Biology of parasitic protozoa
VIII. Pneumocystis (Opisthokonta, Fungi)
Andrea Bardůnek Valigurová
andreav@sci.muni.cz
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5 supergroups = megagroups
• Pneumocystis jirovecii (previously
Pneumocystis carinii) was previously
classified as a protozoan
• traditionally studied by protozoologists
• fungus based on molecular and
biochemical analysis
• cause of pneumonia = Pneumocystis
pneumonia (PCP) in people with
compromised immune system and in
premature, malnourished infants
• source of opportunistic infection especially
in people with cancer
undergoing chemotherapy, HIV/AIDS, and
the use of medications (e.g.
corticosteroids) that suppress the immune
system
Pneumocystis and pneumocystosis
Carlos Chagas in 1909 inoculated human blood infected with Trypanosoma cruzi into
guinea pigs:
• detected cystic, multinucleated bodies lungs of these experimental animals but
confused it with part of the lifecycle of T. cruzi
• later named both organisms Schizotrypanum cruzi
Subsequent observations rule out the association with T. cruzi:
• Carini in 1910 described Pneumocystis in lungs of rats without T. cruzi
• Delanoe and Delanoe in 1912 performed study of rats in Paris and described
Pneumocystis carinii
• in 1938 German pathologists described a special disease of malnourished infants interstitial
plasma cell pneumonia (IPP) (but the causative agent remained unknown)
• in 1942 Dutch pathologists described the finding of Pneumocystis in human lungs
(discovery did not elicit any scientific response)
• in 1951-1952 Czech researchers Vaněk and Jírovec identified Pneumocystis as the
etiological agent of IPP in infants
Pneumocystis: history of the discovery
genus Pneumocystis
• extracellular fungus
• worldwide, in humans and animals
• found in the lungs of mammals where it resides without causing overt infection until
the host’s immune system becomes debilitated, which can lead to fatal pneumonia
• complete life cycle of any of the Pneumocystis species not known
• life cycle of P. jirovecii is thought to include both asexual and sexual phases
• both stages known so far (= morphologically distinct), i.e. amoeboid trophozoites
and globular cysts, can be found in the lungs and cannot be cultured ex vivo
• 8 spores form within cyst, these are released by rupture of the cyst wall
• terminology follows zoological terms, rather than mycological ones
P. carinii
P. wakefieldiae
• rats
P. oryctolagi
• rabbitts
P. murina
• laboratory mice
Pneumocystis jirovecii
• the only species found in humans
• number of genotypes
• spreading from person to person through the air
• PCP is extremely rare in healthy people, but the fungus causing it can live in their
lungs without causing symptoms
• serologic evidence (70-80% positivity) indicates that most healthy children have been
exposed by age 3 to 4
• up to 20% of adults might carry this fungus at any given time and their immune
system removes it after several months
• host immune status of the determines whether the host clears Pneumocystis or
becomes a permanent asymptomatic carrier or becomes ill
• e.g. young rabbits and piglets commonly develop pneumocystis pneumonia infection
after weaning, which resolves after 2-3 weeks
• AIDS, transplantation, malignancy patients - necessary prevention or treatment
(pentamidine, trimethoprim + co-trimoxazole
https://www.youtube.com/watch?v=cuZb539SaaY
Asexual phase: Trophic forms (1) replicate by mitosis (2) to (3). Sexual phase: Haploid trophic forms conjugate (1) and produce
a zygote or sporocyte (early cyst) (2). The zygote undergoes meiosis and subsequent mitosis to produce 8 haploid nuclei (late
phase cyst) (3). Spores exhibit different shapes (such as, spherical and elongated forms). It is postulated that elongation of the
spores precedes release from the spore case. It is believed that the release occurs through a rent in the cell wall. After release, the
empty spore case usually collapses, but retains some residual cytoplasm (4). A trophic stage, where the organisms probably
multiply by binary fission is also recognised to exist.
Generalised life cycle proposed for Pneumocystis spp.
Proposed Pneumocystis life cycle
The life cycle of pneumocystis is complex, and
several forms are seen during infection.
Electron micrograph in A shows a trophic form
that is tightly adherent to the alveolar epithelium
by apposition of its cell membrane with that of
the host lung cell membrane. During infection,
trophic forms are more abundant than cysts
(approximately 9:1), and the majority of the
trophic forms are believed to be haploid during
normal growth, with a smaller fraction that are
diploid. Trophic forms attach to one another, as
shown in the electron micrograph in B, and
clusters of clumped trophic forms can be seen
during infection. The events that lead to the
formation of the cyst, shown in the electron
micrograph in C, are unclear, but we
hypothesize that the trophic forms conjugate
and mature into cysts, which contain 2, 4, or 8
nuclei as they mature.
