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4. The picture below is for PCP (Pneumocystis pneumonia caused by Pneumocystis Jirovecii) diagnosed by lung biopsy
- What are the modalities used in the diagnosis of this disease?
- What are their sensitivities and specificities?
- What is the place of lung biopsy in the diagnosis of PJP?
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Chest x ray ,HRCT with specific characteristics for PCP subpleural spaces ,reticulations and some cysts
Sputum culture And BAL
B D glucan assay
lung biopsy
to see the organism this is the golden standard for diagnosis
-Direct microscopic visualization of PCP ( most sensitive but less specific ).
-Bronchoalveolar lavage .
-Open lung biopsy specimens
-PCR is more specific than DFA, but can’t differentiate between infection and colonization.
-Serum-β-D-glucan assay (BDG) sensitivity was 91%.
Modalities use to diagnose PJP are:
Mainly: chest x-ray, arterial blood gas, serum LDH level, induced sputum analysis.
Then followed by: high-resolution computed tomography (HRCT) chest, pulmonary function testing, bronchoscopy, bronchoalveolar lavage (BAL), and lung tissue biopsy.
Emerging tests as polymerase chain reaction (PCR), plasma S-adenosylmethionine level and serum (1,3)-beta-D-glucan level.
Diagnosis is made by detection of the organism in either induced sputum or bronchoalveolar lavage.
PCR using BAL fluid and sputum had a high sensitivity (97.1% and 91.4%, respectively) but relatively low specificity (86.1% and 86.1%, respectively). The combination of the sputum PCR OR serum cfDNA PCR yielded a sensitivity of 97.1%
Lung biopsy is the final tool for detection of PJP within lung tissue by gimesa stain being invasive procedure, it also aids to exclude other pathologies as malignancy, fungal infection and TB or CMV infection.
● What are the modalities used in the diagnosis of this disease?
☆ Sputum induction is the quickest and least-invasive method for definitively diagnosing PJP. Expectorated sputum has a very low sensitivity and should not be submitted for diagnosis.
☆ Conventional PCR
This test has low specificity, high false-positive rates, and low positive predictive values, and cannot differentiate PCP from asymptomatic Pneumocystis colonization.
☆ PCR directed against major surface glycoproteins has sensitivities from 93% to 100% and specificities higher than those observed for standard PCR (83%–100%).
☆ BDG assays have sensitivities ranging from 90% to 100% and specificities of 88%–96% in non-HIV-infected immunosuppressed patients with pneumonia.
☆ Bronchoalveolar lavage and transbronchial biopsy diagnosed in > 95%
● What is the place of lung biopsy in the diagnosis of PJP?
When There is non specific findings to exclude other etiologies as malignancy and fungal or mycobacterial infections which is also common in immunocompromised patients .
What are the modalities used in the diagnosis of this disease?
What are their sensitivities and specificities?
What is the place of lung biopsy in the diagnosis of PJP?
· Diagnosis of PCP needs identification of cystic or trophic forms in respiratory secretions
Laboratory diagnosis:
o LDH levels:
· It is elevated (>220 U/L) in 90% of patients with (PJP) who are infected with HIV.
· Has high sensitivity (78%-100%) but low specificity
o β-D-Glucan (BDG)
· It us a sensitive test to detect PJP in patients with HIV infection.
· In non-HIV cases, the results should be interpreted
· The use of this assay in supporting the diagnosis of PCP.
o Quantitative PCR for PJP:
· PCR of respiratory fluid particularly obtained by (BAL), is increasingly used to make the diagnosis of PCP in both patients with and without HIV.
· Disadvantage: cannot distinguish between colonization and disease
Imaging Modalities
· HRCT of the chest is helpful when as chest radiography findings are equivocal.
· It has high sensitivity for PJP in patients with HIV infection.
· It shows patchy areas of ground-glass attenuation with a background of interlobular septal thickening.
Sputum Induction:
· Sputum is induced by inhalation of a hypertonic saline solution.
· It is the quick and least-invasive method
· very low sensitivity compared to BAL
· It allows for histo-pathologic testing and Pneumocystis antigen detection assays
Invasive Procedures:
Bronchoalveolar lavage (BAL)
· The most common invasive procedure used to diagnose PJP.
· The diagnostic yield > 90% (and may be higher if multiple lobes are sampled).However, it has a lower sensitivity in patients receiving aerosolized pentamidine, in which case a trans-bronchial biopsy may be performed in conjunction with BAL.
· Lung biopsy
The most invasive procedure and yields 100% sensitivity and specificity(reserved for cases in whom BAL testing was negative despite having a high index of suspicion for PCP
· staining techniques for respiratory tract secretions like Crystal violet, Giemsa, are available. Direct Immunofluorescence using monoclonal antibodies to detect Pneumocystis organisms can be used (more sensitive than histologic staining).
Reference:
1. Shelley A Gilroy; Pranatharthi Haran Chandrasekar.Pneumocystis jiroveci Pneumonia (PJP) Overview of Pneumocystis jiroveci Pneumonia Nov 04, 2022
2. Crans CA, Jr., Boiselle PM. Imaging features of Pneumocystis carinii pneumonia. Critical reviews in diagnostic imaging. 1999 Aug;40(4):251-84.
3. Thomas CF, Jr., Limper AH. Pneumocystis pneumonia. The New England journal of medicine. 2004 Jun 10;350(24):2487-98.
What are the modalities used in the diagnosis of this disease?
Microscopy with staining –
Detection of the organism in respiratory specimens is most commonly achieved by microscopy with staining of an induced sputum specimen or BAL fluid . Staining is necessary . Direct fluorescent antibody staining using a fluorescein-conjugated monoclonal antibody can visualize both trophic forms and cysts and is the most common technique used. The most rapid and least invasive method of diagnosing PCP is by analysis of sputum induced by the inhalation of hypertonic saline.If PCP is not identified by this modality, then bronchoscopy with BAL should be performed.
The diagnostic yield of microscopy with staining of induced sputum is 50 to 90 percent in patients with HIV and PCP but is thought to be lower in patients without HIV due to a decreased organism burden . A similar difference has been noted with BAL. The diagnostic yield is over 90 percent in patients with HIV but may be lower in patients without HIV
Polymerase chain reaction —
A number of PCR assays have been developed for the detection of Pneumocystis in induced sputum or BAL fluid, blood, or nasopharyngeal aspirates. These assays may be of particular use in patients without HIV, in whom the sensitivity of microscopy with staining is substantially lower than in patients with HIV. Overall, PCR-based detection of PCP adds roughly 7 percent yield over stains alone when applied to BAL specimens
Beta-D-glucan assay –
Serum beta-D-glucan assay can be used as an adjunct to the diagnosis of PCP. Generally, the test has good sensitivity and a high negative predictive value, making it unlikely that a patient with a negative beta-D-glucan result has PCP . Using a cut-off of 80 pg/mL, the assay had a sensitivity and specificity of 70 and 81 percent, respectively.
What is the place of lung biopsy in the diagnosis of PJP?
Open lung biopsy is the most invasive procedure and yields 100% sensitivity and specificity because it provides the greatest amount of tissue for diagnosis. However, this procedure is reserved for rare cases when bronchoscopy findings are nondiagnostic.
Polymerase chain reaction
PCR has been shown to be more sensitive for detection of PCP than staining methods in patients with and without HIV.
Some studies suggest that it may be 104 to 106 times as sensitive
Bronchoalveolar lavage fluid (BALF)
The current gold standard sample for diagnosis of PCP is BALF, which is considered to be the highest quality respiratory sample.
lack of a standardized sampling technique can impact test performance. Bronchoscopy, when performed after negative induced sputum testing, has been noted to yield a diagnosis in 51% of cases and, if negative for PCP, allows for discontinuation of treatment.
Limitations include:
-invasive procedure, expensive, carries more of a risk to the patient, may not always be feasible for patients with severe pulmonary disease, and may not be available in some places .
Induced Sputum
less invasive techniques for obtaining samples for testing.
found to have a >95% negative predictive value in low prevalence situations (<10% prevalence), making a negative test adequate for ruling out PCP.
However, in high prevalence areas and cases of high suspicion of PCP, a bronchoscopy with BAL should be performed when negative IS results are obtained.
IS has also been found to have 85–100% sensitivity and good concordance with BALF results when methods of detection such as PCR are utilized.
Oral washing
Pneumocystis jirovecii may be found in oral washes if the organism has been coughed or recently inhaled into the oropharyngeal tract. Oral washes can be obtained quickly and noninvasively, and positive tests may reflect an even higher fungal burden in the lower respiratory tract. However, there are theoretical disadvantages such as increased degree of PCR inhibition due to dilution from pharyngeal secretions, the inability of organisms to reach the oral cavity in low fungal burden infections.
When compared with sputum and BAL, oral wash PCR has been noted to have a sensitivity of 75–91% and a specificity of 68–100%.
Oral wash samples may be most useful in supporting a diagnosis of PCP if positive, but a negative result cannot reliably rule out PCP in symptomatic patients.
Nasopharyngeal aspirate
Lower respiratory tract specimens such as BALF and sputum are difficult to obtain in children. In one study, MSG PCR on nasopharyngeal samples was found to have an 86% sensitivity and 95% specificity for detecting PCP when compared to BALF and sputum samples.
PCR was specifically noted to have a higher detection rate than immunofluorescence staining techniques, which may explain the low sensitivities, PCR on nasopharyngeal samples, if positive, may obviate the need to obtain more invasive samples.
Blood/serum
Blood/serum has the significant advantage of being easily obtained and inexpensive. The presence of Pneumocystis jirovecii in the blood reflects disease progression, as the pathogen is no longer limited to the respiratory tract.
use of serum PCR is not currently recommended for detection of PCP. Other serum diagnostic tests including serum enzyme-linked immunosorbent assay (ELISA) for antibodies and antigens associated with PCP are more promising and are further described below.
Nonimmunofluorescent staining
The cyst life-form can be detected with many stains. Giemsa, Diff-Quik, and Wright stains can detect the cyst but do not stain its wall.
The Gomori-methenamine-silver (GMS) stain, Gram-Weigert, cresyl echt violet, toluidine blue O (TBO), and calcofluor white (CW) stains stain the cell wall of the cyst.
The stains for the cyst cell wall have traditionally been preferred due to the ability for rapid analysis and minimal expertise needed for interpretation. These stains will stain both live and dead cysts.
only CW and GMS had positive and negative predictive values >90% when performed on BALF.
Immunofluorescent stainingImmunofluorescent stains via monoclonal antibodies to Pneumocystis jirovecii have a higher sensitivity and specificity than conventional stains. Sensitivity ranges from 48 to 100%, and specificity from 82 to 100%
Lung biopsy is the gold standard test for accurate diagnostic test but being invasiveness procedure it is used if highly indicated.
Bateman M, Oladele R, Kolls JK. Diagnosing Pneumocystis jirovecii pneumonia: A review of current methods and novel approaches. Med Mycol. 2020 Nov 10;58(8):1015-1028.
Modalities are :
PCR of respiratory fluid .
Bronchialvelolar lavage .
Beta _d glucan assay .
Cytology assays.
Lung biopsy are rarly done in pcp infection.
What are the modalities used in the diagnosis of this disease?
To diagnose PCP, quantitative real-time PCR (qPCR) is more sensitive than microscopic examination. However, qPCR can also detect colonization by the organism, not just active infection. The current diagnostic approach involves examining bronchoalveolar lavage fluid (BALF) under a microscope using suitable staining methods, such as May-Grünwald-Giemsa, Gomori-Grocott, or immunofluorescence assay, to identify trophic forms or cysts of P. jirovecii.
Specimens for analysis can be obtained from the bronchi using techniques like bronchoalveolar lavage (BAL), induced sputum with saline, video-assisted thoracoscopic surgery (VATS), or endotracheal aspirates.
The beta-D-glucan assay can be used to detect the presence of beta-D-glucan, which is indicative of conditions such as candidemia, invasive aspergillosis, or PCP. It is more sensitive in immunocompromised individuals but may yield false-positive results in cases of Pseudomonas infection or recent intravenous immunoglobulin (IVIG) use. The assay can be useful in guiding treatment until PCP PCR results become available.
What are their sensitivities and specificities?
Different diagnostic modalities exhibit varying sensitivities and specificities. Induced sputum has a sensitivity of 99% and specificity of 96%. Cytological staining shows a sensitivity of 50% and specificity of 100%. PCR has a sensitivity of 87% and specificity of 92%.
What is the place of lung biopsy in the diagnosis of PJP?
Lung biopsy is rarely performed for the diagnosis of PCP. It may be considered in situations where the diagnosis is uncertain or when investigating other conditions.
Following investigations should be done to confirm the diagnosis of pneumocystis .
· Chest Xray is abnormal in around 92% of cases .HRCT chest is better modality .
· Sputum analysis with special stains like methenamine silver, cresyl violet, Gram-Weigert and toluidine blue for staining cystic forms of the organism and Wright-Giemsa stain for both cystic and trophic forms. Sputum can be production via hypertonic saline and this has sensitivity of 55-90%.Bronchoalveolar lavage in cases in which there is no sputum production with similar staining techniques as mentioned above –it has sensitivity of sensitivity was 90-100%.PCR can also be done on BAL which has sensitivity of 100% and specificity 97.2%. Direct fluorescent antibody staining using a fluorescein-conjugated monoclonal antibody has better sensitivity .
· Beta D- glucan assay can be done which indicates invasive fungal infection .It has sensitivity of 70-92%, while specificity is 80-85% with different cut off values used.
· Non-invasive test like Pulmonary function tests which indicates reduced DLCO in PJP (89 -100%) with poor specificity of 53%.
What is the place of lung biopsy in the diagnosis of PJP?
Lung biopsy which can be done by VATS or thoracotomy has sensitivity of 95 to 100 per cent. Not routinely performed, usually done in cases in which above tests inconclusive or if there is another cause of pulmonary disease.
REFERENCES:
Fauchier T, Hasseine L, Gari-Toussaint M, Casanova V, Marty PM, Pomares C. Detection of Pneumocystis jirovecii by Quantitative PCR to Differentiate Colonization and Pneumonia in Immunocompromised HIV-Positive and HIV-Negative Patients. Journal of Clinical Microbiology. 2016 Jun. 54(6):1487-1494
Q1: Modalities:
1. Induced sputum test or BAL fluid test is used for microscopic and staining by fluorescein-conjugated monoclonal antibodies (both forms). Wright-Geimse, Papanicolaou, Diff-Quik, and Gram-Weigert staining for trophozoites. Cysts are stained by Gomari methenamine silver (GMS), and toluidine blue O (TBO).
PCR test using these specimens is useful.
2. Serum 1,3 D glucan
3. Serum LDH
4. Lung biopsy
Q2:
Induced sputum: 30-35% sensitivity. Immunofluorescence staining has 80-100% specificity and NPV≤95%.
BAL fluid PCR: Sensitivity≤ 98% and specificity ≤ 90%.
Beta D glucan: Sensitivity ≤90% and specificity up to 75%.
Serum LDH: In HIV positive patients 100% sensitivity and in the other patients 63%, specificity 36-52%.
Q3: Lung biopsy is required only in patients with negative BAL fluid results and high suspicious. It has 100% specificity. But, it is invasive and causes pneumothorax in 30% of cases.
We can use for the diagnosis the following methods:
1 – Citologic:
– Citologic staing
– Fluorescent antibody
2 – Molecular:
– Polymerase chain reaction (PCR):
3 – Biomarkers
– Lactate dehydrogenase
– B-D-glucan
– Cytologic staining: moderate sensitivity at 50% (95%CI 39-61%) and 25 high specificity at 100% (95%CI 100-100%)
– Fluorescent antibody: sensitivity 26 -74% (95%CI 62-87%) and specificity 100% (95%CI 91-100%)
– Polymerase chain reaction (PCR): sensitivity of 97 – 99%, with a pooled specificity of 90- 94%
-Lactate dehydrogenase: was not helpful to rule in or out the diagnosis
– B-D-glucan : sensitivity is moderate (86%; 95%CI 78-91%
Bronchoalveolar lavage fluid with transbronchial biopsy, which undergoes microscopy with conventional or immunofluorescent staining to identify Pneumocystis is the current criterion standard. However, it has fallen into disuse due to the risk of pneumothorax and other complications.
REFERENCES:
1 – Non-invasive diagnosis of Pneumocystis jirovecii pneumonia: a systematic review and meta-analysis
Senécal, Julien et al.
Clinical Microbiology and Infection, Volume 28, Issue 1, 23 – 30
2 – Marjorie Bateman, Rita Oladele and Jay K Kolls. Diagnosing Pneumocystis jirovecii pneumonia: A review of current methods and novel approaches. Medical Mycology, 2020, 0, 1–14 doi:10.1093/mmy/myaa024
· What are the modalities used in the diagnosis of this disease?
Samples are either: Broncho-alveolar lavage, induced sputum, oral washings, naso-pharyngeal aspirates.
a- Stains of respiratory specimens:
Stains for cystic form: Flourescein-conjugated monoclonal antibody, GMS (Gomori Methenamine Silver), TB0 (Toluidine Blue 0), Cresyl Echt Violet and CW (Calcoflour white). Stains for trophic form: Flourescein-conjugated monoclonal antibody, Wright-Geimsa, Modified Papanicolaou, Diff Quik and Gram-Weigert.
b- PCR for PJP in respiratory specimens.
Blood samples:
1- Blood PCR for PJP.
2- Serum 1,3 Beta D Glucan.
3- Serum LDH will be very high in PJP.
· What are their sensitivities and specificities?
Sensitivity of stains: CW, GMS, and TB0 around 57-78% , 31-67% and 49-94% respectively.
IF stains have sensitivity 48-100% and specificity 82-100%.
Induced Sputum: sensitivity 30-55%.
IF stain of induced sputum: sensitivity 85-100%.
BAL: 51% sensitivity in those with negative induced sputum, PCR has 98% sensitivity and 90% specificity.
Oral Washings: PCR 75-91% sensitivity & 68-100% specificity.
Nasopharyngeal aspirates: PCR 86% sensitivity and 95% specificity.
Blood PCR: 100% sensitivity.
Serum 1,3 Beta D glucan: 90% sensitivity, 75% specificity.
Serum LDH: 66-91% sensitivity, 36-52% specificity.
· What is the place of lung biopsy in the diagnosis of PJP?
Is needed if BAL is negative
References:
Bateman M, Oladele R, Kolls JK. Diagnosing Pneumocystis jirovecii pneumonia: A review of current methods and novel approaches. Med Mycol. 2020 Nov 10;58(8):1015-1028. doi: 10.1093/mmy/myaa024. PMID: 32400869; PMCID: PMC7657095.
What are the modalities used in the diagnosis of this disease?
1-Bronchoalveolar lavage fluid (BALF);The current gold standard sample for diagnosis of PCP is BALF, which is considered to be the highest quality respiratory sample.
2- Sputum less invasive techniques for obtaining samples for testing.
3- Oral washingOral washes can be obtained quickly and noninvasively, and positive tests may reflect an even higher fungal burden in the lower respiratory tract.
4- Nasopharyngeal aspirateLower respiratory tract specimens such as BALF and sputum are difficult to obtain in children
5-Blood/serumBlood/serum has the significant advantage of being easily obtained and inexpensive. The presence of Pneumocystis jirovecii in the blood reflects disease progression, as the pathogen is no longer limited to the respiratory tract.
6-UrineUrine antigen testing has proven valuable in the diagnosis of histoplasmosis, and preliminary studies have shown that other fungal infections such as cryptococcosis, blastomycosis, and aspergillosis may be detectable by urine lateral-flow assay testing.
Novel methods of detection;
1-Polymerase chain reaction
2-Loop-mediated isothermal amplification (LAMP)Loop-mediated isothermal amplification (LAMP) provides an alternative to PCR as it can amplify a target gene with only a heating device and isothermal conditions.113 Sensitivity ranges from 87.5 to 95.4%, and LAMP has been shown to be relatively specific with no cross-reactivity to other fungal species.
3-Flow cytometryFlow cytometry can detect single or multiple microbes in an easy, reliable, and fast way. This method allows for detection of Pneumocystis jirovecii in clinical BAL and bronchial samples with 100% sensitivity and specificity when compared to immunofluorescent staining. While the applications are vast, the data are limited, and this is not currently recommended as a diagnostic method.
4-Antibody assaysA promising diagnostic approach is to use an antigenic tool in an ELISA technique to detect immunoglobulin (Ig), IgM, and IgG antibodies against Pneumocystis jirovecii
5-Antigen and biomarker assays;Beta D-Glucan (BG) is a cell wall constituent in the ascus life-form of Pneumocystis jirovecii and multiple other fungal pathogens.
6-Lactate dehydrogenase (LDH);
Serum levels of LDH have been found to be significantly elevated in patients with PCP relative to patients negative for PCP.
–
What are their sensitivities and specificities?
Nasopharyngeal aspiratePCR on nasopharyngeal samples was found to have an 86% sensitivity and 95% specificity for detecting PCP when compared to BALF and sputum samples.
