Opportunistic Atypical Fungus: Pneumocystis jiroveci

Published on 08/02/2015 by admin

Filed under Basic Science

Last modified 22/04/2025

Print this page

This article have been viewed 2430 times

Opportunistic Atypical Fungus

Pneumocystis jiroveci

1. Describe the symptoms of Pneumocystis jiroveci infection and the cells affected by this organism.

2. List the appropriate specimen types collected for diagnosis of pneumocystis pneumonia.

3. Discuss the laboratory tests used in the diagnosis of P. jiroveci infection, including the methodology and biochemical principles.

4. List the four stains most commonly used in the diagnosis of P. jiroveci infection.

Genus and Species to be Considered

Current Name	Previous Name
Pneumocystis jiroveci	Pneumocystis carinii

General Characteristics

In 1999 the name of the organism that causes a pneumonia in immunocompromised humans, commonly called pneumocystis pneumonia (PCP), was changed from Pneumocystis carinii to Pneumocystis jiroveci. (The causative organism for the rodent form of pneumocystis is still called P. carinii.) P. jiroveci is an opportunistic, atypical fungus that infects immunocompromised hosts and mostly manifests as PCP.

P. jiroveci originally was thought to be a trypanosome, but its precise taxonomic categorization remains challenging. Several factors supported the notion that P. jiroveci was a protozoan parasite: its morphology is similar to that of microbes and protozoa, and clinically it responds to antiprotozoal drugs but not to antifungal drugs in patients with pneumocystosis. Inability to maintain and propagate the organism in routine culture has further limited its characterization, although cultivation is possible under special conditions. P. jiroveci exists as three forms in its life cycle: trophozoite, precyst (sporocyte), and cyst (the latter is the diagnostic form).

Although P. jiroveci has been shown to be a fungus, it differs from other fungi in various aspects. Its cell membrane contains cholesterol rather than ergosterol. The flexible-walled trophozoite is susceptible to osmotic disturbances. Also, P. jiroveci contains only one or two copies of the small ribosomal subunit gene, whereas most other fungi contain numerous copies of this gene. DNA sequence analysis of the small ribosomal subunit gene in P. jiroveci has disclosed a greater sequence homology with the fungi than with the protozoa. Two independent analyses that compared the DNA sequences of P. jiroveci with those of other fungi confirmed the placement of P. jiroveci in the fungal kingdom, somewhere between the ascomycetes and the basidiomycetes (the closest yeast is the fission yeast, Schizosaccharomyces pombe). DNA analysis also confirmed the difference between the rodent and human forms of pneumocystis.

Epidemiology

P. jiroveci has a worldwide distribution and most commonly presents as pneumonia in an immunocompromised host. As mentioned, infection appears to be species specific, with P. carinii causing disease in rodents and P. jiroveci causing human disease. The exact transmission of disease is still not known. Some speculate that pneumocystis is transmitted person to person. Immunocompetent mammals may be the reservoir for P. jiroveci, which is transmitted to immunodeficient individuals as a pathogen. Most children by age 2 to 4 have antibodies to pneumocystis. Vargas et al. showed that pneumocystis DNA was present in 24 of 72 infants, as determined from nasopharyngeal specimens, and that seroconversion occurred in 85% of infants by 20 months of age.

Since the onset of the human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) epidemic in the 1980s, pneumocystis has been defined as the most common opportunistic infection among those with HIV or AIDS in the United States. The introduction of highly active antiretroviral therapy (HAART) for patients with HIV has reduced the incidence of disease. However, PCP remains a significant medical problem, because numerous patients with HIV do not respond to therapy, do not comply with therapy, or do not know they are infected.

Pathogenesis and Spectrum of Disease

After inhalation of P. jiroveci, the pathogen is thought to adhere to type I pneumocytes. The organisms replicate extracellularly while bathed in alveolar lining fluid. With successful replication of the organism, the alveolar spaces fill with foamy material, which can be detected with hematoxylin and eosin staining. These changes result in impaired oxygen-diffusing capacity and hypoxemia. A predominantly interstitial mononuclear inflammatory response is associated with this type of pneumonia. When first described, this pneumonia was known as interstitial plasma cell pneumonia.

Symptoms of PCP include a nonproductive cough, low-grade fever, dyspnea, chest tightness, and night sweats. In patients without HIV infection, the underlying conditions most commonly seen as risk factors for this opportunistic infection are asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, systemic lupus erythematosus (SLE), pregnancy, rheumatoid arthritis, infection with Epstein-Barr virus, ulcerative colitis, and high-dose corticosteroid therapy.

