1. PCP: Diagnosis Dr. Rita O. Oladele Consultant Clinical Microbiologist
University of Lagos, Nigeria

2. Intended Learning Objectives
To be aware of the different diagnostic tests available for the diagnosis of PCP
To be able to appreciate and compare the diagnostic performances of the different modalities
To be familiar with the different adjunctive tests required to establish the diagnosis of PCP

3. Available diagnostic tests
Microscopy
- Conventional
- Immunofluorescence
Serum markers
PCR

4. Types of specimens Bronchoscopy with Bronchoalveolar lavage
Gold standard. Requires intubation.
Induced sputum
Obtained after nebulisation with 3% saline
Difficult but can still be obtained in < 2 years
Upper respiratory tract specimens:
Evidence for nasopharyngeal aspirates with better yield when PCR is used (nasogastric aspirates also used)
Lung tissue biopsy can also be used.

5. MICROSCOPY Microscopy is the low cost gold standard why?
Pneumocystis does not routinely grow in culture &
often presents with non specific features
Visualize Pneumocystis cysts & trophozoites by:
a) Immunofluorescence using monoclonal antibodies
b) Conventional microscopy on stained specimens

6. Conventional microscopy
Stained specimens are observed under a
microscope for cyst or trophozoite identification.
Types of stains used:
a) Trophic form stains
-modified Papanicolaou
- Diff-Quick Wright-Giemsa
-Gomori methenamine silver (GMS) stains the cyst walls
b) Cyst wall stains
-toluidine blue O
- calcoflour white
- cresyl echt violet

7. Comparison of the different diagnostic methods for Pneumocystis detection currently used
PCR
IFA
GMS
Toluidine
Blue O
Gram
Weigert
Giemsa
Diff
Quick
B-d
glucan
Target
Cysts/
trophic forms
Cyst
Cysts
Cyst/
Trophic
forms
Sensitivity
+++++
++++
+++ ++
Specificity
Procedure
time (min)
150
200
100 50 60 80 33
>200
Cost ↑ ↓

8. Microscopy with Giemsa
source link:
Staining with Giemsa is cheaper and less time consuming.
May be carried out in a routine laboratory.

9. Conventional Microscopy: silver stains
cysts
Alveoli space
Grocott-Gomori methenamine silver stain

10. Microscopy with methenamine silver
Lung tissue stained
with MS and H& E.
Cysts appear black
Source :

11. Immunofluorescence microscopy (1)
Image courtesy of Brigham & Women's Hospital, Boston, MA.
Direct immunofluorescence antibody stain using monoclonal antibodies that target Pneumocystis jirovecii from a bronchoalveolar lavage (BAL) specimen.

12. Immunofluorescence microscopy (2)
cysts

13. Adjuvant tests Serum lactate dehydrogenase (LDH)
Elevated LDH is a marker of tissue damage
Frequently elevated in PCP, with higher levels than found in other LRTIs (TB & bacterial pneumonia)
LDH levels >220 IU/L are found in 90% of adults with PCP. No studies to validate levels in children
Sensitivity is % but poor specificity
β 1,3-d glucan
Cell component of most fungi with limited data for PCP diagnosis in paediatric patients
(1→3) β-d-glucan is produced in the cyst cell wall and detection in the serum has been associated with underlying infection (also positive in other invasive fungal infections.Clinical trials data lacking

14. Molecular methods: PCR
PCR analysis of DNA extracted
from respiratory specimens
Higher sensitivity than microscopy especially of upper airway specimens like nasopharyngeal aspirates

15. Summary
Microbiological diagnosis of PCP is essential since the disease presents with non specific clinical features
Several types of respiratory specimens can be submitted for diagnosis
Microscopy is the gold standard of diagnosing PCP

16. END