Blood/serumA recent report from Sweden revealed that real-time PCR analysis on serum samples had a very high sensitivity (100%) and negative predictive value (99%) for the diagnosis of PCP in HIV-infected patients.
Loop-mediated isothermal amplification (LAMP)Sensitivity ranges from 87.5 to 95.4%, and LAMP has been shown to be relatively specific with no cross-reactivity to other fungal species.
Flow cytometryThis method allows for detection of Pneumocystis jirovecii in clinical BAL and bronchial samples with 100% sensitivity and specificity when compared to immunofluorescent staining.
Antibody assaysOne study has shown that ELISA IgM anti-P. jirovecii has a sensitivity of 100% and a specificity of 81% when testing serum samples from 88 patients.
Antigen and biomarker assays;
In several meta-analyses, this assay was found to be 91%, 96%, and 95% sensitive with high sensitivity being demonstrated in both HIV positive and negative patients. However, BG was only 75%, 84%, and 86% specific for definite PCP because the assay could be positive in other fungal infections in patients with gram-negative endotoxinemia, in patients on certain antibiotics, in patients on albumin or globulin therapy, and in patients undergoing HD due to cellulose membranes and filters.
Lactate dehydrogenase (LDH);
The sensitivity and specificity of this marker for PCP have been estimated to be 66%–91% and 36–52%, respectively.
What is the place of lung biopsy in the diagnosis of PJP?
ENB-guided FNA with ROSE is commonly used in the evaluation of thoracic malignancies, but there is no established use for it in the setting of the infectious disease workup. While bronchoscopy with BAL is a reasonable method for evaluating a cavitary lung lesion, a lavage sample would be unlikely to render a diagnosis of malignancy. In addition, many of these cavitary lesions may be located in the periphery of the lung and are difficult to access with conventional bronchoscopy and sampling via a transthoracic needle approach with radiologic guidance can lead to a pneumothorax (complication rates from 17% to 26%).
Reference ;
1-Marjorie Bateman, Rita Oladele, et al. Diagnosing Pneumocystis jirovecii pneumonia: A review of current methods and novel approaches. Med Mycol. 2020; 58(8): 1015–1028.
2. Yeow KM, Tsay PK, Cheung YC, Lui KW, Pan KT, Chou AS. Factors affecting diagnostic accuracy of CT-guided coaxial cutting needle lung biopsy: Retrospective analysis of 631 procedures. J Vasc Interv Radiol. 2003;14:581–8. [PubMed] [Google Scholar]
3. Khan MF, Straub R, Moghaddam SR, Maataoui A, Gurung J, Wagner TO, et al. Variables affecting the risk of pneumothorax and intrapulmonal hemorrhage in CT-guided transthoracic biopsy. Eur Radiol. 2008;18:1356–63. [PubMed] [Google Scholar]
4. Covey AM, Gandhi R, Brody LA, Getrajdman G, Thaler HT, Brown KT. Factors associated with pneumothorax and pneumothorax requiring treatment after percutaneous lung biopsy in 443 consecutive patients. J Vasc Interv Radiol. 2004;15:479–83. [PubMed] [Google Scholar]
Modalities for diagnosing pcp include
1.examination of respiratory specimens optained from sputum, BAL, or nasopharyngeal aspirate stained by fluorescein conjugated monoclonal antibody, which stains both cysts and the trophic form of the organism. Trophic forms can also be seen by using Wright-Geimsa, modified Papanicolaou, Diff-Quik, and Gram-Weigert stains. Cysts can be seen with Gomori methenamine silver (GMS), toluidine blue O (TBO), cresyl echt violet, and calcofluor white (CW) stain.
2. PCR of blood or respiratory secretions
3. Elevation of inflamatory markers CRP and also high LDH
Nonimmunofluorescent stainingThe cyst life-form can be detected with many stains. Giemsa, Diff-Quik, and Wright stains can detect the cyst but do not stain its wall. The Gomori-methenamine-silver (GMS) stain, Gram-Weigert, cresyl echt violet, toluidine blue O (TBO), and calcofluor white (CW) stains stain the cell wall of the cyst. The stains for the cyst cell wall have traditionally been preferred due to the ability for rapid analysis and minimal expertise needed for interpretation. These stains will stain both live and dead cysts. The trophozoite life-form can be detected with Giemsa, Diff-Quik, Wright-Giemsa stain, modified Papanicolaou, or Gram-Weigert stains.1 However, due to its small size and nonspecific staining pattern, this is not the life-form typically used in diagnosis.
Studies comparing staining methods report that the highest sensitivity methods are CW, GMS stain, and TBO stain. Per Procop et al., only CW and GMS had positive and negative predictive values >90% when performed on BALF. The sensitivity of the CW stain has ranged from 57 to 78%. The sensitivity of GMS ranges from 31 to 97% with lower sensitivities being present in studies including poor quality samples or a large number of noninvasive samples such as IS and nasopharyngeal aspirates. TBO staining has also been reported to have lower sensitivity ranging from 49 to 94%.These staining methods are specific for the presence of organisms but if negative, do not rule out the presence of PCP. While these tests are easy to perform, they are reliant on the quality of the sample and subjective due to dependence on stain interpretation.
Immunofluorescent staining Immunofluorescent stains via monoclonal antibodies to Pneumocystis jirovecii have a higher sensitivity and specificity than conventional stains. Sensitivity ranges from 48 to 100%, and specificity from 82 to 100%. They are easier to perform, more repeatable, and less reliant on technical skill for performance and interpretation. They can also stain both trophozoites and cysts.1 Comparisons of GMS and immunofluorescent stains.
Polymerase chain reaction PCR for Pneumocystis jirovecii was initially developed in the 1980s with primers testing for the gene for pneumocystis mitochondrial large-subunit ribosomal RNA (mtLSU rRNA).1 Nested PCR was one of the first techniques developed but has since been shown to be more labor intensive, more expensive, less quantitative, and less specific than real-time PCR, which will be the focus of this review. mtLSU real-time PCR has remained one of the most popular methods with other gene assays being developed including Kex-1, dihydroperoate synthase (DHPS), 5S rRNA, mitochondrial ribosomal rRNA, major surface glycoprotein (MSG), and internal transcribed spacer.PCR has been shown to be more sensitive for detection of PCP than staining methods in patients with and without HIV. Some studies suggest that it may be 104 to 106 times as sensitive.18 Three meta-analyses published in recent years reported a pooled sensitivity of 98%, 99%, and 97% with a pooled specificity of 91%, 90%, and 94% with most samples being BALF This high sensitivity and specificity persisted in both the HIV-positive and HIV-negative populations.Notably, the highest sensitivity and specificity were found in studies using quantitative PCR methods. Because the sensitivity is high, a false negative test result is rare. Therefore, a negative PCR on BALF means that PCP is an unlikely diagnosis, and other diagnoses should be considered as the etiology for the patient’s symptoms. In contrast, the high specificity means that a positive PCR on BALF is highly suggestive of the presence of Pneumocystis jirovecii. Loop-mediated isothermal amplification (LAMP) Loop-mediated isothermal amplification (LAMP) provides an alternative to PCR as it can amplify a target gene with only a heating device and isothermal conditions. Sensitivity ranges from 87.5 to 95.4%,and LAMP has been shown to be relatively specific with no cross-reactivity to other fungal species. In small studies, LAMP has been shown to have higher rates of detection of PCP thanconventional stains and rates similar to those of PCR.In some cases, visual detection with LAMP is possible as a particularly rapid and easy assay with only a black light and heating block.Flow cytometry Flow cytometry can detect single or multiple microbes in an easy, reliable, and fast way. These organisms can be identif ied by cytometric parameters, fluorochromes such as CW, or
monoclonal antibodies to Pneumocystis jirovecii. Flow cytometers can also detect antibodies against Pneumocystis and comment on antifungal susceptibility. Barbosa et al. have developed a method that uses immunofluorescent staining with the Detect IF kit (Axis-Shield Diagnostics Limited, UK) followed by flow cytometry.This method allows for detection of Pneumocystis jirovecii in clinical BAL and bronchial samples with 100% sensitivity and specificity when compared to immunofluorescent staining.While the applications are vast, the data are limited, and this is not currently recommended as a diagnostic method. Antibody assays A promising diagnostic approach is to use an antigenic tool in an ELISA technique to detect immunoglobulin (Ig), IgM, and IgG antibodies against Pneumocystis jirovecii. Multiple potential immunogenic antigens have been described, including natural antigens such as Meu10 and recombinant synthetic antigens designed from the MSG gene. One study has shown that ELISA IgM anti-P. jirovecii has a sensitivity of 100% and a specificity of 81% when testing serum samples from 88 patients. Notably,the immune response may be variable depending on the nature of the immunocompromise and may affect the sensitivity of this assay in certain populations. Previous studies have shownalterations in immuneresponseinpatients withHIV, patients with a history of transplant, patients with cancer, patients who fail to adhere to prophylactic therapy, patients who smoke, patients with chronic obstructive pulmonary disease, patients with hazardous alcohol use, patients with injection drug use, and even patients from different geographic areas. Previous clinical infection or subclinical exposure to Pneumocystis may also impact immune response and lead to false positive tests. Additional elucidation of the complex host and environmental factors that affect antibody formation will be required before this and similar tests are considered for widespread utilization. Antigen and biomarker assays (1,3)-Beta D-Glucan (BG) is a cell wall constituent in the ascus life-form of Pneumocystis jirovecii and multiple other fungal pathogens. Various assays that detect BG in the serum have beendevelopedwiththemostpopularintheWesternhemisphere being the Fungitell test, a chromogenic kinetic test approved in 2003 by the US Food and Drug Administration. In several meta-analyses, this assay was found to be 91%, 96%, and 95% sensitive with high sensitivity being demonstrated in both HIVpositiveandnegativepatients. However,BGwasonly 75%, 84%, and 86% specific for definite PCP because the assay could be positive in other fungal infections in patients with gram-negative endotoxinemia, in patients on certain antibiotics, in patients on albumin or globulin therapy, and in patients undergoing HD due to cellulose membranes and filters. Notably, the true value for specificity is more likely closer to 75% because the other two meta-analyses excluded the patients diagnosed with other invasive fungal diseases which likely exaggerated specifity. The significant advantage is the noninvasive nature of the test and the ability of a negative test to make PCP highly unlikely. The European Conference on Infections in Leukemia even stated that a negative serum BG was adequate to rule out PCP. The disadvantage is that a positive BG is not specific for the diagnosis of PCP, so further testing would need to be performed for validation of the diagnosis. Lactate dehydrogenase (LDH) is an intracellular enzyme found in almost all tissues. Serum levels of LDH have been found to be significantly elevated in patients with PCP relative to patients negative for PCP. The sensitivity and specificity of this marker for PCP have been estimated to be 66%–91%and36–52%,respectively. In one study,the sensitivity of LDH elevation was found to be 100% in HIV-positive patients but only 63% in HIV-negative patients,indicating that this marker may only be useful in detecting PCP in HIV-positive patients. OxygenationandBALneutrophillevels have also been found to correlate with LDH levels. Therefore, LDH levels are likely a reflection of the underlying lung inflammation and injury and are not specific to PCP. Other antigens such as KL-6 and S-adenosylmethionine have also been evaluated as prospective markers. KL-6 antigen is a mucin-glycoprotein expressed on type 2 alveolar pneumocytes and bronchiolar epithelial cells.Serum levels of KL-6 have been found to be elevated in patients with PCP, but this marker has low specificity due to its elevation in any interstitial lung disease and other infectious diseases. Sadenosylmethionine (SAM) is an intermediate in multiple cellular functions that Pneumocystis cannot synthesize and must extract from the plasma of its host. Some studies have demonstrated significantly lower serum SAM levels in patients infected with PCP relative to patients infected with other pathogens and control patients. In other studies, this marker failed to discriminate patients with PCP from those without PCP. These biomarkers cannot be recommended for use at this time.
1. What are the modalities used in the diagnosis of this disease?
– HRCT of chest (subpleural spearing, honey combing, ground glass opacity, reticulation, stripes)
– Diagnostic specimen – BAL, Transbronchial biopsy, open lung biopsy/ VATS, induced sputum.
– Ancillary blood tests – CBC, CRP, LDH, ABG, RFT, LFT
– Diagnostic technique- immunofluorescence assay, real time PCR, nucleic acid testing, silver staining.
– Lung biopsy specimen – lymphocytic infiltration; Gonori methenamine silver staining (dark brown oval / cup shaped organism in alveolar spaces)
2. What are their sensitivities and specificities?
-Microscopic detection of P. Jirovecii in respiratory specimen following chemical or immunofluorescence staining- low sensitivity & specificity.
– Serum Beta- D glucan- sensitive but lack specificity.
– LDH – not specific
– CXR- may be normal in 30% patients
– HRCT- 80% sensitive
– Lung biopsy is gold standard. 95-100% sensitivity & specificity
3. What is the place of lung biopsy in the diagnosis of PJP?
– In patients with high clinical suspicion of PJP, but BAL testing is negative
– In patients with other reason to proceed to lung biopsy for diagnosis of a pulmonary disease process.
What are the modalities used in the diagnosis of this disease?
What are their sensitivities and specificities?
What is the place of lung biopsy in the diagnosis of PJP?
Sputum Induction
histopathologic testing.
in sputum induced by inhalation of a hypertonic saline solution.
It is the quickest and least-invasive method
But it has very low sensitivity
Pneumocystis antigen detection assays on sputum
may also be helpful but have a low sensitivity.
The sensitivity of sputum induction varies widely (< 50% to >90%)
Specificity is high (99%-100%).
This study may be less sensitive in patients without HIV infection.
Invasive Procedures:
Bronchoalveolar lavage (BAL)
It is the most common invasive procedure used to diagnose (PJP).
It has a diagnostic yield that exceeds 90% (and may be higher if multiple lobes are sampled).
BAL yields a lower sensitivity in patients receiving aerosolized pentamidine, in which case a transbronchial biopsy may be performed in conjunction with BAL.
Lung biopsy
Open lung biopsy is the most invasive procedure and yields 100% sensitivity and specificity
Histologic Findings
The following staining techniques available for respiratory tract secretions.
Crystal violet,Giemsa,Diff-Quik,Wright stainSome facilities prefer to use direc ( IF ) Immunofluorescence using monoclonal antibodies to detect Pneumocystis organisms because it may be more sensitive than histologic staining.
Imaging Modalities
(HRCT) scanning of chest is helpful when the chest radiography findings are equivocal.
It has high sensitivity for (PJP) in patients with HIV infection.
The typical appearance is patchy areas of ground-glass attenuation with a background of interlobular septal thickening.
Negative (normal or unchanged) CT scan findings alone do not rule out PJP
Laboratory modalities:
LDH levels
elevated (>220 U/L) in patients with (PJP).
They are elevated in 90% of patients with PJP who are infected with HIV.
high sensitivity (78%-100%);
its specificity is much lower
β-D-Glucan (BDG)
a cell-wall component of many fungi, including Candida, Aspergillus, and Pneumocystis (but not the Zygomycetes).
It us a sensitive test to detect PJP in a meta-analysis of 13 studies assessing the sensitivity, specificity, only in patients with HIV infection.
In non-HIV cases, the results should be interpreted
The use of this assay in supporting the diagnosis of PCP.
Quantitative PCR for pneumocystis may be useful in distinguishing between colonization and active infection.
(PCR) of respiratory fluid, in particular (BAL), is increasingly used to make the diagnosis of PCP in both patients with and without HIV.
A disadvantage is that PCR cannot distinguish between colonization and disease
REFERENCE
Shelley A Gilroy; Pranatharthi Haran Chandrasekar.Pneumocystis jiroveci Pneumonia (PJP) Overview of Pneumocystis jiroveci Pneumonia Nov 04, 2022
Investigations-
– CBC, CRP, ABG
– Renal function test, LFT
– LDH
– HRCT of chest( subpleural spearing, honey combing, ground glass opacity, reticulation, stripes)
– Diagnostic specimen – BAL, Transbronchial biopsy, open lung biopsy/ VATS, induced sputum.
– Diagnostic technique- immunofluorescence assay, real time PCR, nucleic acid testing, silver staining.
– Lung biopsy specimen -lymphocytic infiltration, Gonori methenamine silver staining ( dark brown oval or cup shaped organism in alveolar spaces)
-Microscopic detection of P. Jirovecii in respiratory specimen following chemical or immunofluorescence staining- low sensitivity & specificity.
– Serum Beta- D glucan- sensitive but lack specificity.
– LDH – not specific
– CXR- may be normal upto 30%
– HRCT- 80% sensitive
– Lung biopsy- Gold standard. 95-100% sensitivity & specificity
-patients with high suspicion of PCP and BAL testing has been negative
– In those who have another reason to proceed to lung biopsy for diagnosis of a pulmonary process.
Sputum: Induced sputum, sensitivity of 85-100% when PCR is used
Immunofluorescent staining: Has a sensitivity of 90% and a specificity of 94%
PCR: Has a sensitivity of 97-99% and a specificity of 91-94%
Lung biopsy Gold standard, mostly done when high suspicion of PCP but BAL negative.
· What are the modalities used in the diagnosis of this disease?
Chest X ray;
May be asymptomatic vs diffuse bilateral infiltrates from hilar region up-to extending, pneumothorax, pneumatoceles, pleural effusion,
Sputum;
Noninvasive method for early detection of PCP.
High resolution computed tomography;
It yeildes a high sensitivity for PCP. They present with interlobular septal thickening, ground glass attenuation.
B-D-glucan;
Elevated level of BDG is sensitive marker for diagnosis of many fungal infection, it’s the cell wall components of many fungi.
PCR quantitative;
It has been increasingly used for diagnosis of fungal infection. The respiratory fluid is taken via BAL and tested.
LDH level;
It’s usually elevated above 200, its sensitivity is around 78 to 100% while its specificity is lower. Actually it shows the lung parenchymal injury, it can be monitored for treatment and prognosis.
BAL;
Its diagnostic yield is around 90%, it’s mandatory when patient is not cooperative for sputum induction.
Tissue biopsy;
This is an invasive procedure and yields around 100% sensitivity and specificity. Its last resorts when the clinical and other labs cannot rule out the final diagnosis.
Following staining techniques are used for histological diagnosis, Geimsa stain, Dff-Quik, wright stain, crystal violet stain, in addition, immunofluorescence issued for antibodies detection.
A lactic dehydrogenase (LDH) study is performed as part of the initial workup. LDH levels are usually elevated (>220 U/L) in patients with P jiroveci pneumonia (PJP). The study has a high sensitivity (78%-100%); its specificity is much lower because other disease processes can result in an elevated LDH level.
LDH levels appear to reflect the degree of lung injury. They should decline with successful treatment. Consistently elevated LDH levels during treatment may indicate therapy failure and a worse prognosis.
Quantitative PCR for pneumocystis may be useful in distinguishing between colonization and active infection. Polymerase chain reaction (PCR) of respiratory fluid, in particular bronchoalveolar lavage (BAL), is increasingly used to make the diagnosis of PCP in both patients with and without HIV, and several clinical laboratories offer this test. A disadvantage is that PCR cannot distinguish between colonization and disease.
Sputum P jirovecii PCR testing may be a viable alternative to invasive testing. This could be a more timely method for sample collection and would provide a safer alternative to bronchoscopic evaluation in patients who already have respiratory failure.
Further studies comparing the sensitivity, specificity, and positive and negative predictive values for each sample type are needed.
β-D-Glucan (BDG) is a cell-wall component of many fungi, including Candida, Aspergillus, and Pneumocystis (but not the Zygomycetes). It has been shown to be a sensitive test to detect PJP in a meta-analysis of 13 studies assessing the sensitivity, specificity, and overall accuracy of the test. A negative serum BDG result is sufficient for excluding PJP only in patients with HIV infection. In non-HIV cases, the results should be interpreted in parallel with clinical and radiologic findings. Elevated plasma levels of 1-3-beta-D-glucan, a component of the cell wall of P. jirovecii, have been found in patients with HIV and PCP. In a study of 282 patients with HIV, those with a diagnosis of PCP had significantly higher median beta-D-glucan levels than patients without the disease.
Chest RadiographyThe chest radiographic findings may be normal in patients with early mild disease. Diffuse bilateral infiltrates extending from the perihilar region are visible in most patients with P jiroveci pneumonia (PJP). Less-common findings include patchy asymmetric infiltrates, pneumothorax, and pneumatoceles. A radiographically normal chest radiograph has also been described. Pleural effusions and intrathoracic adenopathy are rare.
Pneumothorax may also develop in patients using aerosolized pentamidine. Apical disease may also be found in patients using aerosolized pentamidine for prophylaxis, as
Computed TomographyHigh-resolution computed tomography (HRCT) scanning of chest is helpful when the chest radiography findings are equivocal. HRCT yields a high sensitivity for P jiroveci pneumonia (PJP) in patients with HIV infection.
The typical appearance is patchy areas of ground-glass attenuation with a background of interlobular septal thickening. Negative (normal or unchanged) CT scan findings alone do not rule out PJP .