Laboratory Diagnosis

Specimen Collection and Transport

Respiratory specimens from the deep portions of the lung, such as bronchoalveolar lavage fluid (BALF), are best for detection of P. jiroveci. A sputum specimen submitted for direct examination should be an induced sputum obtained by a trained respiratory therapist; otherwise, the rate of false-negative results may be unacceptably high.

Specimen Processing

See the following section for specific details for specimen processing required for different test methods.

Direct Detection Methods

Stains

The diagnosis of P. jiroveci pneumonia currently is based on the clinical presentation, radiographic studies, and direct and/or pathologic examination of bronchoalveolar lavage fluid or biopsy material. The flexible-walled trophozoites are the predominant form of the organism, but these are difficult to visualize. They are somewhat discernible in Giemsa-stained material, but their pleomorphic appearance makes this form of the organism difficult to identify. A firm-walled cystic form also exists, although the cysts are outnumbered by the trophozoites 10 to 1. Cysts are more easily recognized than trophozoites and may be definitively identified using a variety of stains, such as calcofluor white, methenamine silver, and immunofluorescent staining (Figure 62-1). The cysts are spherical to concave, uniform in size (4 to 7 μm in diameter), do not bud, and contain distinctive intracystic bodies.

Figure 62-1 Cystic forms of Pneumocystis jiroveci (arrows) stain well with methenamine silver and hematoxylin and eosin stain (×500).

A comparison of the four most common staining methods used for P. jiroveci (i.e., Giemsa, immunofluorescent, calcofluor white, and methenamine silver) has demonstrated that immunofluorescent staining (Merifluor Pneumocystis; Meridian Bioscience, Cincinnati, Ohio), calcofluor white staining (Fungifluor; Polysciences, Warington, Pennsylvania), and methenamine silver staining (GMS and DiffQuick; Baxter Scientific, McGraw Park, Illinois) likely represent the best balance between sensitivity and specificity and have the best overall positive and negative predictive values. The immunofluorescent method showed greater sensitivity than the other three but a smaller negative predictive value. Therefore, if this method is used as a screening tool for pneumocystis, a confirmatory method should be performed because of the high number of false-positive results.

Antigen-Protein

Commercial kits use monoclonal antibodies directed against P. jiroveci to stain both the cysts and the trophozoites. These tests are a highly sensitive microscopic method of detection, but they are expensive. Also, because they use nonspecific staining, which may be thought to represent the pleomorphic trophozoite forms, the specificity of this assay may be limited.

Nucleic Acid Amplification

A variety of nucleic acid amplification assays for P. jiroveci have been developed, including, most recently, real-time polymerase chain reaction (PCR) methods. These methods are making their way from the research setting into the clinical molecular microbiology laboratory, but commercial kits are not yet available.

Cultivation

P. jiroveci is very difficult to cultivate outside the lung; therefore, routine culture methods are not performed.

Approach to Identification

See Direct Detection Methods.

Serodiagnosis

Serology is not useful for the diagnosis of pneumocystosis.

1. A 65-year-old male with HIV presents to the emergency department with night sweats, a nonproductive cough, and a low-grade fever. PCP is suspected. Which of the following is the best specimen for diagnostic staining for Pneumocystis jiroveci?

c. Bronchial alveolar lavage fluid

e. Nasopharyngeal swab

2. Short Answer

(1) List at least three stains used to detect Pneumocystis jiroveci.

Case Study 62-1

A 69-year-old male is diagnosed with follicular lymphoma and placed on a 4-week regimen of combination chemotherapy. During the fourth week of treatment, the patient develops a fever that lasts 3 days. On the third day, he is admitted to the hospital. The findings from the initial physical exam are normal except for the fever. The following laboratory test results are obtained:

WBC count	2500/µL	Normal: 4800-10,800/µL
Absolute neutrophil count	1100/µL	Normal: 50% to 60% of WBCs
Serum beta-D glucan	182 pg/mL	Normal: <20 pg/mL
CT scan of the lungs	Ground-glass opacities in the lung

These findings result in a diagnosis of interstitial pneumonia. The patient is treated with intravenous (IV) panipenem/betamipron, micafungin, and ganciclovir.

The patient does not seem to be recovering. After additional testing, he is switched to oral trimethoprim-sulfamethoxazole (TMP-SMX), and the lung infiltrates disappear within 2 weeks.

1. What additional testing would have been performed to confirm the diagnosis of Pneumocystis jiroveci?

2. What patient characteristics would have contributed to the man’s susceptibility to infection with P. jiroveci?

Related

Bailey _ Scotts Diagnostic Microbiology 13e