{"id":22934,"date":"2025-03-14T14:51:22","date_gmt":"2025-03-14T14:51:22","guid":{"rendered":"https:\/\/clinlabint.com\/?p=22934"},"modified":"2025-03-14T14:54:42","modified_gmt":"2025-03-14T14:54:42","slug":"laboratory-diagnosis-of-pneumocystis-jirovecii-pneumonia","status":"publish","type":"post","link":"https:\/\/clinlabint.com\/laboratory-diagnosis-of-pneumocystis-jirovecii-pneumonia\/","title":{"rendered":"Laboratory diagnosis of Pneumocystis jirovecii pneumonia"},"content":{"rendered":"
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Laboratory diagnosis of Pneumocystis jirovecii pneumonia<\/h1>\/ in Featured Articles<\/a> <\/span><\/span><\/header>\n<\/div><\/section>
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by Dr Osaretin Emmanuel Asowata<\/em><\/p>\n

The detection of Pneumocystis jirovecii in immunocompromised patients is crucial for the treatment of Pneumocystis jirovecii pneumonia (PCP) and to prevent invasive infection. However, PCP can be challenging to diagnose clinically, as symptoms are non-specific. There are several laboratory methods that can be used to diagnose PCP and their advantages and limitations are discussed here.<\/h3>\n

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Background<\/h4>\n

Pneumocystis jirovecii pneumonia (PCP) is a disease that is caused by the fungus Pneumocystis jirovecii. P. jirovecii is an opportunistic pathogen, which means that it does not normally cause disease in healthy individuals. P. jirovecii was originally known as P. carinii, hence the name PCP (Pneumocystis carinii pneumonia) which is still often used. PCP mainly affects immunocompromised individuals, which include HIV-infected individual and non-HIV-infected individuals. The mode of transmission of Pneumocystis is not fully understood but the hypothesis is that people<\/p>\n

are exposed at a very early age, for example as early as 7 months old. This was reported in a study where 83% of 7-month-old children had antibodies to P. jirovecii. The presence of P. jirovecii in healthy individuals at this early stage of life is mostly latent until there are favourable conditions for this organism to proliferate, which is mainly immunocompromised status associated with HIV, solid organ transplant, stem cell transplant or other immunocompromised conditions, which reactivates the organisms, increasing the risk of disease.<\/p>\n<\/div><\/section>
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Pneumocystis jirovecii is an opportunistic fungus that can cause pneumonia and invasive infection in immunocompromised patients\u00a0 (Adobe Stock.com)<\/em><\/p>\n<\/div><\/section>
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Importance and challenges of PCP diagnosis<\/h4>\n

It’s important to diagnose P. jirovecii infection because of its potential to cause severe morbidity and mortality, especially among immunocompromised patients. The infection exhibited by these patients could lead to severe consequences if not treated. Specifically, among untreated HIV-infected individuals, P. jirovecii could lead to complications such as extrapulmonary Pneumocystis pneumonia, which is a condition where this organism affects various tissues in the body such as the thymus, the liver, the lymph nodes, the spleen and other extrapulmonary regions.<\/p>\n

PCP is very difficult to diagnose clinically due to its non-specific presentation. For example, some of the common symptoms are persistent or intermittent fever, progressive dyspnea, hypoxemia (low blood oxygen), nonproductive cough, fatigue, and weight loss, which could be linked to other diseases. Clinical diagnosis is mainly radiologically by high-resolution computed tomography (HRCT), X-ray or ultrasound. However, chest X-radiographs can appear normal, especially in the early stages of the disease.<\/p>\n

A challenge of laboratory diagnosis of PCP is the lack of routine method for culturing P. jirovecii; it is more difficult to conduct research and development of antimicrobial susceptibility test on this organism without a routine in vitro culture method.<\/p>\n

Laboratory diagnosis of PCP<\/h4>\n

Microscopy<\/strong><\/p>\n

Before the advent of nucleic acid amplification testing (NAAT), laboratory diagnosis of PCP was mainly via microscopy, which involves direct observation of a respiratory specimen or lung biopsy for P. jirovecii ascus (cyst; 5\u20138 \u00b5m) or the trophic forms (1\u20134 \u00b5m). Although microscopy remains the gold standard, especially in many resource-limited clinical laboratories, its lower sensitivity compared to the NAAT is a challenge, and most clinical microbiology laboratories are gradually incorporating the NAAT through acquisition of this infrastructure or sending specimens to reference laboratories for NAAT as an additional test for PCP. Microscopy includes different stains such as the Calcofluor white and Giemsa-like stains, as well as the immunofluorescent stains, which are available in the clinical microbiology laboratories. In pathology laboratories, stains such as the hematoxylin and eosin (H&E), periodic Acid-Schiff, Papanicolaou\u2019s, and Grocott\u2019s Methenamine Silver are routinely performed. The advantages of these stains are the fast turnaround time, which is very important in PCP diagnosis in immunocompromised patient.<\/p>\n