Pulmonary function testsPulmonary function tests should be obtained as part of the initial noninvasive workup in patients with suspected P jiroveci pneumonia (PJP). Results may demonstrate a decreased diffusion capacity of carbon monoxide (DLCO) of less than 75% predicted. Decreased DLCO has a high sensitivity (89%-100%) but poor specificity (53%). PJP is unlikely if DLCO is normal.
When combined with normal or unchanged HRCT findings, pulmonary function tests may be used to identify patients unlikely to have PJP; such patients may be managed with observation alone.
Sputum Induction If P jiroveci pneumonia (PJP) is strongly suspected, obtain a sputum sample by sputum induction for histopathologic testing. Pneumocystis organisms are frequently found in sputum induced by inhalation of a hypertonic saline solution. Sputum induction is the quickest and least-invasive method for definitively diagnosing PJP. Expectorated sputum has a very low sensitivity and should not be submitted for diagnosis. Pneumocystis antigen detection assays on sputum may also be helpful but may have a low sensitivity.
The sensitivity of sputum induction varies widely (< 50% to >90%) and depends on proficiency in using the technique and the experience of the laboratory. Specificity is high (99%-100%). This study may be less sensitive in patients without HIV infection, as the immunodeficiency caused by HIV infection typically leads to a greater alveolar load of Pneumocystis organisms. It may also be less sensitive in patients receiving aerosolized pentamidine for prophylaxis.
Bronchoalveolar lavageBronchoalveolar lavage (BAL) is the most common invasive procedure used to diagnose P jiroveci pneumonia (PJP). It has a diagnostic yield that exceeds 90% (and may be higher if multiple lobes are sampled). BAL yields a lower sensitivity in patients receiving aerosolized pentamidine, in which case a transbronchial biopsy may be performed in conjunction with BAL.
Obtain BAL if PJP is strongly suspected and the induced sputum sample findings are negative. BAL may be used in patients who are unable to cooperate with an induced sputum sample (eg, because of altered mental status). BAL may be less useful in cases of suspected PJP relapse. Consultation with a pulmonologist is required for BAL.
Lung biopsyOpen lung biopsy is the most invasive procedure and yields 100% sensitivity and specificity because it provides the greatest amount of tissue for diagnosis. However, this procedure is reserved for rare cases when bronchoscopy findings are nondiagnostic.
Histologic FindingsBecause clinical and radiologic findings are not specific for PJP and because P jiroveci cannot be grown in vitro, histopathologic demonstration is necessary before a definitive diagnosis is established.
References :
1) https://emedicine.medscape.com/article/225976-overview#showall
.
The microscopic image shown is a Gomori Methenamine stain (GMS) stain of lung biopsy in a PCP Patient
Respiratory specimen:
Include sputum, BAL, induced sputum are used in diagnosis…. The specimens are stained by fluorescen conjugated antibodies which stain both the cysts and trophic form of the bug… Other stains used in the diagnosis are Wright Geimsa stain, Modified papaniculou stain, Toulidien blue stain, calcoflour white stain… The stains can identify the trophic forms also
PCR assays although not uniformly available are specific and may increase the diagnostic yield….
Serum LDH, increased serum 1,3 beta D glucan can be used as surrogate markers for PCP….
The sensitivity of induced sputum is low around 30%… BAL fluid gives an yield of 51% in patients with negative sputum… GMS and calcowhite flour stain have sensitivity of 90% … PCR has a sensitivity and specificity of >95%… Blood PCR for PCP has been reported to have 100% sensitivity and 95% specificity in HIV individuals…The testing is uniformly not available ….Serum 1,3 beta D glucan is a marker with 90% sensitivity and specificity of 75% ..high negative predictive value is described with serum beta D glucan….Serum LDH has sensitivity of 60% with low specificity…
The stains with highest sensitivity are Gmori methenamie stain, followed by calco white flour stain and last the toulidine blue stain in that order
Lung biopsy is indicated to diagnose PCP if the BAL is negative…it is very sensitive but high risk in terms of bleeding and pneumothorax
Q1-What are the modalities used in the diagnosis of this disease?
Q2 What are their sensitivities and specificities?
Q3 What is the place of lung biopsy in the diagnosis of PJP?
1- Chest Radiography
The chest radiographic findings may be normal in patients with early mild disease. Diffuse bilateral infiltrates extending from the perihilar region are visible in most patients with P jiroveci pneumonia (PJP). Less-common findings include patchy asymmetric infiltrates, pneumothorax, and pneumatoceles. Pneumothorax may also develop in patients using aerosolized pentamidine.
2- Sputum Induction
obtain a sputum sample by sputum induction for histopathologic testing. Sputum induction is the quickest and least-invasive method for definitively diagnosing PJP. Expectorated sputum has a very low sensitivity and should not be submitted for diagnosis. Pneumocystis antigen detection assays on sputum may also be helpful but may have a low sensitivity.The sensitivity of sputum induction varies widely (< 50% to >90%) and depends on proficiency in using the technique and the experience of the laboratory. Specificity is high (99%-100%).
3- Laboratory Studies
A lactic dehydrogenase (LDH) study is performed as part of the initial workup. LDH levels are usually elevated (>220 U/L) in patients with P jiroveci pneumonia (PJP). They are elevated in 90% of patients with PJP who are infected with HIV. The study has a high sensitivity (78%-100%); its specificity is much lower because other disease processes can result in an elevated LDH level.
4- Quantitative PCR for pneumocystis may be useful in distinguishing between colonization and active infection. [26] Polymerase chain reaction (PCR) of respiratory fluid, in particular bronchoalveolar lavage (BAL), is increasingly used to make the diagnosis of PCP in both patients with and without HIV, and several clinical laboratories offer this test. A disadvantage is that PCR cannot distinguish between colonization and disease.
5- β-D-Glucan (BDG) is a cell-wall component of many fungi, including Candida, Aspergillus, and Pneumocystis (but not the Zygomycetes). It has been shown to be a sensitive test to detect PJP A negative serum BDG result is sufficient for excluding PJP only in patients with HIV infection. The use of this assay in supporting the diagnosis of PCP.
6- Microbiology of Pneumocystis jiroveci Pneumonia.
Micro-organism is difficult to culture .
7- Computed Tomography
High-resolution computed tomography (HRCT) scanning of chest is helpful when the chest radiography findings are equivocal. HRCT yields a high sensitivity for P jiroveci pneumonia (PJP) in patients with HIV infection. The typical appearance is patchy areas of ground-glass attenuation with a background of interlobular septal thickening. Negative (normal or unchanged) CT scan findings alone do not rule out PJP.
Invasive Procedures
8- Bronchoalveolar lavage
Bronchoalveolar lavage (BAL) is the most common invasive procedure used to diagnose P jiroveci pneumonia (PJP). It has a diagnostic yield that exceeds 90% (and may be higher if multiple lobes are sampled). BAL yields a lower sensitivity in patients receiving aerosolized pentamidine, in which case a transbronchial biopsy may be performed in conjunction with BAL.
9- Lung biopsy
Open lung biopsy is the most invasive procedure and yields 100% sensitivity and specificity because it provides the greatest amount of tissue for diagnosis. However, this procedure is reserved for rare cases when bronchoscopy findings are nondiagnostic.
Ref:
1- Shelley A Gilroy, Overview of Pneumocystis jiroveci Pneumonia, MEDSCAPE .
What are the modalities used in the diagnosis of this disease?What are their sensitivities and specificities?What is the place of lung biopsy in the diagnosis of PJP?
Sputum Induction
histopathologic testing.
in sputum induced by inhalation of a hypertonic saline solution.
It is the quickest and least-invasive method
But it has very low sensitivity
Pneumocystis antigen detection assays on sputum
may also be helpful but have a low sensitivity.
The sensitivity of sputum induction varies widely (< 50% to >90%)
Specificity is high (99%-100%).
This study may be less sensitive in patients without HIV infection.
Invasive Procedures
Bronchoalveolar lavage (BAL)
It is the most common invasive procedure used to diagnose (PJP).
It has a diagnostic yield that exceeds 90% (and may be higher if multiple lobes are sampled).
BAL yields a lower sensitivity in patients receiving aerosolized pentamidine, in which case a transbronchial biopsy may be performed in conjunction with BAL.
Lung biopsy
Open lung biopsy is the most invasive procedure and yields 100% sensitivity and specificity
Histologic Findings
The following staining techniques available for respiratory tract secretions.
Crystal violet,Giemsa,Diff-Quik,Wright stainSome facilities prefer to use direc ( IF ) Immunofluorescence using monoclonal antibodies to detect Pneumocystis organisms because it may be more sensitive than histologic staining.
Imaging Modalities
(HRCT) scanning of chest is helpful when the chest radiography findings are equivocal.
It has high sensitivity for (PJP) in patients with HIV infection.
The typical appearance is patchy areas of ground-glass attenuation with a background of interlobular septal thickening.
Negative (normal or unchanged) CT scan findings alone do not rule out PJP
Laboratory modalities
LDH levels
elevated (>220 U/L) in patients with (PJP).
They are elevated in 90% of patients with PJP who are infected with HIV.
high sensitivity (78%-100%);
its specificity is much lower
β-D-Glucan (BDG)
a cell-wall component of many fungi, including Candida, Aspergillus, and Pneumocystis (but not the Zygomycetes).
It us a sensitive test to detect PJP in a meta-analysis of 13 studies assessing the sensitivity, specificity, only in patients with HIV infection.
In non-HIV cases, the results should be interpreted
The use of this assay in supporting the diagnosis of PCP.
Quantitative PCR for pneumocystis may be useful in distinguishing between colonization and active infection.
(PCR) of respiratory fluid, in particular (BAL), is increasingly used to make the diagnosis of PCP in both patients with and without HIV.
A disadvantage is that PCR cannot distinguish between colonization and disease
REFERENCE
Shelley A Gilroy; Pranatharthi Haran Chandrasekar.Pneumocystis jiroveci Pneumonia (PJP) Overview of Pneumocystis jiroveci Pneumonia Nov 04, 2022
4. A picture for PCP (Pneumocystis pneumonia caused by Pneumocystis Jirovecii) diagnosed by lung biopsy
What are the modalities used in the diagnosis of this disease?
– LDH reflects the degree of lung injury but it has little utility in the non-HIV population since it can be elevated due to other causes e.g., hematologic malignancy, acute lung injury
– beta-D-glucan (BDG) is a cell wall component of most fungi hence can be elevated due to other fungal infections
– chest radiograph – typical findings include diffuse, bilateral, interstitial infiltrates; atypical findings include solitary or multiple nodules which may become cavitary, lobar infiltrates, pneumothorax, pneumatoceles (1)
– HRCT Chest has a high sensitivity for pneumocystis and can reveal ground glass opacities or cystic lesions with interlobular septal thickening (1)
– microbiology analysis using an induced sputum or bronchoalveolar lavage (BAL) sample
– definitive diagnostic tests include dye-based testing, fluorescent antibody staining and PCR-based assays of respiratory samples (induced sputum or BAL sample)
– sputum is induced by inhalation of hypertonic saline, if PCP is not identified using this modality, then bronchoscopy with BAL should be performed (2)
– sputum induction is the most rapid and least invasive method of diagnosing PCP
– pneumocystis cannot be cultured making staining a very relevant test
– calcofluor white, cresyl echt violet, Gomori methenamine silver, or toluidine blue stains help visualize the cell wall of the cysts
– Gram-Weigert, Wright-Giemsa, or modified Papanicolaou stains help visualize the trophic forms
– direct fluorescent antibody staining using fluorescent-conjugated monoclonal antibody is the most commonly used technique, it helps visualize both the trophic forms and the cysts
– PCR assays of induced sputum, BAL, blood or nasopharyngeal aspirates can be done to diagnose pneumocystis especially in the non-HIV population in whom the sensitivity of microscopy staining is lower than that in HIV positive patients
– quantitative PCR helps distinguish between colonization and active pneumocystis infection but it cannot distinguish between colonization and disease (3)
– lung biopsy can also be performed
What are their sensitivities and specificities?
– LDH sensitivity 78-100% but has a much lower specificity since other disease processes can result in an elevated LDH
– diagnostic yield of microscopy staining of induced sputum is 50-90% in the HIV population but is thought to be lower in the non-HIV due to a decreased organism burden (4)
– with BAL, the diagnostic yield is >90% in the HIV+ population but lower in the non-HIV population due to reduced organism burden (4)
– in immunocompromised patients without HIV, PCR of induced sputum or BAL fluid increases the diagnostic yield over the conventional staining alone (5)
– beta-D-glucan (BDG) assay is used as an adjunct in PCP diagnosis
– beta-D-glucan is a sensitive test but lacks specificity for pneumocystis
– in HIV positive patients with PCP, BDG assay has good sensitivity and a high negative predictive value i.e., a negative serum BDG is sufficient to rule out pneumocystis in HIV positive patients
– unfortunately, in the non-HIV population, the BDG assay cut-offs have not been well defined
– in the non-HIV population, the BDG assay may have reduced sensitivity due to the reduced organism burden (6)
– lung biopsy is rarely required, but lung biopsy with tissue stains and PCR has an excellent (100%) sensitivity and specificity for PCP diagnosis since it provides the greatest amount of tissue for diagnosis
– pulmonary function tests e.g., a decreased DLCO (diffusion lung capacity of carbon monoxide) has a high sensitivity (89-100%) but poor specificity (53%) meaning pneumocystis is unlikely if DLCO is normal
What is the place of lung biopsy in the diagnosis of PJP?
– lung biopsy is reserved for patients in whom BAL testing was negative despite having a high index of suspicion for PCP
– lung biopsy is also indicated in patients who require a lung biopsy for diagnosis of a pulmonary pathology
References
1. Crans CA, Jr., Boiselle PM. Imaging features of Pneumocystis carinii pneumonia. Critical reviews in diagnostic imaging. 1999 Aug;40(4):251-84. PubMed PMID: 10514937. Epub 1999/10/09. eng.
2. Thomas CF, Jr., Limper AH. Pneumocystis pneumonia. The New England journal of medicine. 2004 Jun 10;350(24):2487-98. PubMed PMID: 15190141. Epub 2004/06/11. eng.
3. Fauchier T, Hasseine L, Gari-Toussaint M, Casanova V, Marty PM, Pomares C. Detection of Pneumocystis jirovecii by Quantitative PCR To Differentiate Colonization and Pneumonia in Immunocompromised HIV-Positive and HIV-Negative Patients. Journal of clinical microbiology. 2016 Jun;54(6):1487-95. PubMed PMID: 27008872. Pubmed Central PMCID: PMC4879311. Epub 2016/03/25. eng.
4. Shelhamer JH, Gill VJ, Quinn TC, Crawford SW, Kovacs JA, Masur H, et al. The laboratory evaluation of opportunistic pulmonary infections. Annals of internal medicine. 1996 Mar 15;124(6):585-99. PubMed PMID: 8597323. Epub 1996/03/15. eng.
5. Azoulay É, Bergeron A, Chevret S, Bele N, Schlemmer B, Menotti J. Polymerase chain reaction for diagnosing pneumocystis pneumonia in non-HIV immunocompromised patients with pulmonary infiltrates. Chest. 2009 Mar;135(3):655-61. PubMed PMID: 19265086. Epub 2009/03/07. eng.
6. Theel ES, Jespersen DJ, Iqbal S, Bestrom JE, Rollins LO, Misner LJ, et al. Detection of (1, 3)-β-D-glucan in bronchoalveolar lavage and serum samples collected from immunocompromised hosts. Mycopathologia. 2013 Feb;175(1-2):33-41. PubMed PMID: 22945270. Epub 2012/09/05. eng.
Modalities used in the diagnosis of this disease with their sensitivities and specificities:
· High LDH (>220 U/L). 90% of HIV-positive PJP patients have elevated levels. high sensitivity (78%-100%) but low specificity.
· PCR of bronchoalveolar lavage (BAL). PCR cannot distinguish between colonization and disease.
· Sputum PCR: low sensitivity and specificity
· β-D-Glucan (BDG) is a cell-wall component of many fungi. is non-specific but if positive it supports diagnosis of fungal infection.
· Chest radiograph: up to 90% of in patients with Pneumocystis pneumonia are abnormal, appearances are sometimes non-specific. Between 10-15% of patients have normal chest radiographs and close to 30% have non-specific or inconclusive findings.
· HRCT is more sensitive and may be used to exclude PCP in patients with clinical suspicion for PCP but normal or inconclusive chest radiographs.
· Lung biopsy
Open lung biopsy is the invasive procedure but yields 100% sensitivity and specificity because it provides the greatest amount of tissue for diagnosis.
References:
(i) Hartman TE, Primack SL, Müller NL et-al. Diagnosis of thoracic complications in AIDS: accuracy of CT. AJR Am J Roentgenol. 1994;162 (3): 547-53. AJR Am J Roentgenol (abstract) – Pubmed citation)
(ii) Hidalgo A, Falcó V, Mauleón S et-al. Accuracy of high-resolution CT in distinguishing between Pneumocystis carinii pneumonia and non- Pneumocystis carinii pneumonia in AIDS patients. Eur Radiol. 2003;13 (5): 1179-84. doi:10.1007/s00330-002-1641-6 – Pubmed citation)
Bronchoalveolar lavage (BAL) is the most common invasive procedure used to diagnose P jiroveci pneumonia (PJP)
(iii) UpToDate
What are the modalities used in the diagnosis of this disease?
What are their sensitivities and specificities?
What is the place of lung biopsy in the diagnosis of PJP?
Fishman JA, Gans H; AST Infectious Diseases Community of Practice. Pneumocystis jiroveci in solid organ transplantation: Guidelines from the American Society of Transplantation Infectious Diseases Community of Practice. Clin Transplant. 2019 Sep;33(9):e13587. doi: 10.1111/ctr.13587. Epub 2019 Jul 1. PMID: 31077616.
modality of diagnsis of PJP
culture is not poassible
source of specimen-
Induced sputum
BAL -COMMON
Transbrochial bopsy
open / VATS lung biopsy – HIGHEST SENSITIVITY AND SPECIFICITY REPORTED FOR DUAGNOSIS
test performed on any samples
staining like GMS , toludine O Blue 30% to 90% sensitive
PCR 97 % sensitive and more specific BUT can not differentiate colonisation from infection
Immunofluorescence 50-100 % sensitive
LDH LEVEL MORE THAN 300 IU/ML
Beta D Glucan level are high 95% sensitive and specific
The given microscopic picture of PJP can be due to GMS staining or DFA
I feel it is DFA slide as GMS looks like SILVER colour
# CXR( may be normal up to 30% case), HRCT, induced sputum/ BALF for GMS/ IF study or PCR, flow cytometry, BDG, S. LDH( mainly for prognosis), lung biopsy.
#. BALF is gold standard.
# lung biopsy rarely needed, when above test failed.
Bronchoalveolar lavage fluid
current gold standard sample for diagnosis of PCP
Sputum
has been found to have a >95% negative predictive value in low prevalence situations
making a negative test adequate for ruling out PCP. It has 85–100% sensitivity when PCR is used.
Oral washing
have a sensitivity of 75–91% and a specificity of 68–100%.
Blood/serum PCR
a very high sensitivity (100%) and negative predictive value (99%) for the diagnosis of PCP in HIV-infected patients.
Conventional stains
Gomori methenamine silver (GMS), toluidine-blue O, calcofluor white and Gram–Weigert, which stain the cell wall of the cysts.
The sensitivity of GMS ranges from 31 to 97%.
IF staining:
Sensitivity ranges from 48 to 100%, and specificity from 82 to 100%.
Polymerase chain reaction:
Sensitivity of 97% with a specificity of 91%, with most samples being BALF.
PCR cannot differentiate between infection and colonization, leading to higher rates of false positive assay
Loop-mediated isothermal amplification (LAMP):
Sensitivity ranges from 87.5 to 95.4%, and LAMP has been shown to be relatively specific with no cross-reactivity to other fungal species.
Flow cytometry:
can also detect antibodies against Pneumocystis and comment on antifungal susceptibility.
Detection of Pneumocystis jirovecii in clinical BAL and bronchial samples with 100% sensitivity and specificity when compared to IF staining.
The serum (1,3)-β-D-glucan assay
was found to be 95% sensitive with high sensitivity being demonstrated in both HIV positive and negative patients.
Serum LDH is usually elevated (>300 IU/mL)
sensitivity and specificity of this marker for PCP have been estimated to be 66%–91% and 36–52%,respectively.
Open lung biopsy
an invasive procedure with sensitivity and specificity of 100%, since it provides the highest quality respiratory sample for diagnosis.
Reference:
Marjorie Bateman et al, Diagnosing Pneumocystis jirovecii pneumonia: A review of current methods and novel approaches, Medical Mycology, 2020, 58,1015–1028
· LDH levels are usually elevated (>220 U/L) in patients with PJP(elevated in 90% of PJP patients who are infected with HIV). It has a high sensitivity (78%-100%) but not specific
· PCR of respiratory fluid, mainly BAL, used to make the diagnosis of PCP in both patients with and without HIV, but that PCR cannot distinguish between colonization and disease.