https://pubmed.ncbi.nlm.nih.gov/15190141
https://doi.org/10.1038/nrmicro1621
Clinical presentation of Pneumocystis pneumonia
• symptoms of PCP include dyspnoea, non-productive cough, chest pain, chills, fatigue,
fever
• chest radiography demonstrates bilateral infiltrates
• extrapulmonary lesions occur in a minority (<3%) of patients, involving most frequently
the lymph nodes, spleen, liver, and bone marrow
• in untreated PCP increasing pulmonary involvement leads to death
• collection of bronchioalveolar fluid with an endoscope
• identification of P. jirovecii in bronchopulmonary secretions obtained as induced (or
noninduced) sputum or bronchoalveolar lavage (BAL)
• if the above techniques cannot be used, transbronchial or open lung biopsy may prove
necessary
• Giemsa, Gram-Weigert or silver staining
• immuno(fluorescence) microscopy using monoclonal antibodies
• PCR
• serological tests: for screening only
Diagnosis of PCP
Diagnosis of PCP
Detection of Pneumocystis forms with the use of different stains. A) Typical pneumocystis cyst forms in a bronchoalveolarlavage
specimen stained with Gomori methenamine. Thick cyst walls and some intracystic bodies are evident. B) Wright–Giemsa
staining can be used for rapid identification of trophic forms of the organisms within foamy exudates (arrows) in bronchoalveolarlavage
fluid or induced sputum but usually requires a high organism burden and expertise in interpretation. C) Calcofluor white is
a fungal cyst-wall stain that can be used for rapid confirmation of the presence of cyst forms. D) Immunofluorescence staining
can sensitively and specifically identify both Pneumocystis trophic forms (arrowheads) and cysts (arrows).
Diagnosis of PCP
Pneumocystis stain kit
(formalin-fixed-paraffin
embedded specimen)
Diagnosis of PCP
https://doi.org/10.1177/1753465810380102
Histological features of Pneumocystis pneumonia. A)
Pneumocystis pneumonia in a surgical lung biopsy
specimen. The characteristic frothy exudates harbouring the
organisms fills an alveolus (small arrows) lined by reactive
type II cells (large arrow). Internal structures of the organisms
can be discerned at high magnification (inset). HE, B) Silver
stain of Pneumocystis organisms in a surgical lung biopsy.
Pneumocystis organisms appear round or cup-shaped, some
show intracystic bodies (lower right). Gomori’s methenamine
silver staining. C) Immunohistochemical staining of surgical
lung biopsy for Pneumocystis.
Pathology and
diagnosis of PCP
A) Posteroanterior chest radiograph showing
diffuse bilateral predominantly interstitial opacities
in 71-year-old male with Pneumocystis pneumonia
in the setting of diffuse large B-cell lymphoma on
R-CHOP therapy. B) Selected non-contrast CT
axial image revealing bilateral diffuse groundglass
opacities and associated interlobular and
intralobular septal thickening. C) Contrast CT axial
image revealing diffuse ground-glass pulmonary
infiltrates and innumerable cystic changes
predominantly in the upper lobes in a 53-year-old
male with Pneumocystis pneumonia in the setting
of recently diagnosed HIV.
Posteroanterior chest radiograph
of a 68-year-old patient with
Pneumocystis pneumonia that
developed as a consequence of
long-term corticosteroid therapy
for an inflammatory neuropathy.
Mixed alveolar and interstitial
infiltrates are more prominent on
the right side.
Pathology and diagnosis of PCP
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7121032/?report=classic
Gross photograph of lung from a patient who died
within 5 days of onset of respiratory failure. The lung
weighed 1750 g and showed a tan solid cut surface,
consistent with diffuse alveolar damage (DAD).
Necrotizing P. jirovecii pneumonia. Apical subpleural
cavity due to necrotizing PCP in a patient with AIDS.
Pathology and diagnosis of PCP
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7121032/?report=classic
A) Lung (gross photograph shown in previous slide) showing typical histologic pattern of exudative stage of DAD. Alveolar ducts are dilated
and lined by hyaline membranes, and surrounded by collapsed alveoli. Intraalveolar foamy exudates of Pneumocystis are also visible. B)
Higher magnification of hyaline membranes showing embedded Pneumocystis cysts. GMS. C) Immunostaining for Pneumocystis may also
be used to demonstrate the cysts and trophozoites in the hyaline membranes. D) Lung from a patient with long-standing Pneumocystis
infection shows enlarged and atypical type II pneumocytes with high nuclear cytoplasmic ratio, as seen in atypical alveolar hyperplasia.
Pathology and diagnosis of PCP
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7121032/?report=classic
A) Low-magnification photomicrograph shows thickened
alveolar septa with mild interstitial inflammation and fibrosis,
lined by hyperplastic type II pneumocytes. Alveoli are filled with
typical foamy, eosinophilic exudate of Pneumocystis. B) Higher
magnification shows an alveolus with the foamy/bubbly
eosinophilic exudates and a few mononuclear cells. Basophilic
dots are visible within the exudate. Alveolar septa have a few
inflammatory cells and collagen, and are lined by hyperplastic
type II pneumocytes. C) Immunostain demonstrates the cyst
forms of Pneumocystis, surrounded by inflammatory cells.
Trophozoites also stained with the immunostain; however, these
are not as clearly visualized as the cyst forms.
Thank you for your attention ☺
Lectures
✓ Introduction: BPP 2022 I
✓ Euglenozoa (Excavata): BPP 2022 II
✓ Fornicata / Preaxostyla / Parabasala (Excavata): BPP 2022 III
✓ Apicomplexa I (SAR): BPP 2022 IV
✓ Apicomplexa II (SAR): BPP 2022 V
✓ Amoebae (Excavata, Amoebozoa): BPP 2022 VI
✓ Ciliophora, Opalinata (SAR): BPP 2022 VII
✓ Pneumocystis (Opisthokonta, Fungi): BPP 2022 VIII
 Microsporidia (Opisthokonta, Fungi): BPP 2022 IX
• Myxozoa (Opisthokonta, Animalia): BPP 2022 X