damages the alveolar pneumocytes but does not penetrate the alveolar wall [4].

Figure 7 : H&E-stained lung section depicting pulmonary alveoli filled with eosinophilic, proteinaceous debris. Note minimal inflammation present (20X).
Figure 8 : Papanicolaou-stained BAL showing characteristic foamy proteinaceous alveolar cast (400X).

A Gomori-methenamine silver (GMS) stain, which highlights the cup-shaped cyst forms, is most commonly used to visualize the organism (Figure 9) [5]. As in the present case, GMS stains may not be available on off-shifts, so Romanowsky stains (e.g. Wright, Geimsa, Diff-quik, etc.) may be used to observe the trophozoite forms localized within “negative-image” cyst spaces throughout the foamy exudate (Figure 2). Fluorescence microscopy may also be utilized in conjunction with calcofluor white stain (a fluorescent brightener that highlights most fungi by binding to cellulose and chitin constituents of the cell wall) or fluorophore conjugated antibodies specific for Pneumocystis (i.e. direct fluorescence antibody, DFA). In histologic sections or cytology cell blocks, immunohistochemistry is also available.

Figure 9 : GMS-stained BAL showing a group of 6-7 µm round to cup-shaped cysts of Pneumocystis (1000X).

Possible differential diagnoses of a positive GMS stain may include Histoplasma capsulatum, Cryptococcus neoformans, Blastomyces dermatiditis or Candida spp. [1] though each can be differentiated as follows:

  • Choice A – Histoplasma yeast forms are small (2-4 µm), ovoid with crescent-shaped nuclei and are usually located within macrophages (Figure 3).
  • Choice C – Cryptococcus does not typically form aggregates on cytologic examination, but rather appears as single, round yeasts with occasional narrow-based budding and extreme variability in size (4-20µm) (Figure 4) with our without a surrounding halo produced by polysaccharide capsular material.
  • Choice D – Blastomyces is large (~20 µm) with refractile cell walls and often exhibits broad-based budding (Figure 5).
  • Choice E – Candida spp. typically demonstrate budding yeast forms with formation of pseudohyphae (Figure 6). Their presence in BAL fluid usually represents contamination from the upper oropharynx rather than an infectious process.

Samples can also be evaluated using PCR methods.

Figure 3 : Wright-stained BAL fluid demonstrating intracellular yeast forms of Histoplasma capsulatum (1000X).
Figure 4 : Pap-stained BAL fluid showing variably-sized, round yeast forms of Cryptococcus neoformans (1000X).
Figure 5 : Pap-stained BAL fluid demonstrating large, retractile yeast forms of Blastomyces dermatiditis (400X).
Figure 6 : Wright-stained BAL fluid demonstrating oblong, budding yeast forms with pseudohyphae (1000X).

Possible differential diagnoses of a positive GMS stain may include Cryptococcus neoformans, Histoplasma capsulatum, Blastomyces dermatiditis or Candida spp. [1] though each can be differentiated as noted in the above question. Samples can also be evaluated using PCR methods. Although this approach may be more sensitive, it also is prone to false positivity (due to non-pathogenic colonization). Further, molecular detection is more costly and is not as widely available [6]. If less invasive methods of detection fail, a transbronchial lung biopsy may be performed in order to identify the organism. Histologically, lung alveoli are filled with eosinophilic foamy debris which contains trophozoites and sporozoites and are PAS and GMS positive. The major histologic differential diagnosis is alveolar proteinosis, which is nearly identical histologically except there is generally no inflammation, and organisms will not be seen with a GMS stain. Pnemocystis jiroveci is present worldwide in the environment and the transmission pattern is not completely understood. The two main theories for organism acquisition are from environmental (air) sources versus spread between human carriers. Studies to elucidate the transmission cycle have shown varied results. Some demonstrate immunocompetent patients to be carriers through use of PCR techniques [6]. Others have shown the ability of immunocompetent patients to clear the organism. Still others have examined clustering of cases within areas, which likely indicate airborne spread. Most likely, transmission occurs by a variety of these routes [2].

Treatment of P. jiroveci infection is relatively straightforward once a definitive identification of the organism takes place. Typically, trimethoprim-sulfamethoxazole or inhaled pentamidine isothionate are used for treatment. These agents may also be used for prophylaxis in HIV patients with CD4 counts < 200 µL or in persons at high risk including cancer and transplant patients [1]. Pentamidine is less often used and can sometimes interfere with identification of the organism in break-through infections (infections while on prophylaxis). A corticosteroid is often added to the regimen. Several other drugs are available for patients who cannot take TMP-SMX or pentamidine, and treatment generally proceeds for around three weeks.


  1. Tomashefski JF, et. al. Dail and Hammar’s Pulmonary Pathology: Volume 1: Nonneoplastic Lung Disease. New York City: Springer Science+Business Media, LLC., 2008.
  2. Tietjen P. (2012). Clinical presentation and diagnosis of Pneumocystis infection in HIV- infected patients. In J. Bartlett (Ed.), UpToDate. Available from http://www.uptodate.com.
  3. Watts JC, Chandler FW. Pneumocystosis. Connor DH and Chandler FW (Eds.) Pathology of Infectious Diseases: Volume II. Appleton and Lange, 1997.
  4. Pathology Outlines. (2012). Retrieved October 13, 2012, from http://www.pathologyoutlines.com/topic/lungnontumorPCP.html.
  5. Procop GW, Haddad S, Quinn J, et. al. Detection of Pneumocystis jiroveci in Respiratory Specimens by Four Staining Methods. J Clin Microbiol 2004; 42(7):3333-3335.
  6. Medrano FJ, Montes-Cano M, Conde M, et. al. Pneumocystis jiroveci in the General Population. Emerg Infect Dis 2005; 11(2):245-249.