· Sputum P jirovecii PCR testing may be a viable alternative to invasive testing, it is faster and safer than BAL, but still need more study.
· β-D-Glucan (BDG) is sensitive test to detect PJP. A negative serum BDG result is sufficient for excluding PJP only in patients with HIV infection.
· Sputum induction is the quickest and least-invasive method for definitively diagnosing PJP.
The sensitivity of sputum induction varies widely (< 50% to >90%), Specificity is high (99%-100%). but less sensitive in patients without HIV infection
Invasive Procedures
1) Bronchoalveolar lavage
· BAL is the most common invasive procedure used to diagnose PJP.
· It has a diagnostic yield that exceeds 90% (and may be higher if multiple lobes are sampled).
· It can be used in patients who are unable to induce sputum sample (unconscious)
2) Lung biopsy
· Open lung biopsy is the most invasive procedure and yields 100% sensitivity and specificity as it provides the greatest amount of tissue for diagnosis.
· It reserved for rare cases when bronchoscopy findings are nondiagnostic.
Chest radiography
· Can be normal in patients with early mild disease.
· Diffuse bilateral infiltrates extending from the perihilar region are visible in most patients with PJP.
· Less-common findings include patchy asymmetric infiltrates, pneumothorax, and pneumatoceles.
· Pleural effusions and intrathoracic adenopathy are rare
Computed Tomography
· HRCT yields a high sensitivity for PJP in patients with HIV infection.
· Negative (normal or unchanged) CT scan findings alone do not rule out PJP
https://emedicine.medscape.com/article/225976-overview#a8:~:text=Pneumocystis%20jiroveci%20Pneumonia%20(PJP)%20Overview%20of%20Pneumocystis%20jiroveci%20Pneumonia
Modalities used in diagnosis
Sensitivity and specificity
Pneumocystis Jivorecii can not be cultured hence diagnosis relies on clinical symptoms and radiological findings with confirmation via visualisation of the organism done through:
1.Traditional methods
Giemsa and Wrights stain for the trophozoite.
Gomori methionine stains the cyst.
However they stain for both colonisation and infection
Stains for monoclonal antibodies against P.jivorecii.
Sensitivity ranges between 48-100% and specificity 82-100%.
Direct immunofluorescence done on induced sputum or BAL is the modality of choice.
2.Novel methods
More sensitive than staining methods and immunofluorescence.
Pooled sensitivity of 98% and specificity of 91%.
Can’t differentiate infection from colonisation
An alternative to PCR.
Sensitivity ranges between 87.5-95.4%
Highly specific with no cross reaction with other fungal species.
Able to detect the organism and antibodies directed against it.
Has sensitivity and specificity of 100%.
Serum β D glucan levels-can be tested in the BAL or serum
Sensitivity 90-95% specificity 75-86%
False positive can occur with cross reaction with other fungal species.
Serum LDH-sensitivity ranges between 66-91% specificity 36-52%
What is the place of lung biopsy in the diagnosis of PJP?
Open lung biopsy is an invasive procedure with sensitivity and specificity of 100%, since it provides the highest quality respiratory sample for diagnosis. However its use is reserved for the diagnosis in rare cases where other diagnostic approaches are inconclusive or there is suspicion for other concomitant disease. Thus has now been replaced with minimally invasive sampling techniques.
References.
A Serologic Test to Diagnose Pneumocystis Pneumonia: Are We There Yet? Alison M. Morris, Henry Masur
Pneumocystis Pneumonia in Solid Organ Transplantation. The AST Infectious Diseases Community of Practice
The Diagnosis of PCP requires one having a high index of suspicion and using different available Modalities in a stepwise fashion to confirm the diagnosis.
Del Corpo O., Butler-Laporte G., Sheppard D.C., Cheng M.P., McDonald E.G., Lee T.C. Diagnostic accuracy of serum (1-3)-β-D-glucan for Pneumocystis jirovecii pneumonia: A systematic review and meta-analysis. Clin. Microbiol. Infect. 2020;26:1137–1143. doi: 10.1016/j.cmi.2020.05.024.
Procop G.W., Haddad S., Quinn J., Wilson M.L., Henshaw N.G., Reller L.B., Artymyshyn R.L., Katanik M.T., Weinstein M.P. Detection of Pneumocystis jiroveci in respiratory specimens by four staining methods. J. Clin. Microbiol. 2004;42:3333–3335. doi: 10.1128/JCM.42.7.3333-3335.2004.
Apostolopoulou A, Fishman JA. The Pathogenesis and Diagnosis of Pneumocystis jiroveci Pneumonia. J Fungi (Basel). 2022 Nov 5;8(11):1167. doi: 10.3390/jof8111167. PMID: 36354934; PMCID: PMC9696632.
Moreno A, Epstein D, Budvytiene I, Banaei N. Accuracy of Pneumocystis jirovecii Plasma Cell-Free DNA PCR for Noninvasive Diagnosis of Pneumocystis Pneumonia. J Clin Microbiol. 2022 May 18;60(5):e0010122. doi: 10.1128/jcm.00101-22. Epub 2022 Apr 7. PMID: 35387472; PMCID: PMC9116171.
DIAGNOSTIC MODALITIES,SENSITIVITY ,SPECIFICITY AND ROLE OF LUNG BIOPSY.
-Definitive diagnosis of PCP needs identification of cystic or trophic forms in respiratory secretions. Obtaining samples in PCP is quite problematic.
LABS;
INVASIVE;
REFERENCES.
As the PJP cannot grow in vitro, diagnosis depends on organism detection in respiratory specimens with chemical and fluorescent staining however these techniques lack sensitivity and specificity ;
testing in induced sputum yields a diagnosis in 50 % of cases
testing in BAL yields up to 80 %
transbronchial biopsy showing lymphocytic infiltrates, foamy or granular eosinophilic exudates yields a diagnosis in 90 % OF cases
open lung biopsy or VATS is the gold standard method yet not required
staining of obtained specimens depends mainly on silver polychrome stains however immunofluorescence Assayas are more sensitive
B D glucan serum detection seems sensitive but not specific and can be used as an adjuvant diagnostic tool, LDH is a good prognostic tool
a significant improvement in sensitivity and specificity is achieved after adding PCR-based techniques in organism detection in BAL however it cannot discriminate between infection and carrier stage so the results should be correlated with clinical and radiological finding
genotype sequencing could be done for negative results
metagenomics is a recent accurate method
Clinical presentation and diagnostic challenges in Pneumocystis jirovecii pneumonia (PJP)
PJP is an opportunistic fungal infection where clinically significant disease occurs almost exclusively in those with compromised immune systems, including HIV, solid organ transplant recipients and patients receiving immunosuppressive medications for autoimmune conditions. There are several diagnostic challenges which must be overcome in order to make a definitive diagnosis: this is imperative as unnecessary treatment with trimethoprim-sulfamethoxazole (TMP-SMX) can result in serious side effects whilst also delaying effective treatment in cases of misdiagnosis.
Diagnostic challenges:
· Pneumocystis jirovecii cannot be reliably cultured in vitro
· Radiological findings are diverse with no pathognomic findings specific for PJP. Additionally, PJP pneumonia is often of more rapid-onset in solid organ transplant (SOT) recipients compared to HIV-positive individuals and consequently more pronounced radiological features may not have developed in imaging performed early in the disease course
· Exposure to Pneumocystis jirovecii in childhood is common and colonisation must be distinguished from pathogenic cases
· Only 71-81% of patients have a cough as part of their clinical presentation (1) and obtaining a sputum sample for analysis can be challenging
· A respiratory sample must be obtained in order to establish a definitive diagnosis: the methods required for this are predominantly invasive such as bronchiolar lavage (BAL) +/- transbronchial biopsy or open lung biopsy: these methods require significant resources and trained personnel which may not be available in certain centres or under-resourced settings. Additionally, they carry risks to the patient such as pneumothorax and in some cases lying flat for the procedure can precipitate a decline in the patient’s condition prompting them to be subsequently intubated and mechanically ventilated (2)
Modalities of diagnosis in PJP
A respiratory sample is needed for definitive diagnosis. This can be obtained via:
1. Inducted sputum
– advantage: non-invasive
– disadvantage: may not be possible to obtain, less likely to yield diagnosis than BAL/biopsy
– can be processed using 3 different assays: (2)
i) polymerase chain reaction (PCR): sensitivity 99%, specificity 96%
ii) immunofluorescence: sensitivity 74%, specificity 100%
iii) cytological staining: sensitivity 50%, specificity 100%
2. Bronchiolar lavage (BAL) +/- transbronchial biopsy
– advantage: effective method to obtain respiratory samples required for definitive diagnosis, more sensitive than induced sputum (particularly useful in non-HIV patients where organism burden may be lower (1))
– disadvantage: potential for patient to deteriorate in needing to lie flat for procedure
– sensitivity 80-95% (1), specificity 91% (2)
3. Open lung biopsy or video-assisted thoracoscopy (VATS)
– this method is regarded as the gold standard test in terms of likelihood of accurate diagnostic yield (1); however, due to the invasiveness of the procedure with associated risks other methods of obtaining a respiratory specimen are used whenever possible (1)
Adjunct tests
Given the varying range of clinical presentations of PJP pneumonia it is imperative for clinicians to have a high index of suspicion in immunocompromised patients; delays in initiating appropriate treatment can lead to further patient deterioration and the disease is associated with high morbidity and mortality. Whilst arranging to obtain a respiratory sample (as above) for definitive diagnosis, the following adjunct tests may support the likelihood of PJP pneumonia (1)
· Arterial blood gas: hypoxia with PO2 <60mmHg, respiratory alkalosis
· Radiological imaging: CXR with para-hilar shadowing, CT scan with ground glass appearances and subpleural sparing
· Blood tests: lactate dehydrogenase (LDH) often >300 IU/ml, (1-3)beta-D-glucan assay: not specific but if it is negative the diagnosis is unlikely to be PJP (1)
References:
1. Fishman JA, Gans H; AST Infectious Diseases Community of Practice. Pneumocystis jiroveci in solid organ transplantation: Guidelines from the American Society of Transplantation Infectious Diseases Community of Practice. Clin Transplant. 2019 Sep;33(9). Available at: https://pubmed.ncbi.nlm.nih.gov/31077616/
2. Senécal J, Smyth E, Del Corpo O, Hsu JM, Amar-Zifkin A, Bergeron A, Cheng MP, Butler-Laporte G, McDonald EG, Lee TC. Non-invasive diagnosis of Pneumocystis jirovecii pneumonia: a systematic review and meta-analysis. Clin Microbiol Infect. 2022 Jan;28(1):23-30. Available at: https://pubmed.ncbi.nlm.nih.gov/34464734/
What are the modalities used in the diagnosis of this disease?
Pneumocystis jirovecii pneumonia (PCP) is a life-threatening infection in immunocompromised patients. Mode of transmission is usually air born. in 75% patient it may just be reactivation of latent organism and in 1/4th it may be new infection by airborne route. It mainly resides in alveoli and bronchi are spared. In case of infection it is usually alveolitis.
Quantitative real-time PCR (qPCR) is more sensitive than microscopic examination for the detection of P. jirovecii but also detects colonized patients also. PCP diagnosis currently relies on the demonstration of trophic forms or cysts after microscopic examination of bronchoalveolar lavage fluid (BALF) using adequate staining methods (May-Grünwald-Giemsa, Gomori-Grocott, or immunofluorescence assay)
Specimen from bronchi can be achieved by BAL, cough induction with saline , by VATS, or endotracheal aspirates.
Beta D Glucan Assay
The positive assay indicates candidiemia, invasive aspergillosis or PCJ. Sensitivity is higher in immunocompromised.
Sensitivity- 70-90%
Specificity- 80-85%
Those having of Peudomonas infection or recent IVIG use may be false positive.
It can be used to guide treatment till PCP PCR results are available.
What are their sensitivities and specificities?
Induced sputum- Sensitivity-99% Specificity- 96%
Cytological staining- Sensitivity- 50% Specificity- 100%
PCR- Sensitivity- 87% Specificity- 92%
What is the place of lung biopsy in the diagnosis of PJP?
It is rarely used. It can be considered if diagnosis is in doubt or for another condition.
1-Florence Robert-Gangneux, Sorya Belaz, e tal. Diagnosis of Pneumocystis jirovecii Pneumonia in Immunocompromised Patients by Real-Time PCR: a 4-Year Prospective Study. J Clin Microbiol. 2014 Sep; 52(9): 3370–3376.
2-Thomas C, Jr, Limper A. 2004. Pneumocystis pneumonia. N. Engl. J. Med. 350:2487–2498.
3- Jouneau S, Poineuf JS, Minjolle S, Tattevin P, Uhel F, Kerjouan M, Le Ho H, Desrues B. 2013. Which patients should be tested for viruses on bronchoalveolar lavage fluid? Eur. J. Clin. Microbiol. Infect. Dis. 32:671–677.
Pneumocystis jirovecii In the early days of diagnosis, lung biopsy procedures were used to obtain large specimens of tissue to stain for organism identification.
biopsy has been replaced with more minimally invasive sampling techniques as noted below.48
–Bronchoalveolar lavage fluid (BALF)
-SputumNasopharyngeal aspirate
– Blood/serum-Traditional diagnostic tests: Nonimmunofluorescent staining ,Immunofluorescentstaining, Polymerase chain reaction,Flow cytometry, Antibody assays,Antigen and biomarker assays
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7657095/
. Limper AH. Diagnosis of Pneumocystis cariniipneumonia: does use of only bronchoalveolar lavage suffice? Mayo Clin Proc. 1996; 71: 1121–1123. [PubMed] [Google Scholar]
The four testing modalities :
1- cytological staining,
2- fluorescent antibody,
3-PCR
4-lactate dehydrogenase.
Induced sputum had the most data available; this modality was both highly sensitive at 99% (95% CI 51%-100%) and specific at 96% (95% CI 88%-99%). Induced sputum cytological staining had moderate sensitivity at 50% (95% CI 39%-61%) and high specificity at 100% (95% CI 100%-100%), as did fluorescent antibody testing with sensitivity 74% (95% CI 62%-87%) and specificity 100% (95% CI 91%-100%)
Molecular methods of detection such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and antibody-antigen assays These techniques are very sensitive and have the potential to detect Pneumocystis life-forms in noninvasive samples such as sputum, oral washes, nasopharyngeal aspirates, and serum.
Trans bronchial biopsy is strongly recommended and increased yield of BAL .
Open lung biopsy or video associated thoarco scopy (VAST) is gold standard for diagnosis ,strongly recommended but low quality of evidence
REFERNCE:
1-Diagnosing Pneumocystis jirovecii pneumonia: A review of current methods and novel approaches.
Bateman M, Oladele R, Kolls JK.Med Mycol. 2020 Nov 10;58(8):1015-1028. doi: 10.1093/mmy/myaa024.PMID: 32400869 Free PMC article. Review.
2- week 5 lecture
What are the modalities used in the diagnosis of this disease and their sensitivities and specificities.
1. Sample considerations
Bronchoalveolar lavage fluid (BALF): Is the current gold standard sample for diagnosis of PCP is BALF.
Sputum: Performance of IF staining on induced sputum (IS) has been found to have a >95% negative predictive value in low prevalence situations, making a negative test adequate for ruling out PCP. It has 85–100% sensitivity when PCR is used.
Oral washing: Oral wash PCR has been noted to have a sensitivity of 75–91% and a specificity of 68–100%.
Blood/serum: PCR analysis on serum samples had a very high sensitivity (100%) and negative predictive value (99%) for the diagnosis of PCP in HIV-infected patients. Use of serum PCR is not currently recommended for detection of PCP.
2. Traditional diagnostic tests
Conventional stains include Gomori methenamine silver (GMS), toluidine-blue O, calcofluor white and Gram–Weigert, which stain the cell wall of the cysts. The sensitivity of GMS ranges from 31 to 97%.
Immunofluorescent staining:
3. Novel methods of detection
Polymerase chain reaction:
Loop-mediated isothermal amplification (LAMP):
Flow cytometry:
Antibody assays:
Antigen and biomarker assays:
2 . What is the place of lung biopsy in the diagnosis of PJP?
REFRENCES
1 . Marjorie Bateman et al, Diagnosing Pneumocystis jirovecii pneumonia: A review of current methods and novel approaches, Medical Mycology, 2020, 58,1015–1028
2 Anna Apostolopoulou and Jay A. Fishman, The Pathogenesis and Diagnosis of Pneumocystis jiroveci Pneumonia, Fungi 2022, 8, 1167
3 Isabelle Durand J et al, Molecular diagnosis of Pneumocystis Pneumonia, FEMS Immunology & Medical Microbilogy , Volume 45, 3, Septemberv2005, 405-410
4 YavaraceV ,Saowanee Yenrudi, Histopathological featurs of Pneumocystis Carenii pneumonia in 54 Thai AIDS patients- Chula Med J Vol.48 No. 2004
What are the modalities used in the diagnosis of this disease?
What are their sensitivities and specificities?
Microscopy with staining — Detection of the organism in respiratory specimens is most commonly achieved by microscopy with staining of an induced sputum specimen or BAL fluid
Staining is necessary because Pneumocystis cannot be cultured.
Direct fluorescent antibody staining using a fluorescein-conjugated monoclonal antibody can visualize both trophic forms and cysts and is the most common technique used.
Trophic forms can also be seen with tinctorial stains such as Gram-Weigert, Wright-Giemsa, or modified Papanicolaou stains.
The cell wall of the cysts can be visualized with calcofluor white, cresyl echt violet, Gomori methenamine silver, or toluidine blue.
Type of respiratory specimen
The most rapid and least invasive method of diagnosing PCP is by analysis of sputum induced by the inhalation of hypertonic saline
If PCP is not identified by this modality, then bronchoscopy with BAL should be performed.
What is the place of lung biopsy in the diagnosis of PJP?
Lung biopsy with tissue stains and PCR has excellent sensitivity for diagnosing PCP but is rarely required
. Lung biopsy for diagnosis is generally reserved for patients in whom there is a high suspicion of PCP and in whom BAL testing has been negative or in those who have another reason to proceed to lung biopsy for diagnosis of a pulmonary process.
The diagnostic yield of microscopy with staining of induced sputum is 50 to 90 percent in patients with HIV and PCP but is thought to be lower in patients without HIV due to a decreased organism burden
A similar difference has been noted with BAL. The diagnostic yield is over 90 percent in patients with HIV but may be lower in patients without HIV
However, in one report, BAL was positive for PCP in 47 of 48 patients
Polymerase chain reaction — A number of PCR assays have been developed for the detection of Pneumocystis in induced sputum or BAL fluid, blood, or nasopharyngeal aspirates. These assays may be of particular use in patients without HIV, in whom the sensitivity of microscopy with staining is substantially lower than in patients with HIV.
Relative contraindications to lung biopsy should be evaluated on a case-by-case basis to be sure that the procedure is likely to reveal a treatable diagnosis at an acceptable level of risk and that less invasive methods will not yield a diagnosis.
immunosuppressed patients may have a diagnosis that can be determined by less invasive methods (eg, bronchoalveolar lavage).
Beside clinical history, examination and radiological modalities to diagnose PJP there are many other laboratory investigations to do so. These include:
1-Direct microscopic visualization of Pneumocystis in induced sputum.
2-Bronchoalveolar lavage specimens.
3-Transbronchial or
4-Open lung biopsy specimens
The DFA ( direct immunofluorescence antibodies) was most sensitive (90.8% as opposed to 50–80% for the conventional stains) but less specific (94.7% as opposed to >99% for the conventional stains) .
PCR is more specific than DFA, but can’t differentiate between infection and colonization. But if we used real-time PCR with cut of 1450 or 1900 copies may overcome this with a positive predictive value of 98-100%.
LDH) elevated (>300 IU/mL) in patients with PJP reflecting diffuse pneumonia.
Serum-β-D-glucan assay (BDG), non-specific fungal marker that has high sensitivity but low specificity for PJP. The overall sensitivity of BDG was 91% (94% in patients with HIV vs. 86% in patients without HIV). A negative predictive value of BDG at <80 pg/mL (the manufacturer’s recommended cut-off) was 95% when the pre-test probability was intermediate (50%)
Rarely we use lung biopsy to diagnose PJP as it is an invasive procedure and there is safer alternative . The complication rate of the Electromagnetic navigational bronchoscopy (ENB‑guided biopsies has been reported to be approximately 4% ENB both with and without ROSE has a diagnostic yield for malignancy that range from 38% to 97%.
References;
1- A novel diagnostic approach for Pneumocystis jirovecii pneumonia using fine‑needle aspiration, electromagnetic navigational bronchoscopy and rapid on‑site evaluation.
