Pneumocystis jirovecii: Fungus or Protozoan, Pathogenesis, and Lab Diagnosis

A patient with advanced HIV and a CD4 count below 200 develops a dry cough and worsening breathlessness, and the chest X-ray shows a diffuse haze but the sputum grows nothing on ordinary culture. A silver stain of a bronchoalveolar lavage reveals clusters of small cup-shaped cysts. This is Pneumocystis jirovecii, an organism that behaves like a protozoan, is classified as a fungus, cannot be grown in culture, and ignores the usual antifungal drugs. Understanding why it breaks all these rules is the key to diagnosing and treating it.

Pneumocystis jirovecii is an atypical opportunistic fungus that causes pneumonia in immunocompromised people. It is of low virulence and spreads through the air. The disease it causes is called Pneumocystis pneumonia, abbreviated PJP (or the older PCP). It is most often seen in patients with advanced HIV/AIDS, but also in other immunocompromised patients.

A note on the name. The human pathogen is Pneumocystis jirovecii (spelled with two i's at the end), named after the Czech parasitologist Otto Jírovec. It was previously called Pneumocystis carinii, a name now reserved for the species that infects rats. The spelling changed from "jiroveci" to "jirovecii" when the organism was reclassified from a protozoan to a fungus, because the naming rules for fungi differ from those for animals. So the double-i spelling is the correct current form.

Pneumocystis jirovecii is common in the environment and does not cause illness in healthy people. The fungus can live in their lungs without causing symptoms.  Asymptomatic infection is also quite common.  Serologic evidence suggests that most children are exposed to this organism by age 3 to 4.

Pneumocystis jirovecii pneumonia was a relatively rare infection before the AIDS epidemic. Infection with P.jirovecii is an AIDS-defining illness. People with a weakened immune system (i.e. people having medical conditions like an organ transplant, blood cancer, autoimmune diseases, stem cell transplant, etc) are at high risk of developing Pneumocystis pneumonia.

Is Pneumocystis a fungus or a protozoan?

This is one of the most famous reclassification stories in microbiology, and it is a common exam question.

Pneumocystis was originally classified as a protozoan, because under the microscope its forms (trophic forms and cysts) look like protozoa, and it behaves like one in important ways. In 1988, analysis of its ribosomal RNA and DNA showed it is actually a fungus, closely related to yeasts such as Saccharomyces. It is now classified in the fungal phylum Ascomycota.

So why is it called an atypical fungus? Because it breaks several fungal rules:

1. Its cell membrane contains cholesterol instead of ergosterol. Ergosterol is the target of the common antifungal drugs (azoles and amphotericin B). Because Pneumocystis lacks it, these antifungals do not work. This single fact explains why an antifungal-resistant fungus is treated with a drug that works on protozoa.
2. It cannot be grown in ordinary culture. Unlike most fungi, it does not grow on fungal culture media.
3. It responds to antiprotozoal drugs. It is treated with co-trimoxazole, the same kind of drug used against some protozoal infections, not with antifungals.

The way to hold this together: Pneumocystis is a fungus by its genes, but it looks and behaves enough like a protozoan that it was misclassified for decades, and it is still treated like one.

Life cycle and forms

Figure: Life cycle of Pneumocystis jirovecii (Source: Dr. John J. Ruffolo)

Pneumocystis jirovecii lives entirely in the lung and is an extracellular organism, meaning it sits on the surface of the alveolar lining rather than inside cells. It exists in two main forms:

1. The trophic form (trophozoite): a small, thin-walled form that attaches to the alveolar lining and multiplies.
2. The cyst form: a larger, thick-walled, round to cup-shaped form. A mature cyst contains eight intracystic bodies, which are released to become new trophic forms.

The organism is inhaled and reaches the alveoli. Because Pneumocystis is common in the environment and most people are exposed in early childhood, disease is thought to arise either from new infection or from reactivation of organisms already carried at low levels, when the immune system weakens.

Virulence and pathogenesis

Pneumocystis jirovecii is a low-virulence organism, so its disease depends almost entirely on the host's immune state. Its key features work together to cause pneumonia when defenses fail.

1. Attachment to the alveolar lining. What: the trophic form binds tightly to type I alveolar epithelial cells, aided by its major surface glycoprotein. Why: this attachment lets the organism establish itself in the alveoli and triggers its change from the trophic to the cystic form.
2. Immune evasion by surface variation. What: the organism varies its major surface glycoprotein. Why: this helps it avoid the host immune response.
3. Dependence on host immunity. What: it causes disease only when cell-mediated immunity is weak. Why: in healthy people the organism is cleared or carried harmlessly, so it is the failure of the host, not the strength of the organism, that produces disease.

Putting it together

Inhaled organisms reach the alveoli and the trophic forms attach to the alveolar epithelium (attach). In a person with intact immunity they are cleared, but when cell-mediated immunity is weak, the organism multiplies unchecked (resist and proliferate). The growing organisms and the host's inflammatory response fill the alveoli with a foamy material and injure the alveolar lining (damage). This blocks gas exchange, causing low blood oxygen (hypoxia) and, in severe cases, respiratory failure. Pneumocystis stays in the lungs and does not usually spread to other organs.

Cell-mediated immunity, especially CD4 T cells, is the key defense, which is why disease appears when CD4 counts fall.

Clinical features

Pneumocystis pneumonia (PJP) typically develops in people with impaired cell-mediated immunity. In HIV, the risk rises sharply when the CD4 count falls below 200 cells per cubic millimeter, which is why this count is used as a threshold for starting preventive treatment.