NAAT<\/strong><\/p>\n

Molecular testing is more sensitive than microscopy but is typically conducted in reference laboratories or laboratories with advanced infrastructure. An example of this is the NAAT using respiratory specimens such as induced sputum, BAL fluid and lung tissues. An important variation of the NAAT is the conventional PCR testing that was developed more than three decades ago. Conventional PCR uses specific primers that target the conserved region of the P. jirovecii genome. We now have the more recent and rapid real-time PCR, which is easier and more accurate to detect and quantify P. jirovecii DNA in clinical specimen. An advantage of both the conventional and real-time PCR assays is the high sensitivity \u2013 where P. jirovecii DNA can be detected in samples that might not have been positive by microscopy.<\/p>\n

Beta-D-glucan biomarker<\/strong><\/p>\n

To support PCP diagnosis, testing serum samples for the presence of beta-D-glucan biomarker is usually performed. Beta-D-glucan is a polysaccharide component of the fungal cell wall used to diagnose invasive fungal infection, including P. jirovecii. A negative microscopy result from respiratory samples with positive beta-D-glucan in serum could suggest the presence of an active invasive fungal infection including Pneumocystis, warranting send out NAAT testing for P. jirovecii in a reference laboratory if not available in the laboratory.<\/p>\n<\/div><\/section>
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Ground-glass appearance <\/em>of Pneumocystis jirovecii <\/em>in human lungs<\/em>
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Limitations of P. jirovecii detection<\/h3>\n

Specimen type<\/strong>
\nThe recommended specimens for PCP diagnosis are broncho-alveolar lavage (BAL) or lung tissue biopsy. However, collection of these specimen involves invasive procedures. These invasive proce-dures might not be feasible in immunocompromised patients, such as thrombocytopenic patients with hematological cancer. Therefore, it might be useful to have validated non-invasive or minimally invasive samples for P. jirovecii detection, such as induced sputum or blood. Sputum is currently being used by clinical microbiology laboratories to detect Pneumocystis both microscopically and via PCR but less sensitive than BAL.<\/p>\n

Microscopy<\/strong><\/p>\n

Although microscopy (including the immunofluorescence techniques) is the gold standard for Pneumocystis detection, a disadvantage is that it has lower sensitivity, particularly
\namong non-HIV-infected individuals with low fungal load.<\/p>\n

NAAT<\/strong><\/p>\n

One disadvantage of conventional PCR is that the presence of PCP DNA does not differentiate active infection from colonization. However, this challenge is solved by the real time qPCR testing which can quantify the burden of PCP in the sample, differentiating active infection from colonization.<\/p>\n

Beta-D-glucan biomarker<\/strong><\/p>\n

The limitation of testing for the presence of beta-D-glucan is that it is not specific for P. jirovecii, as it is also present in other fungal organisms and false-positive results can occur from cross-reaction with other compounds present in the patient\u2019s serum especially among patients undergoing hemodialysis with cellulose membranes, patients treated with immunoglobulin and patients with exposure to glucan-containing gauze. Hence, beta-D-glucan testing is only used as a supportive test for PCP, requiring PCP-specific testing, such as NAAT.<\/p>\n

Future developments to improve diagnosis of PCP<\/h4>\n

The lack of an easy and simple culture system for P. jirovecii continues to impair research and development in P. jirovecii diagnostics, including hampering antimicrobial susceptibility testing and accurate disease definition. Development of ex-vivo lung tissue and in-vivo animal models of P. jirovecii culture has been reported but this is not feasible for routine testing in a clinical microbiology laboratory. Real-time PCR on blood samples including plasma and serum have been evaluated as a reliable surrogate sample for the more invasive BAL. Additionally, the urgent need for an FDA approved IVD NAAT test for PCP cannot be overemphasized. This is important for the diagnosis and management of PCP, especially among non-HIV-infected immunocompromised patients in this era of highly active antiretroviral treatment for HIV-infected individuals.<\/p>\n<\/div><\/section>
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The author<\/strong><\/em><\/p>\n

Osaretin Emmanuel Asowata PhD, Clinical Microbiology Fellow Clinical Microbiology Services, Department <\/em>of Pathology and Laboratory Medicine, Memorial Sloan Kettering Cancer Center, <\/em>New York, NY, USA<\/em><\/p>\n

Email: asowato1@mskcc.org<\/a><\/em><\/p>\n<\/div><\/section>
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