2- The Pathogenesis and Diagnosis of Pneumocystis jiroveci Pneumonia, . Fungi 2022, 8(11), 1167; https://doi.org/10.3390/jof8111167
LDH with sensitivity 70-90%
CXR not specific and poor sensitivity
PCR in brochoalveolar lavage with sensitivity 89%
Beta D glucan test to detect fungal cells
HRCT more specific (perihilar infiltration and groud glass appearance
PFT
Open lung biopsy is diagnostic and sensitivity 100%
This slide showed Gomori staining for PCP, which showed positive.
The laboratory test for PCP
LDH is showing 70 to 100% sensitivity
B-D-glucan, which is part of the fungal cell wall which, is having high negative predictive value for PCP
CXR can give you some finidu=ing in favour of PCP, like bilateral hilar and bilateral chest infiltrative but normal chest x ary not ruled out PCP.
HRCT will show typical of ground glass appearance
Bronchoscopy with BAL with a sensitivity of 90%
references
uptodate
Diagnostic modalities of PJP:
Lab testing:
-LDH: has 70-100% sensitivity.
-PCR: very useful test ;can be done with BAL or tissue specimens but not differentiating between infection and colonization.
-Serum Beta-D glucan: a cell wall component of different fungi; a negative test excludes PJP.
-CXR: could be normal or may show para hilar infiltrates with pleural effusions rarely.
-HRCT:
.Typically reveals ground glass appearance.
.Inter lobar septal thickening with reticulonodular pattern.
.Normal CT findings do not exclude PJP.
-Hypoxemia < 90 % confirmed by ABGs.
-PFTs: have sensitivity of 89 -100% but with poor specificity of 53%.
. Reduced diffusion capacity for carbon dioxide (DLCO) to < 75% of predicted is typical finding.
-BAL: has diagnostic sensitivity up to 90% but with poor sensitivity in case of aerosolized pentamidine.
-Transbronchial biopsy :may be needed and has more sensitivity.
-Open lung biopsy: is an invasive diagnostic test of 100% sensitivity and specificity ;indicated if bronchoscopy is not diagnostic.
– elevated LDH (>220 U/L). 90% of HIV-positive PJP patients have elevated levels. high sensitivity (78%-100%) but low specificity.
– PCR of bronchoalveolar lavage (BAL). A disadvantage is that PCR cannot distinguish between colonization and disease.
– sputum pcr >> low sensitivity & specificity
– β-D-Glucan (BDG) is a cell-wall component of many fungi, including Candida, Aspergillus, and Pneumocystis (but not the Zygomycetes). It has been shown to be a sensitive test to detect PJP in a meta-analysis of 13 studies assessing the sensitivity, specificity, and overall accuracy of the test.
– A negative serum BDG result is sufficient for excluding PJP only in patients with HIV infection. In non-HIV cases, the results should be interpreted in parallel with clinical and radiologic findings
(Hartman TE, Primack SL, Müller NL et-al. Diagnosis of thoracic complications in AIDS: accuracy of CT. AJR Am J Roentgenol. 1994;162 (3): 547-53. AJR Am J Roentgenol (abstract) – Pubmed citation)
– HRCT is more sensitive and may be used to exclude PCP in patients with clinical suspicion for PCP but normal or inconclusive chest radiographs.
( Hidalgo A, Falcó V, Mauleón S et-al. Accuracy of high-resolution CT in distinguishing between Pneumocystis carinii pneumonia and non- Pneumocystis carinii pneumonia in AIDS patients. Eur Radiol. 2003;13 (5): 1179-84. doi:10.1007/s00330-002-1641-6 – Pubmed citation)
Bronchoalveolar lavage (BAL) is the most common invasive procedure used to diagnose P jiroveci pneumonia (PJP). It has a diagnostic yield that exceeds 90% (and may be higher if multiple lobes are sampled). BAL yields a lower sensitivity in patients receiving aerosolized pentamidine, in which case a transbronchial biopsy may be performed in conjunction with BAL.
Lung biopsy
Open lung biopsy is the most invasive procedure and yields 100% sensitivity and specificity because it provides the greatest amount of tissue for diagnosis. However, this procedure is reserved for rare cases when bronchoscopy findings are nondiagnostic
(Shelley A Gilroy , Medscape 2022)
Microscopy with staining ( from induced sputum specimen or BAL fluid ) Direct fluorescent antibody staining using a fluorescein-conjugated monoclonal antibody can visualize both trophic forms and cysts and is the most common technique used.
The diagnostic yield of microscopy with staining of induced sputum is 50 to 90 percent in patients with HIV and PCP but is thought to be lower in patients without HIV due to a decreased organism burden .
A similar difference has been noted with BAL. The diagnostic yield is over 90 percent in patients with HIV but may be lower in patients without HIV.
However, in one report, BAL was positive for PCP in 47 of 48 patients
Polymerase chain reaction — A number of PCR assays have been developed for the detection of Pneumocystis in induced sputum or BAL fluid, blood, or nasopharyngeal aspirates. These assays may be of particular use in patients without HIV, in whom the sensitivity of microscopy with staining is substantially lower than in patients with HIV.
PCR of BAL fluid or induced sputum can increase the diagnostic yield over conventional staining alone in immunocompromised patients without HIV.
Beta-D-glucan assay — Beta-D-glucan is a cell wall component of all fungi, including Pneumocystis. A serum assay for beta-D-glucan is available and can be used to screen for a variety of invasive fungal infections. Although this test has been best studied for Candida and Aspergillus spp, it may also have utility for diagnosing PCP.
Although the beta-D-glucan assay is not specific for PCP, it is useful while awaiting an induced sputum or BAL specimen for microscopy with staining for PCP or in situations in which respiratory sampling such as BAL cannot be performed safely.
Lung biopsy with tissue stains and PCR has excellent sensitivity for diagnosing PCP but is rarely require.
Lung biopsy for diagnosis is generally reserved for patients in whom there is a high suspicion of PCP and in whom BAL testing has been negative or in those who have another reason to proceed to lung biopsy for diagnosis of a pulmonary process.
What are the modalities used in the diagnosis of this disease?
LABORATORY FINDINGS:
– An elevated LDH in the setting of pulmonary infiltrates without another apparent cause should raise suspicion for PCP.
– Serum beta-D-glucan assay.
RADIOGRAPHIC FINDINGS:
1-The typical radiographic features of PCP in patients without HIV are diffuse, bilateral, interstitial infiltrates.
2-High-resolution computed tomography scanning may demonstrate extensive ground-glass opacities or cystic lesions .
Microscopy with staining and Type of respiratory specimen.
-Sputum induced by the inhalation of hypertonic saline .
–Bronchoscopy with BAL should be performed.
-Lung biopsy in patient who is highly suspected and BAL came negative
What are their sensitivities and specificities?
The diagnostic yield of microscopy with staining of induced sputum is 50 to 90 percent in patients with HIV and PCP but is thought to be lower in patients without HIV.
The diagnostic yield of BAL is over 90 percent in patients with HIV but may be lower in patients without HIV.
PCR of BAL fluid or induced sputum can increase the diagnostic yield over conventional staining alone in immunocompromised patients without HIV.
What is the place of lung biopsy in the diagnosis of PJP?
Lung biopsy, either by thoracotomy or by video-assisted thoracoscopic surgery, can be performed with a sensitivity of 95 to 100 percent for the diagnosis of PCP.
References:
1- Kovacs JA, Hiemenz JW, Macher AM, et al. Pneumocystis carinii pneumonia: a comparison between patients with the acquired immunodeficiency syndrome and patients with other immunodeficiencies. Ann Intern Med. 1984;100(5):663-671. doi:10.7326/0003-4819-100-5-663.
2-Wilson JW, Limper AH, Grys TE, Karre T, Wengenack NL, Binnicker MJ. Pneumocystis jirovecii testing by real-time polymerase chain reaction and direct examination among immunocompetent and immunosuppressed patient groups and correlation to disease specificity. Diagn Microbiol Infect Dis. 2011;69(2):145-152. doi:10.1016/j.diagmicrobio.2010.10.021.
3-Panel on Guidelines for the Prevention and Treatment of Opportunistic Infections in Adults and Adolescents with HIV. Guidelines for the Prevention and Treatment of Opportunistic Infections in Adults and Adolescents with HIV. National Institutes of Health, Centers for Disease Control and Prevention, and the HIV Medicine Association of the Infectious Disease Society of America.
PJP may have some challenges because of some atypical presentations like isolated pulmonary cavity,consolidation etc., entailng biopsy (In secnerio seems to be silver staining ??). The typical findings as shared in other scenarios, include the reticaullar pattern and fine ground appearance that we observe on chest x-ray. In some cases we may need the CT scan which is more sensitive. In the picture attached we can see documentation of a case with on site evaluation
There are traditional and novel strategies to detect PJP in suspected cases (2) :
Sampling:
-Bronchoalveolar lavage fluid (BALF): currently the gold standard for diagnosis of PJP
-Sputum/ oral wash : less sesnitive
Traditional modalities:
either immune or non immune staing
— Non IF staing :
— IF staining: Sensitivity reported from 48-100%, while specificity was in range of 82-100%
Novel Modalities:
-PCR
-Loop-mediated isothermal amplification (LAMP): Sensitivity 87.5 to 95.4% , specificity relatively high (no cross reaction)
-Flowcytometry: Can detect PJP from BAL and bronchial samples with 100% sensitivity and specificity when compared to IF staining.
Antibody detection (ELIZA): sensitivity 100% / specificity 81%
(1,3)-Beta D-Glucan (BG): found positive in other fungal infections in patients with gram-negative endotoxinemia, in patients on certain antibiotics so : specificity reported between 75% – 86%
**************
1- Attached picture foot note as taken from the source (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6784444/):
In this case a suspected lesion on the right apex was found but the CT is demonstartive. system did not allow 2 pictures so the radyologic evaluation can be seen from the link)
Pictures documenting appearance of pneumocystis in various cytologic preparations. (a) High-power view of dot-like organisms within the foamy cast (black arrows). (Papanicolaou, ×60). (b) High-power view of dot-like organisms within the foamy cast (black arrows) (Diff-Quick, ×60). (c) Fine-needle aspiration specimen showing Pneumocystis organisms as alveolar casts. Typical appearance of Pneumocystis organism with crescent shape, spheres with a dense dot, and collapsed spheres also known as crushed ping pong balls (Grocott–Gomori’s methenamine silver, ×60). (d) Fine-needle aspiration cell block specimen demonstrating foamy cast adherent to the alveolar cell wall (black arrows) (H and E, ×10)
2- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7657095/pdf/myaa024.pdf
What are the modalities used in the diagnosis of this disease?
Sample sources for the diagnosis of PJP
Non-immunofluorescent staining –
The cyst life-form can be detected with many stains. Giemsa, Diff-Quik, and Wright stains can detect the cyst but do not stain its wall.
The Gomori-methenamine-silver (GMS) stain, Gram-Weigert, cresyl echt violet, toluidine blue O (TBO), and calcofluor white (CW) stains stain the cell wall of the cyst
Immunofluorescent staining via monoclonal antibodies – Se 48-100%, sp 82-100%
Noval methods of detection:
Role Lung biopsy in PJP:
Transbronchial lung biopsy gives poor yeild and hence if biopsy is used for diagnosis then it has to be surgical lung biopsy which is quite invasive and needs good amount of tissue for detection. Its sensitivity and specificity is both 100%.
But with the advent of noval diagnostic techniques, role of biopsy has become very limited.
REF:
◇ This a lung biopsy stained by Gomori methenamine stain which is positive for pneumocystis pneumonia (PCP).
◇ The current samples used for potential diagnosis of Pneumocystispneumonia.
◇ The modalities used in the diagnosis of this PCP and their sensitivities and specificities:
1. Nonimmunofluorescent staining:
2. Immunofluorescent staining:
◇ Novel methods of detection.
1. Polymerase chain reaction
2. Loop-mediated isothermal amplification (LAMP):
3. Flow cytometry:
4. Antibody assays
5. Antigen & biomarker assays
a. Serum beta D-Glucan (BG):
b. Serum levels of LDH
c. Serum levels of KL-6:
e. S-adenosylmethionine (SAM):
◇The place of lung biopsy in the diagnosis of PJP:
This is an invasive diagnostic methods. It can be done when there is pneumonia in a transplant patients without a biological diagnosis. It can be used only if BALF is negative, and in some other indication.s
____________________________
References
1. Marjorie Bateman, Rita Oladele, et al. Diagnosing Pneumocystis jirovecii pneumonia: A review of current methods and novel approaches. Med Mycol. 2020; 58(8): 1015–1028.
2. Karageorgopoulos DE, Qu JM, Korbila IP, et al. Accuracy of beta-D-glucan for the diagnosis of Pneumocystis jirovecii pneumonia: a meta-analysisexternal icon. Clin Microbiol Infect. 2013;19:39-49.
Thank you, Tahani
Thanks. Prof. Ahmed
What are the modalities used in the diagnosis of this disease?
Biochemistry – LDH, Beta-D-glucanChest radiograph – Diffuse, bilateral interstitial infiltratesCT thorax – ground glass appearance and cystic lesionInduced sputum for direct fluorescent antibody stainingBAL fluid for direct fluorescent antibody stainingLung biopsy for tissue stainAnother method is PCR using sputum, VAL fluid and lung biopsy which carries higher sensitivityWhat is the place of lung biopsy in the diagnosis of PJP?
This test has excellent sensitivity. however, because of the nature of the test, this might not always be necessary. However, in the setting of inconclusive diagnosis and the patient is not responded to the empirical therapy, this is extremely useful; on top of that, lung biopsy can diagnose other lung pathology.
References
Jiang J, et al. Multiplex Real-Time Polymerase Chain Reaction on Sputum for the Diagnosis of Pneumocystis jirovecii Pneumonia in Children: A Retrospective Study. Infection and drug resistance
Thank you
Types of specimen used to diagnose PJP are
Ordinary sputum
Induced sputum
BAL fluid
Nasopharyngeal aspirate
Transbronchial tissue biopsy
Lung biopsy
Modalities for diagnosis :
Culture has no role her as PCP can not be cultured.
Conventional staining like:
modified Papanicolaou, May-Grünwald Giemsa, Giemsa, or Gram-Weigert stains that can detect trophic form of PJP.
And the Gomori methenamine silver, toluidine blue, cresyl echt violet, or calcofluor white that can detect the cystic form.
sensitivity 50–80%, specificity; >99%
Immunofluorescence with anti-Pneumocystis antibodies.
sensitivity 90.8% while specificity 94%
PCR:
100% negative predictive value limiting its ability to distinguish between infection and colonization.
Meta genomics NGS: new diagnostic method, expensive, sensitivities and specificities higher than those observed for standard PCR.
LDH (sensitivity 55.6% specificity 71.4%)
Beta d glucan (sensitivity 95-96%, specificity 84-89%).
CXR :Non specific findings, 90% have abnormal CXR and 10-15 have normal CXR .
HRCT: sensitivity and specificity are 100% and 89%.
What is the place of lung biopsy in the diagnosis of PJP?
Invasive procedure used if PJP is highly suspected but the BAL fluid doesn’t show the microorganism, it is the gold standard modality.
It can also used to diagnose other lung diseases as indicated.
Reference:
Lecture of Dr. Jamal Saadi in PJP
Thank you
What are the modalities used in the diagnosis of this disease?
Modalities used are dependent on centre availability:
– Clinical manifestations: Physicians must be vigilant for symptoms and signs of PJP in immunocompromised patients present with pneumonia and suggestive radiographic findings. Presence of hypoxemia at rest or with exertion or an increase in the alveolar-arterial oxygen tension gradient accompanied with sparce physical findings.
-Laboratory findings:
Elevated lactate dehydrogenase (LDH)
Beta-D-glucan, which is a cell wall component of most fungi.
Microbiologic diagnosis of the organism with a sputum sample obtained by induced sputum or bronchoalveolar lavage [BAL] if can be obtained safely. Using of either tinctorial (dye-based) staining, fluorescent antibody staining, or
PCR-based assays of respiratory specimens.
Lung biopsy is a diagnostic technique of last resort. Stains used in organanism identification include cresyl violet, Diff-Quick, Wright, methamine silver, Papanicoloau and monoclonal antibodies.
The current image showing Gomori’s methenamine silver (GMS) stain shows the typical round and collapsed crescent or boat-shaped cyst forms of Pneumocystis.
Radiological findings:
The typical radiographic features of PCP are diffuse, bilateral, interstitial infiltrates. Extensive bilateral ground-glass opacities in HRCT
Thank you
– What are the modalities used in the diagnosis of this disease?
– What are their sensitivities and specificities?
Broncho-alveolar lavage (BAL): the gold standard for diagnosis of PJP, if negative excludes the disease, but 51% positive test have a disease.
Sputum PCR: negative test sufficient to exclude the disease, 85-100% sensitive.
Oral washing: 75-91% sensitive, and 68-100%, negative test does not exclude PJP.
Nasopharyngeal aspirate: MSG PCR on nasopharyngeal samples was found to have an 86% sensitivity and 95% specificity for detecting PCP when compared to BALF and sputum samples.
Blood/ serum PCR: high sensitivity 100% and negative predictive value of 99% in HIV patients, but not yet recommended to use.
Urine testing for PJP: a new noninvasive molecular diagnostic techniques, but studies are needed is it feasible.
Novel detection methods:
(1) Polymerase chain reaction (PCR): sensitivity of 97-99% with a pooled specificity of 90- 94%, with most samples being BALF.
(2) Loop mediated isothermal amplification: amplify a target gene with only a heating device and isothermal conditions, sensitivity ranges from 87.5-95.4%, and LAMP has been shown to be relatively specific with no cross-reactivity to other fungal species.
(3) Flow cytometry: 100% sensitivity and specificity.
(4) Antibody assay: ELISA detecting antibodies against PJP, with a sensitivity of 100%, and specificity of 81%.
Antigen assay: Beta D-Glucan (BG) is a cell wall constituent in the ascus life-form of Pneumocystis jirovecii, 91-96% sensitive and 75-86% specific, KL-6 and S-adenosylmethionine antigens are not recommended for use by now.
Lactate dehydrogenase (LDH): 66–91% sensitive and 36–52% specific, almost 100% in HIV associated PJP, but only 61% in non-HIV associated PJP.
Nonimmunoflurescence stains: The Gomori-methenamine-silver (GMS) stain, Gram-Weigert, cresyl echt violet, toluidine blue O (TBO), and calcofluor white (CW) stains have positive and negative predictive value of >95% in BAL, the sensitivity of CW is 57-78%, the sensitivity of 31-97%, and 49-94% in TBO, these test are specific but if negative do not exclude disease. The result depends on skills and detects cysts only, cannot discriminate between active or dead ones.
Immunoflurecent stains: sensitivity of 48-100%, and specificity of 82-100%, detects both cysts and trophozoites.
– What is the place of lung biopsy in the diagnosis of PJP?
lung biopsy specimens did not indicate any diagnostic advantage over routine methenamine silver stains.
Atypical features (interstitial and intraluminal fibrosis, absence of alveolar exudate, numerous alveolar macrophages, granulomatous inflammation, hyaline membranes, marked interstitial pneumonitis, parenchymal cavities, interstitial microcalcification, and vascular invasion with vasculitis) requiring further evaluation, and stratification.
References:
(1) Bateman M, Oladele R, Kolls JK. Diagnosing Pneumocystis jirovecii pneumonia: A review of current methods and novel approaches. Med Mycol. 2020 Nov 10;58(8):1015-1028. doi: 10.1093/mmy/myaa024. PMID: 32400869; PMCID: PMC7657095.
(2) Travis WD, Pittaluga S, Lipschik GY, Ognibene FP, Suffredini AF, Masur H, Feuerstein I, Kovacs J, Pass HI, Condron KS, et al. Atypical pathologic manifestations of Pneumocystis carinii pneumonia in the acquired immune deficiency syndrome. Review of 123 lung biopsies from 76 patients with emphasis on cysts, vascular invasion, vasculitis, and granulomas. Am J Surg Pathol. 1990 Jul;14(7):615-25. doi: 10.1097/00000478-199007000-00002. PMID: 2192568.
Thank you
What are the modalities used in the diagnosis of this disease?
1. The picture: it’s Gomori Methenamine-Silver (GMS) stain of lung biopsy shows Pneumocystis jirovecii organisms.
2. Current approaches to Pneumocystis jirovecii (PCJ) screening on bronchioalveolar lavage samples (BAL) include(1):
· Gomori/Grocott methenamine silver stain (GMS).
· Toluidine blue O stain.
· Wright-Giemsa stain.
· Immunofluorescent antibody stain: PCJ IHC performed in cell block is more sensitive and specific than GMS and is a reliable marker when a low number of PCJ organisms are present(1).
· Polymerase chain reaction.
What are their sensitivities and specificities?
1. Sensitivity of the GMS stain for Pneumocystis and for fungi detection was 100%. Sensitivity for Pneumocystis and for fungi detection by Papanicolaou stain alone was 79% and 88%, respectively; by Diff-Quik stain alone it was 68% and 88%, respectively; and by combined Papanicolaou and Diff-Quik stains it was 79% and 100%, respectively(2).
