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

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

Available diagnostic tests Microscopy - Conventional - Immunofluorescence Serum markers PCR

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.

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

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

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 ↑ ↓

Microscopy with Giemsa source link: https://www.cdc.gov/dpdx/pneumocystis/ Staining with Giemsa is cheaper and less time consuming. May be carried out in a routine laboratory.

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

Microscopy with methenamine silver Lung tissue stained with MS and H& E. Cysts appear black Source : https://www.cdc.gov/dpdx/pneumocystis/

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

Immunofluorescence microscopy (2) cysts

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 78-100% 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

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

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

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