The usual features are a gradually worsening dry cough, shortness of breath (especially on exertion), and fever. A characteristic finding is that the drop in blood oxygen is often more severe than the chest examination or X-ray would suggest. The chest X-ray classically shows diffuse, bilateral haziness spreading out from the hila. Untreated, PJP can progress to respiratory failure and death, which is why it is a serious AIDS-defining illness.

Laboratory Diagnosis

Pneumocystis jirovecii can not be cultured in vitro, therefore, laboratory diagnosis relies on cytological staining, immunofluorescent assay, or molecular diagnosis in bronchopulmonary secretions.

Sample

Expectorated sputum has a very low sensitivity and should not be submitted for diagnosis. Bronchoalveolar lavage (BAL) and induced sputum have been demonstrated to be the most useful clinical samples. In situations where these two techniques cannot be used, transbronchial biopsy or open lung biopsy may be used.

Figure: Cysts of P. jirovecii in lung tissue, stained with methenamine silver and hematoxylin and eosin (H&E). (Image source:CDC)

Microscopy and staining

Microscopy uses specific stains. Silver stains (such as Gomori methenamine silver) and immunofluorescence show the cyst wall, while Giemsa and Wright stains show the nuclei of the trophic and intracystic forms. Note that Pneumocystis is not identified by Gram stain, so if you are looking for it, reach for silver or Giemsa, not Gram.

Giemsa stain is used to demonstrate the nuclei of trophozoites and intracystic stages and silver stain is used to demonstrate the cyst wall. Immunofluorescence microscopy using monoclonal antibodies can detect both cystic and trophic forms with higher sensitivity than conventional microscopy.

Culture

Does not grow in fungal culture media and even in cell culture. In 2014, Verena Schildgen et al. reported a successful culture of P. jirovecii from BAL specimen but Liu et.al, could not reproduce the result (i.e. they failed to culture P. jirovecii).

Molecular Diagnosis

Molecular methods have shown very high sensitivity and specificity and are regarded as the gold standard technique for the detection of P. jirovecii. The genome of P. jirovecii present in the sample is amplified by Polymerase Chain Reaction (PCR) and is detected after agarose-gel analysis using a suitable DNA ladder.

Others tests

Blood test to detect β-D-glucan (a part of the cell wall of many different types of fungi) can be used to diagnose Pneumocystis pneumonia.

Treatment and Prevention

Treatment

The drug of choice for Pneumocystis pneumonia is co-trimoxazole (trimethoprim-sulfamethoxazole). It works by blocking the organism's folate synthesis. Note that this is an antibacterial/antiprotozoal drug, not an antifungal, because the usual antifungals (azoles, amphotericin B) do not work against Pneumocystis, which lacks ergosterol.

For patients who cannot take co-trimoxazole, alternative regimens exist, chosen by the treating clinician.

In severe disease with significant low blood oxygen, corticosteroids are added, because they reduce the harmful inflammation that follows when the organisms begin to die.

Prevention (prophylaxis)

Co-trimoxazole is also used to prevent PJP in high-risk patients. In HIV, prophylaxis is started when the CD4 count falls below 200 cells per cubic millimeter and continued until immunity recovers. So the same drug both treats and prevents the disease.

This article gives drug-of-choice and class-level guidance only. Doses and durations are decided by the treating clinician.

How to Remember

Fungus by genes, protozoan by behavior. Its DNA makes it a fungus, but it looks like a protozoan, resists antifungals, and responds to antiprotozoal drugs.

Cholesterol, not ergosterol. No ergosterol means azoles and amphotericin do not work. This one fact explains why an "antifungal-resistant fungus" is treated with co-trimoxazole.

Two i's for the fungus. jirovecii (double i) is the correct fungal spelling. The single-i "jiroveci" is the old protozoan-era form.

CD4 under 200. The number to remember for both risk and prophylaxis in HIV.

Co-trimoxazole treats and prevents. The same drug does both.

Cysts by silver, nuclei by Giemsa, not Gram. Silver stains the cyst wall, Giemsa stains the nuclei, and Gram does not work.

Key exam facts

Where Students Get Confused

"Pneumocystis is a protozoan." Not anymore. It was reclassified as a fungus in 1988 based on its genes. It is called an atypical fungus because it looks and behaves like a protozoan.

"It's a fungus, so treat it with antifungals." No. It lacks ergosterol, the target of azoles and amphotericin B, so those fail. It is treated with co-trimoxazole.

"Pneumocystis jiroveci and Pneumocystis carinii are the same." Not quite. P. jirovecii infects humans; P. carinii now refers to the rat species. The human name also gained a second "i" (jirovecii) with reclassification.

"You can grow it in culture to confirm it." No. Pneumocystis cannot be reliably cultured, which is why diagnosis relies on staining, immunofluorescence, and PCR.

"You identify it with a Gram stain." No. It does not stain reliably with Gram. Silver, Giemsa, and immunofluorescence stains are used.

"It spreads through the body like other serious infections." Usually not. It stays in the lungs and causes pneumonia rather than disseminating.

References and further readings

1. Schildgen, V., et al. (2014). Pneumocystis jirovecii can be productively cultured in differentiated CuFi-8 airway cells. mBio, 5(3), e01186-14.
2. Liu, Y., Fahle, G. A., and Kovacs, J. A. (2018). Inability to culture Pneumocystis jirovecii. mBio, 9(3), e00939-18.
3. Truong, J., and Ashurst, J. V. Pneumocystis jirovecii pneumonia. In StatPearls. StatPearls Publishing.
4. Tille, P. M. (2022). Bailey and Scott's Diagnostic Microbiology (15th ed.). Elsevier.