2. The specificity of both Giemsa and GMS staining of induced sputum samples is high and the methods are simple, but the sensitivity is low. The sensitivity of PCR for P. carinii DNA from induced sputum samples is significantly higher than cytochemical stains, and the method is highly specific when used in the clinical diagnosis of PCP(3).
What is the place of lung biopsy in the diagnosis of PJP?
1. For detection of PJP , the diagnostic yield is significantly higher for direct immunofluorescence monoclonal antibody (DFA)-stained BAL specimens than for GMS-stained bronchoscopic lung biopsy (BLB) specimens(4).
2. The sensitivity of the DFA method on BAL fluid and of the GMS method on BLB was 95% and 43%, respectively(4).
3. Open lung biopsy achieves a higher diagnostic yield earlier in the pneumonitis when pneumocystis pneumonia is confined to the perihilar regions inaccessible to percutaneous needle biopsy(5).
References
1. Gonzalez AA, Hamele-Bena D, Wood T, Valladares-Silva S, Wasserman PG. Pneumocystis jirovecii immunostain versus Gomori/Grocott methenamine silver stain of bronchoalveolar lavage in cell blocks: an institutional experience. J Am Soc Cytopathol. 2017 Nov-Dec;6(6):242-247. doi: 10.1016/j.jasc.2017.06.207. Epub 2017 Jul 6. PMID: 31043294.
2. Raab SS, Cheville JC, Bottles K, Cohen MB. Utility of Gomori methenamine silver stains in bronchoalveolar lavage specimens. Mod Pathol. 1994 Jun;7(5):599-604. PMID: 7524071.
3. Hua L, Qin S, Wang A, Sheng R, Zhang K. [The diagnostic value of polymerase chain reaction for the detection of Pneumocystis carinii DNA from induced sputum samples]. Zhonghua Nei Ke Za Zhi. 2002 Sep;41(9):610-2. Chinese. PMID: 12421494.
4. Fraser JL, Lilly C, Israel E, Hulme P, Hanff PA. Diagnostic yield of bronchoalveolar lavage and bronchoscopic lung biopsy for detection of Pneumocystis carinii. Mayo Clin Proc. 1996 Nov;71(11):1025-9. doi: 10.4065/71.11.1025. PMID: 8917286.
5. Geelhoed GW. Open lung biopsy in the diagnosis of Pneumocystis carinii pneumonia. Natl Cancer Inst Monogr. 1976 Oct;43:141-7. PMID: 1087951.
Thank you
1- The modalities for diagnosis of PCP:
sputum, induced sputum, BAL or even lung biopsy.
2- Sensitivity and specificity:
3- Role of lung biopsy:
By Gomori methamine silver stain—detect dark brown oval or cup shaped organism in alveolar space.
Thank you
The modalities used in the diagnosis include:
Radiological Investigations:
Blood Investigations:
Microbiology:
Immunofluorescent staining: Has a sensitivity of 48-100% and a specificity of 82-100%
PCR: Has a sensitivity of 97-99% and a specificity of 91-94%
A lung biopsy is rarely used unless if the BAL is negative and there is a high index of suspicion of PCP or it is being done for another reason. It yields a 100% sensitivity and specificity
Medical Mycology, 2020, Vol.58, No.8
What are the modalities used in the diagnosis of PJP, the sensitivity and specificities of each, and the place of lung biopsy?
Below are the modalities(1,2,3,4)
CXR:
HRCT:
Serum LDH:
(1→3)β-d-glucan assay:
Silver, polychrome, or calcofluor staining methods:
Immunofluorescence assays on induced sputum or BAL
Real-time quantitative PCR, nucleic acid testing
Trans-bronchial biopsy during BAL
Open lung/video assisted thoracoscopic biopsy
Modalities, recommended usage, strength of recommendation and quality of evidence as in attached table(1)
References:
Thank you
👉 Radiological diagnosis by CXR has low sensitivity and is normal in 30% of cases, while HRCT has better diagnostic sensitivity.
👉Laboratory Diagnosis of PCP depends on obtaining samples either by:
_ Induced sputum as by hypertonic saline (yield is 50%).
_ Bronchoscopy and BAL (yield up to 80%).
_ Suction through ETT in ventilated patients.
👉 Detection if organism by:
_ IF staining the most sensitive.
_Quantitative PCR.
_Polychrome silver staining.
⭐ open lung biopsy or VATS is indicated only if no yield through BAL and high suspicious of PCP clinically
_Remains the gold standard for diagnosis with staining with Gomori silver stain.
Thank you
The modality
1.BALF;
2.Sputum;
3.Oral washing;
4.Nasopharyngeal aspirate
5.PCR
6.Immunoflurescent staining;
7.Flow cytometry;
8.LDH;
9.Lung biopsy;
Refferences
Thomas CF Jr., Limper AH. Pneumocystis pneumonia. N Engl J Med. 2004; 350: 2487–2498. [PubMed] [Google Scholar]
2. Stringer JR, Beard CB, Miller RF, Wakefield AE. A new name (Pneumocystis jiroveci) for Pneumocystis from humans. Emerg Infect Dis. 2002; 8: 891–896. [PMC free article] [PubMed] [Google Scholar]
3. Causes of severe pneumonia requiring hospital admission in children without HIV infection from Africa and Asia: the PERCH multi-country case-control study. Lancet North Am Ed. 2019; 394: 757–779. [PMC free article] [PubMed] [Google Scholar]
Thankyou all for describing all modalities but practically speaking each one should propose a subjective choice of one in each modality with respect to the clinical picture, available facility in his / her work place and a decision of when to start therapy even with non conclusive yield of investigation.
The microscopic image shown is a positive Gomori methenamine stain (GMS) of lung biopsy in a pneumocystis pneumonia (PCP) patient.
Modalities used in the diagnosis of pneumocystis pneumonia include:
1. Microscopy and staining of respiratory specimens: The specimens used include bronchoalveolar lavage fluid (BALF), induced sputum, oral washings, and nasopharyngeal aspirates. The specimens are stained by fluorescein-conjugated monoclonal antibody, which stains both cysts and the trophic form of the organism. Trophic forms can also be seen by using Wright-Geimsa, modified Papanicolaou, Diff-Quik, and Gram-Weigert stains. Cysts can be seen with Gomori methenamine silver (GMS), toluidine blue O (TBO), cresyl echt violet, and calcofluor white (CW) stain (1).
2. Polymerase chain reaction (PCR) assays: PCR of respiratory specimens can further increase the diagnostic yield.
3. Blood PCR.
4. Serum 1,3 Beta D glucan.
5. Serum LDH: Significantly elevated in PCP
2. What are their sensitivities and specificities?
The sensitivities and specificities of the specimen tests vary (2):
With respect to staining with different stains, highest sensitivity is with CW, GMS, and TBO stains. Sensitivity of CW, GMS, and TBO ranges from 57-78%, 31-97%, and 49-94% respectively (2). Immunofluorescent stains have sensitivity of 48-100%, and specificity of 82-100% (2).
3. What is the place of lung biopsy in the diagnosis of PJP?
Invasive diagnostic methods including lung biopsy is required in transplant patients with pneumonia without a biological diagnosis (3). So a lung biopsy is required only if BALF is negative, or if it is being done for some other indication.
References:
Well done
What are the modalities used in the diagnosis of this disease
Giemsa or methenamine silver stain:
Specimens for cysts of P. jiroveci, prepare smears of the sediment and examine after staining .
What are their sensitivities and specificities?
Induced sputum:
Is the quickest and least-invasive method for definitively diagnosing PJP. Expectorated sputum has a very low sensitivity and should not be submitted for diagnosis. Pneumocystis antigen detection assays on sputum may also be helpful but may have a 50% sensitivity
BAL around 80% sensitivity.
Trans bronchial biopsy around 90% sensitivity .
Open lung biopsy or video-assisted thoracoscopy(VAT).
PCR is good test but cannot deferentiate from carrier, around 83%
Lactate dehydrogenase( LDH ) not specific.
B- d- glucan not specific.
Genotype sequencing for outbreak.
What is biopsy in the diagnosis of PJP?the place of lung
Lung biopsy remains a candidate for the diagnostic method of choice.
It is invasive , pneumothorax and bleeding can occure from this procedure.
The sensitivity and specificity 100%.
References
ML Cohen et al. Pneumocystis carinii pneumonia: Percutaneous lung biopsy and review of literature Chest (1971)
Thankyou
Microscopy with staining
· Pneumocystis cannot be cultured.
· Direct fluorescent antibody staining can visualize both trophic forms and cysts
· tinctorial stains such as Gram-Weigert, Wright-Giemsa, or modified Papanicolaou stains. (Trophic forms )
· The cell wall of the cysts can be visualized with calcofluor white, cresyl echt violet, Gomori methenamine silver, or toluidine blue.
Type of respiratory specimen
· sputum induced by the inhalation of hypertonic
· bronchoscopy with BAL
· Lung biopsy with tissue stains and PCR has excellent sensitivity for diagnosing PCP but is rarely required
o The diagnostic yield of microscopy with staining of induced sputum is 50 to 90 percent in patients with HIV and PCP but is thought to be lower in patients without HIV due to a decreased organism burden
o BAL The diagnostic yield is over 90 percent in patients with HIV but may be lower in patients without HIV .
Polymerase chain reaction —
· for the detection of Pneumocystis in induced sputum or BAL fluid, blood, or nasopharyngeal aspirates.
may be of particular use in patients without HIV, in whom the sensitivity of microscopy with staining is substantially lower than in patients with HIV.
· single-copy real-time PCR assays rapidly identify patients with infection rather than colonization and hence are more useful in clinical settings
· PCR-based detection of PCP adds roughly 7 percent yield over stains alone when applied to BAL specimens
Beta-D-glucan assay
The serum beta-D-glucan assay can be used as an adjunct to the diagnosis of PCP. the test has good sensitivity in patients with HIV and PCP and a high negative predictive value, making it unlikely that a patient with a negative beta-D-glucan result has PCP the sensitivity of the assay may be reduced, particularly when the burden of fungal disease is low or in patients without HIV
.
Lung biopsy is reserved for
· patients with high suspicion of PCP and BAL testing has been negative
· or in those who have another reason to proceed to lung biopsy for diagnosis of a pulmonary process.
Thankyou this is excellent
Diagnostic tests, sensitivities & specificities
Laboratory tests:
A negative serum beta-D glucan excludes PCP.
Imaging:
CXR
HRCT
Pulse oximetry
Pulmonary function test
BAL:
transbronchial biopsy
-What is the place of lung biopsy in the diagnosis of PJP?
Source:
Thankyou, Exellent .lung biopsy has 30% chance of pneumothorax .!
What are the modalities used in the diagnosis of this disease? What are their sensitivities and specificities?
Modalities for diagnosis:
I-Microbiological Diagnosis:
A definitive diagnosis of PJP is made by demonstration of organisms in lung tissue or respiratory tract secretions.
*Type of specimen:
Routine sputum; sensitivity Poor
Induced sputum sensitivity 30–55, Rapid and cost-effective
Bronchoalveolar lavage sensitivity 80–95, Sample of choice
Bronchoalveolar lavage and transbronchial biopsy; sensitivity >95
Open-lung biopsy; sensitivity >95
Biopsy is the gold standard
*Culture: Organism cannot be cultured.
*Smear: Negative smear from any single respiratory specimen cannot be used to exclude PJP.
*Staining;
– Conventional staining: sensitivity 50–80%, specificity; >99%
–Trophic forms can be detected with modified Papanicolaou, May-Grünwald Giemsa, Giemsa, or Gram-Weigert stains.
-Cysts(asci): can be stained with Gomori methenamine silver, toluidine blue, cresyl echt violet, or calcofluor white
-Immunofluorescence with anti-Pneumocystis antibodies: higher sensitivity 90.8% while specificity 94%
*PCR
-High negative predictive value of this method, close to 100%,
-Limiting the ability to distinguish between asymptomatic colonization and infection.
*Metagenomics NGS; novel diagnostic method, expensive, using specific primers or probes to determine sequence of all nucleic acids, sensitivities and specificities higher than those observed for standard PCR
*Antibody assays
– ELISA technique to detect anti-P. jirovecii immunoglobulin (Ig); sensitivity of 100% and a specificity of 81%
II-Radiologically:
-No radiographic pattern is pathognomonic for Pneumocystis infection.
-90% of chest radiographs are abnormal, appearances are often non-specific.
-10-15% of patients have normal chest radiographs and
-Features which are highly suggestive of PCP include: fine reticular interstitial changes predominantly perihilar in distribution small pneumatoceles, subpleural blebs,
HRCT: is more sensitive, used to exclude PCP in patients with clinical suspicion for PCP but inconclusive CXR. Features on CT include: Ground glass appearance; reticular opacities, septal thickening, pneumatocele, honeycomb
Subpleural peripheral sparing seen in (~40%)
*sensitivity and specificity are 100% and 89%,
III-Laboratory indicators;
-β-D-glucan antigen in blood sensitivities 90%-100%,specificities of 88%–96%
-LDH can help indicate and correlates with severity of the disease
What is the place of lung biopsy in the diagnosis of PJP?
Obtained large specimen of tissue for diagnosis.
It is invasive diagnosis should be considered only if BAL was inconclusive pneumonia without a microbiological diagnosis or if there is another different reason indicate biopsy
References:
Fishman, JA, Gans, H; on behalf of the AST Infectious Diseases Community of Practice. Pneumocystis jiroveci in solid organ transplantation: Guidelines from the American Society of Transplantation Infectious Diseases Community of Practice. Clin Transplant. 2019; 33:e13587. https://doi.org/10.1111/ctr.13587
Iriart X, Bouar ML, Kamar N, Berry A. Pneumocystis Pneumonia in Solid-Organ Transplant Recipients. J Fungi (Basel). 2015 Sep 28;1(3):293-331. doi: 10.3390/jof1030293. PMID: 29376913; PMCID: PMC5753127.
Thankyou well done
History and examination may give clue to the diagnosis modalities used :
* Bronchoalveolar lavage (BAL) is the gold standard :
-PCR Assay for PCP : Quantitative real-time PCR (qPCR) has progressively supplanted conventional PCR. It is usually used to detect Pneumocystis DNA in different types of samples.
-GMS Stain Assay : Sputum sample was directly centrifuged ,fixed with methanol and stained with GMS PJP cysts are shown black or dark brown, round, or quasi-round.
-mNGS Protocol : The DNA was randomly fragmented and made into a sequencing library using on NextSeq 550Dx platform .
* Plasma β-(1,3)-D glucan.
– In diagnosing PJP mNGS reached a sensitivity of 100%, which is remarkably higher than GMS staining (25.0%) and plasma β-(1,3)-D glucan (67.4%). The specificity of mNGS (96.3%) while β-(1,3)-D glucan has sensitivity of (81.4%)
-The sensitivity and specificity of plasma cfDNA sequencing to detect P. jirovecii infection was 83.3 and 100%, respectively.
Imaging :
CXR: Up to 90% of chest radiographs in patients with Pneumocystis pneumonia are abnormal, appearances are often non-specific. Between 10-15% of patients have normal chest radiographs and close to 30% have non-specific or inconclusive finding.
HRCT : High-resolution computed tomography is more sensitive and may be used to exclude PCP in patients with clinical suspicion for PCP but normal or inconclusive chest radiographs.
Whatever technique can establish this diagnosis safely and early in order to get treatment started quickly should be used. If diagnosis cant be established by other methods lung biopsy though is invasive it remains the essential cornerstone in the diagnosis of pneumocystis pneumonia. The diagnostic sensitivity for PCP was 100%. The negative predictive value of bronchoscopy for PCP was 85%.
Well done but the paragraph of sensitivity and specificity needs rephrasing.
Modalities used in the diagnosis
BAL
Enables microbiologic diagnosis to be obtained with or without transbronchial biopsy.
In 51% of instances, bronchoscopy can identify PCP, enabling medication to be stopped.
Chest CT
Diffuse ground-glass opacity is the most typical high-resolution CT result for Pneumocystis jiroveci pneumonia.
A spontaneous pneumothorax, nodules, cysts, and consolidation are further potential complications
Haematological
Blood tests should check for endemic mycoses and 1,3-d-glucan, which have poor outcomes in SOT recipients, as well as antigen and serologic examination.
Oral secretion and washing
Positive samples may be the most helpful in proving a PCP diagnosis, while a negative result cannot conclusively rule out PCP in symptomatic patients.
Oro-nasopahryngeal aspirate
samples was found to have an 86% sensitivity and 95% specificity for detecting PCP when compared to BALF and sputum samples.
Non immunoflouresecnt staining
Giemsa, Diff-Quik, Wright-Giemsa, modified Papanicolaou, and Gram-Weigert stains can all be used to detect the cyst life form, but the trophozoite life form is rarely employed for diagnosis due to its tiny size and nonspecific staining pattern.
The most sensitive staining techniques are CW, GMS, and TBO, with CW and GMS having positive and negative predictive values >90%.
These tests are simple to carry out, but stain interpretation makes them subjective.
Lung biopsy
Because it delivers the most tissue for diagnosis, an open lung biopsy is the most invasive method and produces 100% sensitivity and specificity. However, this operation is only used in extremely rare situations when the results of bronchoscopy are nondiagnostic.
Pneumocystis jiroveci Pneumonia (PJP) Overview of Pneumocystis jiroveci Pneumonia Updated: Nov 04, 2022
Bateman M, Oladele R, Kolls JK. Diagnosing Pneumocystis jirovecii pneumonia: A review of current methods and novel approaches. Med Mycol. 2020;58(8):1015-1028. doi:10.1093/mmy/myaa024
Alanio A, Hauser PM, Lagrou K, et al. ECIL guidelines for the diagnosis of Pneumocystis jirovecii pneumonia in patients with haematological malignancies and stem cell transplant recipients. J Antimicrob Chemother 2016; 71:2386.
Thankyou but how can oropharyngeal specimen be more yielding than BAL, remember the fungus stays in the alveoli.
thank you prof. correcting me
i want to say oral washing and cough inducing secretions
The picture below is for PCP (Pneumocystis pneumonia caused by Pneumocystis Jirovecii) diagnosed by lung biopsy
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What are the modalities used in the diagnosis of this disease?
===================================================================
What are their sensitivities and specificities?
===================================================================
What is the place of lung biopsy in the diagnosis of PJP?
Lung biopsy
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Reference
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Well done Thankyou but a blood culture will give other organisms if present,not Pcj.
YES ,Many thanks Prof.Dawlat
The picture below is for PCP (Pneumocystis pneumonia caused by Pneumocystis Jirovecii) diagnosed by lung biopsy
==================================================================
What are the modalities used in the diagnosis of this disease?
===================================================================
What are their sensitivities and specificities?
===================================================================
What is the place of lung biopsy in the diagnosis of PJP?
Lung biopsy
==================================================================
Reference
1-What are the modalities used in the diagnosis of this disease?
-An induced sputum sample is usually the initial procedure for the diagnosis of PCP.
-If PCP is not identified by this modality, then bronchoscopy with bronchoalveolar lavage should be performed.
Definitive diagnosis;
-The definitive diagnosis of PCP requires identification of the organism either by tinctorial (dye-based) staining, fluorescent antibody staining, or polymerase chain reaction (PCR)-based assays of respiratory specimens.
-BAL fluid was centrifuged and examined under the microscopes by using a special stain called Gomori methenamine silver (GMS) stain shows the typical round black or brown PJP cysts.
immunofluorescence testing is more sensitive than GMS.
Presumptive diagnosis;
-There are times when a definitive diagnosis cannot be made due to a low burden of organisms and/or the inability to obtain the necessary specimen.
-In these situations, a decision must be made whether or not to continue treatment.
-Clinical and radiographic findings can be highly suggestive of a diagnosis of PCP in patients with risk factors for PCP.
-Increasingly, elevated serum levels of beta-D-glucan are used to help support this diagnosis.
2-What are their sensitivities and specificities?
-The sensitivity of the test is best in immunocompromidsed individuals such as those with HIV with high fungal disease burden, compared to non immunocompromised with lower fungal disease burden.
-Various cut off levels were put, but generally a cut off of > 80 pg/mL is used in USA to predict candidemia in neutropenic patients.
-The sensitivity ranges from 70-92%, while specificity ranges from 80-85% according to cut off used.
-It is a good negative test (patient with negative test are unlikely having PCP), and good positive test reaching up to 100% if using higher cut off level (> 200 pg/mL).
-False positive test can occur in patients with pseudomonas infection, ad after the use of IVIG.
-In conclusion the test can be used to guide treatment (with high negative predictive values and high positive predictive values if the titer is high) till the availability of the result of respiratory sample or if respiratory sample cannot be obtained safely.
3-What is the place of lung biopsy in the diagnosis of PJP?
-Biopsy is an option only if BAL is inconclusive.
Indications for open lung biopsy in patients with HIV:
-Non diagnostic bronchoscopy.
-Failed medical therapy after a diagnostic bronchoscopy.
-Failed empiric medical therapy after a nondiagnostic bronchoscopy.
-Any of the above, in combination with a worsening chest radiograph.
Transbronchial lung cryobiopsy;
-Transbronchial lung cryo biopsy has been reported to be a valid and safe alternative to surgical lung biopsy in patients with diffuse parenchymal lung disease (DPLD).
-Although there are no large studies in persons with HIV, it might be considered a viable alternative to open lung biopsy in those suspected of having noninfectious diffuse pneumonitis and malignancies such as lymphoma, Kaposi sarcoma, lung cancer, and other causes of DPLD.
Surgical lung biopsy;
-Lung biopsy is considered an gold standard invasive test and can be transbronchial or open lung biopsy and both are of limited use due to the risk of bleeding and pneumothorax.
-Surgical lung biopsy remains the procedure with the greatest sensitivity in the diagnosis of parenchymal lung disease.
-Thoracotomy and VATS can both be performed safely in patients with HIV.
-There may be less morbidity associated with VATS, as these patients generally require fewer days in hospital and less time with chest tube drainage.
-If the patient cannot tolerate single lung ventilation, which is necessary during VATS, or if the lesion cannot be reached through a thoracoscope, then an open thoracotomy should be performed.
-In general, the choice of procedure is best left to the surgeon.
-References;
-Up To Date;
Evaluation of pulmonary symptoms in persons with HIV: Feb 10,2023.
Lung biopsy in pneumocystis carnii pneumonia; Aug 02, 2022.
Paragraph 2 is it quantitative value in a specimen (not blood)
Thanks; our Prof. & sorry,if it is not clear;
-Direct visualization of Pneumocystis jiroveci organisms, using Gomori methenamine silver (GMS) staining in bronchoalveolar lavage fluid (BAL), is a historical gold standard that has been widely used for the diagnosis of P jiroveci pneumonia (PJP).
-The BAL GMS stain is insensitive for the diagnosis of PJP in HIV-negative immunocompromised patients and should not be used as the stand-alone diagnostic method in this population.
-Clinicians and pathologists in institutions that use GMS BAL as the primary fungal stain on BAL should be aware of sensitivity limitations for PJP and the potential for missed diagnoses.
-In HIV-negative patients with a high pretest probability of PJP, a negative BAL GMS finding does not rule out the diagnosis of PJP and should be further evaluated with a P jiroveci immunofluorescent antibody test or PCR on BAL.
-Empiric therapy should be initiated once PJP is suspected.
-In this setting, an elevated serum (1-3)-β-D-glucan is a very useful adjunct diagnostic test for PJP
In conclusion;
-BAL GMS has poor sensitivity for PJP in HIV-negative immunocompromised patients.
-Using BAL GMS as a sole method for PJP may result in missed or delayed diagnoses in this population.
Reference;
-Gomori Methenamine Silver Stain on Bronchoalveolar Lavage Fluid Is Poorly Sensitive for Diagnosis of Pneumocystis jiroveci Pneumonia in HIV-Negative Immunocompromised Patients and May Lead to Missed or Delayed Diagnoses; https://doi.org/10.5858/arpa.2019-0394-OA.
What are the modalities used in the diagnosis of this disease, SENSITIVITY AND SPECIFICIY
Place of lung biopsy in the diagnosis of PJP
Most of the time, in such cases, a lung biopsy is performed to guide the diagnosis of pulmonary process in people who have a high chance of having PCP and whose BAL tests came back negative or who have another reason to have a lung biopsy.
Lung biopsy is a risky and invasive process. Depending on which method is used—open or imaging-guided—the sensitivity changes. Tests on affected tissue is highly specific.
Thankyou
4. The picture below is for PCP (Pneumocystis pneumonia caused by Pneumocystis Jirovecii) diagnosed by lung biopsy
–Modalities used in the diagnosis
Chest X-rays
show diffuse bilateral interstitial infiltrates but can also show lobar consolidations and nodules or even normal findings.
HRCT Chest
show diffuse ground-glass opacities .
Induced sputum
Presence of the Pneumocystis jirovecii organism in induced sputum
PCR assays of respiratory specimens
Broncho-alveolar lavage (BAL)
by staining with Gomori methenamine silver (GMS), is considered diagnostic for PCP , toluidine blue O (TBO), and calcofluor white (CW) stains are also used
or using direct immunofluorescent antibodies (DFA)
Loop-mediated isothermal amplification
Elevated level of serum Beta-D-Glucan
can be used to support the diagnosis of PCP, if available.
Metagenomic next-generation sequencing (mNGS)
The mNGS is an efficient technology in diagnosing PCP and has a satisfying performance in the detection of co-pathogens. Both blood and BALF samples for mNGS are suggested for the presumptive diagnosis of PCP.
Transbronchial lung biopsyTBBx
increases the diagnostic yield for PCP in suspected cases
–Sensitivity and specificity of modalities
DFA was most sensitive 90.8% compared to 50–80% for the conventional stains but less specific 94.7% compared to >99% for the conventional stains
The sensitivity of the CW stain ranged from 57 to 78%.
The sensitivity of GMS ranges from 31 to 97% with lower sensitivities in studies with poor quality samples or a large number of noninvasive samples .
TBO staining has lower sensitivity ranging from 49 to 94%.
Real-time PCR analysis on serum samples had a very high sensitivity (100%) and negative predictive value (99%) for the diagnosis of PCP in HIV-infected patients.
Real-time quantitative Pneumocystis PCR (qPCR) assays were used to improve the specificity and positive predictive value of molecular assays, Some studies have proposed cut-off values of >1450 or >1900 pathogens/mL for the diagnosis of PJP, with positive predictive values of 98% and 100%, respectively but those cut offs are not standardized,
Loop-mediated isothermal amplification (LAMP) Sensitivity ranges from 87.5 to 95.4% and relatively specific
P. jirovecii qPCR with microscopy on BAL can improve the diagnostic yield
Pneumocystis jiroveci non-invasive blood cell-free DNA (cfDNA) PCR assay test was 100% sensitive and 93.4% specific in a small cohort with proven disease
Bronchoscopy, when performed after negative induced sputum testing, has been noted to yield a diagnosis in 51% of cases and, if negative for PCP, allows for discontinuation of treatment
Immunoflouresent staining for induced sputum have 85–100% sensitivity and good concordance with BALF results when methods of detection such as PCR are utilized.
β-D-glucan assay (BDG) is non specific but highly sensitive
The mNGS showed a satisfying diagnostic performance with a sensitivity of 100% in detecting P. jirovecii and it’s diagnostic specificity for PCP was higher than that of serum BDG (56.7%) and LDH (71.4%)
The yield from TBBx and BAL was 95%
–Place of lung biopsy in diagnosis of PCP
lung biopsy procedures were used to obtain large specimens of lung tissue to stain for organism identification. As diagnostic modalities developed and technical expertise has improved, biopsy has been replaced with more minimally invasive procedures.
Reference
–Sabbagh W, Darwich NS. Pneumocystis Jiroveci Pneumonia and Newly Diagnosed Human Immunodeficiency Virus (AIDS) in a 63-Year-Old Woman. Am J Case Rep. 2018;19:927-931. Published 2018 Aug 8.
-Apostolopoulou, A.; Fishman, J.A. The Pathogenesis and Diagnosis of Pneumocystis jiroveci Pneumonia. J. Fungi 2022, 8, 1167.
– Bateman M, Oladele R, Kolls JK. Diagnosing Pneumocystis jirovecii pneumonia: A review of current methods and novel approaches. Med Mycol. 2020;58(8):1015-1028.
– Chen, H., Liang, Y., Wang, R. et al. Metagenomic next-generation sequencing for the diagnosis of Pneumocystis jirovecii Pneumonia in critically pediatric patients. Ann Clin Microbiol Antimicrob 22, 6 (2023)
Well done
What are the modalities used in the diagnosis of this disease? and What are their sensitivities and specificities?
Diagnostic specimen
Bronchoalveolar lavage, transbronchial biopsy, open lung biopsy or viedio assited thoracoscopy, induce sputumDiagnostic technique
Sputum
Immunofluorescence assay (BAL sensitivity 55-90%), real time quantitative pcr nucleic acid testing(sensitivity 94-99% specificity 89-91% of BAL), silver polychrome or calcofluor stain, metagenomic next generation sequencing(mNGS)(sensitivity 100%Serum
LDH (sensitivity 55.6% specificity 71.4%), beta d glucan (sensitivity 95-96%, specificity 84-89% , genotyping sequencing, dihydropteroate synthase mutationsImagingx rayHigh resolution CT chest- the sensitivity and specificity of high-resolution CT was 100 and 83%What is the place of lung biopsy in the diagnosis of PJP?
Technique-open lung biopsy or video assisted thoracoscopy.
still the gold standard of diagnosis,when other means are not conclusiveFindings
Lymphocyte infiltrateFoamy or granular eosinophilic exudateGomori methenamine silver staining- dark brown or cup shaped organisms in alveolar spaces1)Lecture:pneumocystis pneumonia in SOT. Prof Gamal Saadi
2)Salzer H, J, F, Schäfer G, Hoenigl M, Günther G, Hoffmann C, Kalsdorf B, Alanio A, Lange C: Clinical, Diagnostic, and Treatment Disparities between HIV-Infected and Non-HIV-Infected Immunocompromised Patients with <b><i>Pneumocystis jirovecii</i></b> Pneumonia. Respiration 2018;96:52-65. doi: 10.1159/000487713
3)Chen, H., Liang, Y., Wang, R. et al. Metagenomic next-generation sequencing for the diagnosis of Pneumocystis jirovecii Pneumonia in critically pediatric patients. Ann Clin Microbiol Antimicrob 22, 6 (2023). https://doi.org/10.1186/s12941-023-00555-5
Thankyou
What are the modalities used in the diagnosis of this disease?
1.Gomori methamine silver stain( GMS Stain) Assay
BAL fluid was centrifuged and examined under the microscopes by using a special stain called Gomori methenamine silver (GMS) stain shows the typical round black or brown PJP cysts
immunofluorescence testing is more sensitive than GMS.
PCR assay
Hypertonic saline induced sputum for q PCR assay a gain of low sensitivity compared to BAL fluid PJP PCR testing is more sensitive 96-100% , specificity > 80% for the diagnosis but invasive procedure as needs bronchoscopy
mNGS Protocol
mNGS method. mNGS is an unbiased approach that Can theoretically detect all pathogens in a clinical sample and
is especially suitable for rare, novel, and atypical etiologies of
complicated infectious diseases like PJP. the testing time of mNGS shorter than GMS. Compared with the traditional GMS method, mNGS has absolute advantages
What are their sensitivities and specificities?
All the available testing for PJP in non-HIV patients are of lower sensitivity, like the GMS with poor therapeutic effects GMS assay of 25% sensitive for PJP diagnosis but mNGS reached a sensitivity of 100%, and specificity of 96% in diagnosing PJP, While plasma β-(1,3)-D glucan test sensitivity of (67.4%) and specificity of 81%.
What is the place of lung biopsy in the diagnosis of PJP?
Lung biopsy is considered an gold standard invasive test and can be transbronchial or open lung biopsy and both are of limited use due to the risk of bleeding and pneumothorax
References
1. Lu X, Zhang J, Ma W, Xing L, Ning H, Yao M. Pneumocystis jirovecii Pneumonia Diagnosis via Metagenomic Next-Generation Sequencing. Front Med (Lausanne). 2022 Mar 9;9:812005. doi: 10.3389/fmed.2022.812005. PMID: 35372422; PMCID: PMC8965517.
2. Salzer HJF, Schäfer G, Hoenigl M, Günther G, Hoffmann C, Kalsdorf B, Alanio A, Lange C. Clinical, Diagnostic, and Treatment Disparities between HIV-Infected and Non-HIV-Infected Immunocompromised Patients with Pneumocystis jirovecii Pneumonia. Respiration. 2018;96(1):52-65.
Modalities of specimen collection for diagnosis
Investigative techniques
Lung biopsy
Lung tissue can be gotten from transbronchial biopsy, open lung biopsy or video assisted thoracoscopic surgery. It is the gold standard. On histology lymphocytic infiltrates, foamy or granular eosinophilic exudates are seen. On Gomori-methenamine silver stain, dark brown oval or cup shaped organisms in alveolar spaces are seen.
Reference
Thomas CF Jr., Limper AH. Pneumocystis pneumonia. N Engl J Med. 2004; 350: 2487–2498. [PubMed] [Google Scholar]
2. Stringer JR, Beard CB, Miller RF, Wakefield AE. A new name (Pneumocystis jiroveci) for Pneumocystis from humans. Emerg Infect Dis. 2002; 8: 891–896. [PMC free article] [PubMed] [Google Scholar]
3. Causes of severe pneumonia requiring hospital admission in children without HIV infection from Africa and Asia: the PERCH multi-country case-control study. Lancet North Am Ed. 2019; 394: 757–779. [PMC free article] [PubMed] [Google Scholar]
4. Kovacs JA, Masur H.. Evolving health effects of Pneumocystis: one hundred years of progress in diagnosis and treatment. JAMA. 2009; 301: 2578–2585. [PubMed] [Google Scholar]
Thankyou well done
CT scan: Showing bilateral ground glass appearance.
Microscopy with fluorescent dye is sensitive in 40-98% and specificity of 80-100% when performed on BAL fluid.
PCR is more specific and sensitive in detecting PJP, particularly when performed on BAL fluid sensitivity of 98% and specificity of 94%.
Beta-D-glucan protein detected in peripheral blood is nonspecific, but sensitive with high concentration.
Antibody essay: for detection of IgM antibodies against PJP with sensitivity of 100% and specificity 81%.
Short but good
lung biopsy is usually performed from apical segment of the lungs
References:
1]Marjorie Bateman, Rita Oladele, and Jay K Kolls. Diagnosing Pneumocystis jirovecii pneumonia: A review of current methods and novel approaches.Med Mycol. 2020 Nov; 58(8): 1015–1028.
Diagnostic tests
Microscopy with staining — Detection of the organism in respiratory specimens is most commonly achieved by microscopy with staining of an induced sputum specimen or BAL fluid
The diagnostic yield of microscopy with staining of induced sputum is 50 to 90 percent
with BAL, the diagnostic yield is over 90 percent
PCR —The utility of PCR for the diagnosis of PCP in immunocompromised patients without HIV was evaluated in a prospective study of 448 such patients with pulmonary infiltrates, the majority of whom had a hematologic malignancy. The study compared conventional staining (Giemsa staining and indirect immunofluorescence) with PCR for P. jirovecii. The following findings were noted:
●
Thirty-nine patients (8.7 percent) were diagnosed with PCP by conventional staining of BAL fluid or induced sputum. Of these, 34 (87 percent) had a positive PCR result.
●
PCR was also positive in 32 patients who had a negative PCP stain. Complete follow-up was available in 21 of these patients, 14 of whom were diagnosed with probable or definite PCP.
Thus, PCR of BAL fluid or induced sputum can increase the diagnostic yield over conventional staining alone in immunocompromised patients without HIV.
Beta-D-glucan assay — In one retrospective case-control study of 295 patients with suspected PCP who had microscopy of BAL fluid for PCP and serum testing with beta-D-glucan, the beta-D-glucan assay had a sensitivity of 92 percent and a specificity of 86 percent for detecting PCP when using a cut-off 31.1 pg/mL. In another study evaluating >400 immunocompromised patients with lower respiratory tract disease, the sensitivity and specificity correlated with the height of the beta-D-glucan value. Using a cut-off of 80 pg/mL, the assay had a sensitivity and specificity of 70 and 81 percent, respectively. The specificity of the assay rose to 100 percent when a >200 pg/mL cut-off was used. As expected, the predictive value of the beta-glucan assay rose when combined with PCP PCR.
Pneumocystis jirovecii is a near obligate alveolar pathogen with only rare cases of dissemination. In the early days of diagnosis, lung biopsy procedures were used to obtain large specimens of tissue to stain for organism identification. As diagnostic methods have become more sophisticated and technical expertise has improved, biopsy has been replaced with more minimally invasive sampling techniques as noted below
Thankyou well done
# The modalities used in the diagnosis of PJP and their sensitivities and specificities
#Bronchoalveolar lavage fluid (BALF)
*The current gold standard sample for diagnosis of PCP is BALF, which highest quality respiratory sample.
*The lack of a standardized sampling technique can impact test performance. *Bronchoscopy, when performed following negative induced sputum, has been noted to gives a diagnosis in 51% of cases.
*If negative for PCP, allows for discontinuation of treatment.
*The limitations:
Invasive procedure is expensive, may not always be feasible for patients with severe pulmonary disease, and may not be available in resource-poor settings.
*Nondirected bronchoalveolar lavage for further study, not requiring bronchoscope.
# Induced Sputum
*Study on IS has been found to have a >95% negative predictive value in low prevalence situations (<10% prevalence), making a negative test adequate for ruling out PCP.
*In high suspicion of PCP, a bronchoscopy with BAL should be performed when negative IS results are obtained.
*IS has 85–100% sensitivity and good concordance with BALF results when methods of detection such as PCR are utilized.
# Oral washing
*PJP may be found in oral washes if the organism has been coughed or recently inhaled into the oropharyngeal tract.
*Obtained quickly and noninvasively, and positive tests may reflect higher fungal burden in the lower respiratory tract.
*Theoretical disadvantages are increased degree of PCR inhibition.
*Sensitivity of 75–91% and a specificity of 68–100%.
*Oral wash samples may be most useful in supporting a diagnosis of PCP if positive, but a negative result cannot reliably rule out PCP in symptomatic patients.
Nasopharyngeal aspirate
*MSG PCR on nasopharyngeal samples was found to have an 86% sensitivity and 95% specificity for detecting PCP when compared to BALF and sputum samples.
*PCR was specifically noted to have a higher detection rate than immunofluorescence staining techniques.
# Blood/serum
*Blood/serum has the significant advantage of being easily obtained and inexpensive. The presence of PJP in the blood reflects disease progression
*A recent report from Sweden revealed that real-time PCR analysis on serum samples had a very high sensitivity (100%) and negative predictive value (99%) for the diagnosis of PCP in HIV-infected patients.
The serum(ELISA) for antibodies and antigens associated with PCP are other methods.
# Urine
*Urine testing for PCP may represent a new frontier for development of noninvasive molecular diagnostic techniques, but studies are needed to ascertain feasibility.
# Non immunofluorescent staining
*The cyst life-form can be detected with many stains. Giemsa, Diff-Quik, and Wright stains can detect the cyst but do not stain its wall. The (GMS) stain, Gram-Weigert, cresyl echt violet, (TBO), and (CW) stains stain the cell wall of the cyst
*Due to its small size and nonspecific staining pattern, this is not the life-form typically used in diagnosis.
*Highest sensitivity methods are CW, GMS, and TBO stain, had positive and negative predictive values >90% when performed on BALF.
*These staining methods are specific for the presence of organisms but if negative, do not rule out the presence of PCP.
# Immunofluorescent staining
*IF stains via monoclonal antibodies to PJP have higher sensitivity and specificity than conventional stains. Sensitivity (48 to 100%), specificity (82 to 100%).
*They are easier, more repeatable, less reliant on technical skill and stain both trophozoites and cysts
#Novel methods of detection PJP
# Polymerase chain reaction
*Nested PCR first techniques developed but more labor intensive, expensive, less quantitative, and less specific.
* mtLSU real-time PCR more specific and popular methods.
*More sensitive for detection of PCP than staining methods in patients with and without HIV.
*Three meta-analyses reported the sensitivity of 98%, 99%, and 97% with specificity of 91%, 90%, and 94% with most samples being BALF, when using quantitative PCR methods
*Due to high sensitivity false negative result is rare, so a negative PCR on BALF means PCP is an unlikely diagnosis, and a high specificity means that a positive PCR on BALF is highly suggestive of the presence of Pneumocystis jirovecii.
# Loop-mediated isothermal amplification (LAMP)
*Provides an alternative to PCR as it can amplify a target gene with only a heating device and isothermal conditions.
* Sensitivity ranges from 87.5 to 95.4%, and LAMP has been shown to be relatively specific with no cross-reactivity to other fungal species.
*In small studies, LAMP has been shown to have higher rates of detection of PCP than conventional stains and rates similar to those of PCR.
# Flow cytometry
*Flow cytometry can detect single or multiple microbes in an easy, reliable, and fast way.
*It can also detect antibodies against Pneumocystis and comment on antifungal susceptibility.
* Itis allows for detection of Pneumocystis jirovecii in clinical BAL and bronchial samples with 100% sensitivity and specificity when compared to IF staining.
*While the applications are vast, the data are limited, and this is not currently recommended as a diagnostic method
# Antibody assays
*A promising diagnostic approach is to use an antigenic tool in an ELISA technique to detect immunoglobulin (Ig), IgM, and IgG antibodies against Pneumocystis jirovecii.
*One study had shown that ELISA IgM anti-P. jirovecii has a sensitivity of 100% and a specificity of 81% when testing serum samples from 88 patients.
*The IR may be variable depending on the nature of the immunocompromise and may affect the sensitivity.
* Additional elucidation of the complex host and environmental factors that affect antibody formation will be required before tests are considered for widespread utilization.
# Antigen and biomarker assays
*(1,3)-Beta D-Glucan (BG) is a cell wall constituent in the ascus life-form of Pneumocystis jirovecii and multiple other fungal pathogens.
*Study found to be 91%, 96%, and 95% sensitive HIV positive and negative patients, but BG was only 75%, 84%, and 86% specific for definite PCP because it could be positive in other fungal infections, antibiotics, albumin or globulin therapy, and in HD.
# Lactate dehydrogenase (LDH):
*The sensitivity and specificity is 66%–91% and 36–52%, respectively.
*LDH levels are likely a reflection of the underlying lung inflammation and injury and are not specific to PCP.
*KL-6 have been found to be elevated in patients with PCP, but this marker has low specificity due to its elevation in any lung disease
# Lung biopsy in the diagnosis of PJP
*Bronchoscopy is the standard approach in evaluation of lung lesions, but it is limited in diagnose malignancy with BAL 33% with excellent in the evaluation of infections (diagnostic yield of 98%)
*Standard bronchoscopy is frequently restricted in accessing these lesions for tissue sampling if they are located in the periphery of lung, may lead to invasive procedures and complication.
*Electromagnetic navigational bronchoscopy (ENB) is an image-guided approach that uses a 3D-reconstructed (CT) and an electromagnetic sensor locator to access peripheral lung lesions beyond the reach of standard bronchoscopy, so tissue can be collected.
*The use of ENB-guided biopsies with Rapid on-site evaluation ROSE in the assessment of cavitary lung lesions in immunocompromised patients is efficient and safe.
References
1. Thomas CF Jr., Limper AH. Pneumocystis pneumonia. N Engl J Med. 2004; 350: 2487–2498.
2. Stringer JR, Beard CB, Miller RF, Wakefield AE. A new name (Pneumocystis jiroveci) for Pneumocystis from humans. Emerg Infect Dis. 2002; 8: 891–896
3. Houshmand F, Aly FZ, Bowling MR. A novel diagnostic approach for Pneumocystis jirovecii pneumonia using fine-needle aspiration, electromagnetic navigational bronchoscopy and rapid on-site evaluation. Ann Thorac Med. 2019 Oct-Dec;14(4):285-287. doi: 10.4103/atm.ATM_171_19. PMID: 31620213; PMCID: PMC6784444.
Well done, Exellent
Clinically speaking choose the practical used methods of diagnosis ,even sometimes by exclusion so as to start therapy.
Thanks alot prof Belal
Important information
Diagnosis:
challenging due to difficulty in reaching confirmatory diagnosis with non-invasive approach because cannot performing culture.
non-invasive methods:
invasive methods:
Thankyou ,open biopsy is not without risks ,maybe via thoracotomy.
MetagenomicsNGS is definately sure but still needs time and expenses.
Laboratory findings:
Low CD4 counts
Oxygenation — Hypoxia occurs with the progression of PCP
Lactate dehydrogenase level
Diffusion capacity — PCP is highly unlikely if the diffusion lung capacity for carbon monoxide (DLCO) is normal
Radiology:
CXR
High-resolution computed tomography
Gallium-67 citrate scanning
Microscopy with staining—Induced sputum specimens or BAL fluids are most often used to detect the organism in respiratory specimens. Staining is needed since Pneumocystis cannot be grown.
Tissue biopsy—If sputum induction, BAL, and transbronchial biopsy are non-diagnostic or cannot be done, more invasive methods may be needed to diagnose PCP. Transthoracic needle biopsy, thoracotomy, or video-assisted thoracoscopic lung biopsy are options. However, these treatments include major hazards that must be balanced against the requirement for a precise diagnosis:
Transthoracic needle biopsy, which is very diagnostic, causes 30% pneumothorax.
A lung biopsy via thoracotomy or video-assisted thoracoscopic surgery may diagnose PCP with 95–100% sensitivity. A lung biopsy shows foamy, eosinophilic alveolar exudate, edema, and interstitial fibrosis in severe instances.
References:
Benfield, T. L., Prentø, P., Junge, J., Vestbo, J., & Lundgren, J. D. (1997). Alveolar damage in AIDS-related Pneumocystis carinii pneumonia. Chest, 111(5), 1193-1199.
Cruciani, M., Marcati, P., Malena, M., Bosco, O., Serpelloni, G., & Mengoli, C. (2002). Meta-analysis of diagnostic procedures for Pneumocystis carinii pneumonia in HIV-1-infected patients. European Respiratory Journal, 20(4), 982-989.
Well done,so in each modality can you choose which will lead to diagnosis in a practical way .
Numbers of Pneumocystis pneumonia (PCP) diagnosed with traditional diagnostic
methods according to the type of samples.
Type of Sample PCP Diagnosed (%)
Routine sputum Poor
Induced sputum 30–55
Bronchoalveolar lavage 80–95
Bronchoalveolar lavage and transbronchial biopsy. >95
Open-lung biopsy >95 .
standard PCR 83%–100%.
Real-time quantitative PCR (qPCR) 93% to 100%.
Open lung biopsy is the most invasive procedure and yields 100% sensitivity and
specificity because it provides the greatest amount of tissue for diagnosis.
this procedure is reserved for rare cases when bronchoscopy findings are non
diagnostic.
References:
XavierIriart, MarineLeBouar,NassimKamar,and AntoineBerry..pneumocystis Pneumonia
in Solid-Organ Transplant Recipients. J Fungi (Basel). 2015 Dec; 1(3): 293–331.
This is a GOMORI METHENAMINE SILVER STAIN.
What is the most sensitive and specific test for diagnosis.
Real time PCR ,sensitivity seems to be generally highest in assays directed at
multicopy targets, such as the major surface glycoproteins (MSG) and the
mitochondrial large subunit ribosomal RNA (mtLSU rRNA) genes (Reid A.B.,
Chen S.C., Worth L.J. )
Diagnostic tests, sensitivities & specificities
1. Gomori-methenamine silver stain : ( either in BAL or induced sputum or lung tissue)it has sensitivity of 81 and specificity of 97%
2. Immunfluorescent staining,: ( either in BAL, induced sputum or tissue ) it has sensitivity of 84% and specificity of 97%
3. Polymerase chain reaction : (either in Sputum BAL, mouth wash or nasopharyngeal secretions or Serum )it has sensitivity of 98% and specificity of 96%
4. (1,3)-beta-D-glucan : ( Serum) sensitivity of 94 sensitivity of 82%
5. lactate dehydrogenase ( Serum ) it has sensitivity of 80.7% and specificity of 40.5%
place of lung Biopsy
It is invasive but provide tissue sample that if examined has the 100 sensitivity and specificity
It comes after bronchoscopy if the bronchoalveolar lavage sample is not conclusive and clinical picture carry high probability of PJP
Ref
Marjorie Bateman, Rita Oladele, and Jay K Kolls: (2020) Diagnosing Pneumocystis jirovecii pneumonia: A review of current methods and novel approaches; Med Mycol. 2020 Nov; 58(8): 1015–1028.
Yes, I appreciate that biopsy is an option ONLY if BAL is inconclusive
-Diagnostic tests, sensitivities & specificities
Lab:
Imaging:
CXR
HRCT
Other non-invasive test
Pulmonary function test
Pulse oximetry
HIV test
Invasive Procedure:
BAL:
-What is the place of lung biopsy in the diagnosis of PJP?
Open lung biopsy:
Source:
Thank you for your reply
wlcm, prof
What are the modalities used in the diagnosis of this disease?/ their sensitivities and specificities?
The lung biopsy showed PJP cysts in lung tissue (see below for stains)
Imaging:
1. Chest x-ray (Abnormal in 92–96%)
2. CT chest: More sensitive than routine chest radiography
Diagnostic specimen:
3. Induced Sputum: the sensitivity is 30-55%. Collected after 20 to 30 minutes of exposure to aerosolized hypertonic saline or water, or after oral hydration
4. Bronchoalveolar Lavage: sensitivity is 80-95%. Lower sensitivity in patients receiving aerosolized pentamidine
5. PCR testing of samples: Sensitivity and specificity vary depending on manner of sampling (sputum vs. BAL) and assay employed
6. Transbronchial biopsy
7. Open Lung biopsy or videoassisted thorocoscopy (VATS): when bronchoscopy findings are nondiagnostic. 100% sensitivity and specificities
8. Other Respiratory specimens: includes sputa and upper respiratory samples (nasopharyngeal aspirates, nasal or oral washes). Not a good alternative, low organism burden
Diagnostic technique:
1. Immunofluorescence assays: most sensitive microscopic diagnostic method
2. Real-time quantitative PCR, nucleic acid testing of BAL: increase sensitivity but cannot distinguish infection from carriage
3. Silver, polychrome, or calcofluor stains: exclusion of PJP by negative BAL only
o Direct fluorescent antibody staining using a fluorescein-conjugated monoclonal antibody can visualize both trophic forms and cysts
o Stains for trophic forms (the most common form of the organism in the alveolus) includes Gram-Weigert, Wright Giemsa or modified Papanicolaou stains
o Cysts can be stained with calcofluor white, cresylecht violet, Grocott Gomori-methenamine silver or toluidine blue
Serum:
1. LDH: elevated in almost all cases of PJP (over 300 IU/ml)
2. β-D-glucan: high sensitivity (>90%) with lower specificity (less than 80%)
Genotyping, sequencing: Investigation of suspected outbreaks
What is the place of lung biopsy in the diagnosis of PJP
o Transbronchial, open lung or videoassisted thorocoscopy (VATS) biopsy
o When other diagnostic fails (induced sputums and BAL)or where other concomitant diseases may be a concern
o Open lung biopsy often considered to be a gold standard, but early patchy disease may decrease yield (Video-assisted thoracoscopic (VATS) biopsies may be appropriate for some patients)
References
1. Shenoy P, Buttigieg J, Zayan T, Sharma A & Halawa A. (2018) Infections after Solid Organ Transplantation. J Renal Transplant Sci, 1(1): 29-42.
2. Fishman JA, Gans H; AST Infectious Diseases Community of Practice. Pneumocystis jiroveci in solid organ transplantation: Guidelines from the American Society of Transplantation Infectious Diseases Community of Practice. Clin Transplant. 2019 Sep;33(9):e13587. doi: 10.1111/ctr.13587. Epub 2019 Jul 1. PMID: 31077616.
3. Martin, S.I., Fishman, J.A. and (2013), Pneumocystis Pneumonia in Solid Organ Transplantation. American Journal of Transplantation, 13: 272-279.
Thank you for your reply
Microscopy with staining — Detection of the organism in respiratory specimens is most commonly achieved by microscopy with staining of an induced sputum specimen or BAL fluid Staining is necessary because Pneumocystis cannot be cultured. (Up to date)
Direct fluorescent antibody staining using a fluorescein-conjugated monoclonal antibody can visualize both trophic forms and cysts and is the most common technique used.
Type of respiratory specimen — The most rapid and least invasive method of diagnosing PCP is by analysis of sputum induced by the inhalation of hypertonic saline. If PCP is not identified by this modality, then bronchoscopy with BAL should be performed. Lung biopsy with tissue stains and PCR has excellent sensitivity for diagnosing PCP but is rarely required
The diagnostic yield of microscopy with staining of induced sputum is 50 to 90 percent in patients with HIV and PCP but is thought to be lower in patients without HIV due to a decreased organism burden.
The diagnostic yield of BAL is over 90 percent in patients with HIV but may be lower in patients without HIV
Polymerase chain reaction — A number of PCR assays have been developed for the detection of Pneumocystis in induced sputum or BAL fluid, blood, or nasopharyngeal aspirates. These assays may be of particular use in patients without HIV, in whom the sensitivity of microscopy with staining is substantially lower than in patients with HIV.
Nested PCR strategies identify both colonization and infection.
In contrast, single-copy real-time PCR assays rapidly identify patients with infection rather than colonization and hence are more useful in clinical settings.
Beta-D-glucan assay — Beta-D-glucan is a cell wall component of all fungi, including Pneumocystis. A serum assay for beta-D-glucan is available and can be used to screen for a variety of invasive fungal infections. Although this test has been best studied for Candida and Aspergillus spp, it may also have utility for diagnosing PCP
The sensitivity and specificity of this test increases for a higher city of values.
Using a cut-off of 80 pg/mL, the assay had a sensitivity and specificity of 70 and 81 percent, respectively. The specificity of the assay rose to 100 percent when a >200 pg/mL cut-off was used.
Although the beta-D-glucan assay is not specific for PCP, it is useful while awaiting an induced sputum or BAL specimen for microscopy with staining for PCP or in situations in which respiratory sampling such as BAL cannot be performed safely.
Lung biopsy for diagnosis is generally reserved for patients in whom there is a high suspicion of PCP and in whom BAL testing has been negative or in those who have another reason to proceed to lung biopsy for diagnosis of a pulmonary process.
Thank you for your reply
What are the modalities used in the diagnosis of this disease? What are their sensitivities and specificities? What is the place of lung biopsy in the diagnosis of PJP?
1- Respiratory specimen
Because the cough is dry, so obtaining an optimal specimen by ordinary cough may be impossible, so it should be done one of the following ways:
2- Beta-D-glucan assay
3- Lung biopsy with tissue stains and PCR
References
1- Pifer LL, Hughes WT, Stagno S, Woods D. Pneumocystis carinii infection: evidence for high prevalence in normal and immunosuppressed children. Pediatrics 1978; 61:35.
2- Catherinot E, Lanternier F, Bougnoux ME, et al. Pneumocystis jirovecii Pneumonia. Infect Dis Clin North Am 2010; 24:107.
3- Willocks L, Burns S, Cossar R, Brettle R. Diagnosis of Pneumocystis carinii pneumonia in a population of HIV-positive drug users, with particular reference to sputum induction and fluorescent antibody techniques. J Infect 1993; 26:257.
4- Alvarez F, Bandi V, Stager C, Guntupalli KK. Detection of Pneumocystis carinii in tracheal aspirates of intubated patients using calcofluor-white (Fungi-Fluor) and immunofluorescence antibody (Genetic Systems) stains. Crit Care Med 1997; 25:948.
5- Levine SJ, Kennedy D, Shelhamer JH, et al. Diagnosis of Pneumocystis carinii pneumonia by multiple lobe, site-directed bronchoalveolar lavage with immunofluorescent monoclonal antibody staining in human immunodeficiency virus-infected patients receiving aerosolized pentamidine chemoprophylaxis. Am Rev Respir Dis 1992; 146:838.
6- Onishi A, Sugiyama D, Kogata Y, et al. Diagnostic accuracy of serum 1,3-β-D-glucan for pneumocystis jiroveci pneumonia, invasive candidiasis, and invasive aspergillosis: systematic review and meta-analysis. J Clin Microbiol 2012; 50:7.
7- Tasaka S, Hasegawa N, Kobayashi S, et al. Serum indicators for the diagnosis of pneumocystis pneumonia. Chest 2007; 131:1173.
8- Morjaria S, Frame J, Franco-Garcia A, et al. Clinical Performance of (1,3) Beta-D Glucan for the Diagnosis of Pneumocystis Pneumonia (PCP) in Cancer Patients Tested With PCP Polymerase Chain Reaction. Clin Infect Dis 2019; 69:1303.
9- Alanio A, Hauser PM, Lagrou K, et al. ECIL guidelines for the diagnosis of Pneumocystis jirovecii pneumonia in patients with haematological malignancies and stem cell transplant recipients. J Antimicrob Chemother 2016; 71:2386.
Excellent Sherif
Thanks a lot
What are the modalities used in the diagnosis of this disease?
· Bronchoalvelor lavage (BAL) Gold standard for PJP.
· Induced sputum
· Trans bronchial biopsy
· Video-assisted thoracoscopy VATs
· Blood
Diagnostic Technique
1. Immunofluorescence assays
2. Real time PCR
3. silver, polychrome stains
What are their sensitivities and specificities?
Modality Sensitivity Specificity
Induced sputum1 99% 96%
Induced sputum cytological staining[1] 50% 100%
Fluorescent antibody testing[1] 74% 100%
beta-D-glucan[2] (using cut off 31.1pg/ml) 92% 86%
PCR[3] 87.2% 92.2%
PCP PCR has high sensitivity but cannot distinguish colonization from infection
What is the place of lung biopsy in the diagnosis of PJP?
Trans-bronchial lung biopsy has lower sensitivity compared modalities
References
1. Senécal J, Smyth E, Del Corpo O, Hsu JM, Amar-Zifkin A, Bergeron A, Cheng MP, Butler-Laporte G, McDonald EG, Lee TC. Non-invasive diagnosis of Pneumocystis jirovecii pneumonia: a systematic review and meta-analysis. Clin Microbiol Infect. 2022 Jan;28(1):23-30. doi: 10.1016/j.cmi.2021.08.017. Epub 2021 Aug 28. PMID: 34464734.
2. UpToDate
3. Fan LC, Lu HW, Cheng KB, Li HP, Xu JF. Evaluation of PCR in bronchoalveolar lavage fluid for diagnosis of Pneumocystis jirovecii pneumonia: a bivariate meta-analysis and systematic review. PLoS One. 2013 Sep 4;8(9):e73099. doi: 10.1371/journal.pone.0073099. PMID: 24023814; PMCID: PMC3762835.
Thank you for your reply
What are the modalities used in the diagnosis of this disease?
A) Radiological, suffice to write that, there is no radiological feature that is pathognomonic of PJP infection. However, the following findings have been documented:
B) Microbiology. The clinical diagnosis depends largely on microscopic visualization of the PJP organism in the respiratory specimens (sputum, BAL, Lung tissues) following chemical or immunofluorescence.
– Conventional stain used on specimen
Other stains that detect sporozoites and trophozoites but do not stain the cyst wall
-Immunofluorescence which uses staining via monoclonal antibodies to pneumocystis, when induced sputum or BAL is used is the diagnostic modality of choice
What are the sensitivities and specificity of the test
A multicentre study done to compare direct immunofluorescence with conventional stain (Gomori methenamine, Calcofluor white, and Diff- Quik) with 313 sputum samples obtained by BAL and the outcomes are:
The PCR test allows a higher sensitivity than immunofluorescence and conventional stains with lower organism load even using induced sputum or oral wash instead of BAL. It is also good for quantification of the fungal load
The limitation of PCR is that, it can not distinguish between infection and colonization.
The next generation sequencing test is 100% sensitive and 93.4% specific
The place of lung biopsy in diagnosis of PJP
It is the gold standard test and it could have a yield in of 90% in infected patient. However, it is an invasive test that will require expert and a lot of logistics to obtain smaples
References
Thank you for your reply
What are the modalities used in the diagnosis of this disease?
Biopsy with slide staining by Gomori Crockott (silver) – this case
Sputum secretion – 50%
Bronchoalveolar lavage – 95%
Transbronchial biopsy – 95 – 100%
What are their sensitivities and specificities?
95 – 100% – this case
What is the place of lung biopsy in the diagnosis of PJP?
Pulmonary alveolus and interstitial space
Thank you, Filipe
What about the antigen test?
Professor, we have no experience with the antigen test for Pneumocystosis. We use it for Histoplasmosis and the more immunosuppressed the patient is, the better the sensitivity and specificity.
·What are the modalities used in the diagnosis of this disease?
Sampling modalities:
2.Induced sputum
3.Nasopharyngeal aspirate
4.Oral wash
5.Blood
Testing modalities:
=============================
·What are their sensitivities and specificities?
=============================
·What is the place of lung biopsy in the diagnosis of PJP?
References
Thank you for your reply
o Pneumocystis cannot be cultured. Therefore, the diagnosis relies upon the visualization of the cystic or trophic forms in appropriate specimens.
o Different stains can be used to visualize the organism, like Gomori-methenamine silver, cresyl violet, Gram-Weigert and toluidine blue O, which stain the cell wall of the cystic form. On the other hand, Wright-Giemsa and Diff-Quick detect both the cystic and trophic forms but do not stain the cell wall.
o Immunofluorescent staining using fluorescein-labelled monoclonal antibodies represents the preferred technique for the diagnosis of PJP and is more sensitive than the general stains (1, 2).
o Polymerase chain reaction (PCR) of respiratory fluid, in particular bronchoalveolar lavage (BAL), is increasingly popular as it has the advantage of higher sensitivity in detecting PJP in clinically suspected cases with negative sputum or BAL smears (sensitivity was 100%, and specificity 97.2%). Furthermore, it can be used to examine other specimens like oral washes and nasopharyngeal aspirates (3). Nevertheless, PCR cannot distinguish between colonization and disease (4).
If sputum induction, BAL, and a transbronchial biopsy are nondiagnostic or cannot be performed, more invasive techniques may be necessary to diagnose PCP. Options include transthoracic needle biopsy or lung biopsy performed either via thoracotomy or by video-associated thoracoscopic surgery. These procedures have significant risks. Therefore, the cost-benefit evaluation should be weighed carefully in each case.
Lung biopsy, either by thoracotomy or by video-assisted thoracoscopic surgery, can be performed with a sensitivity of 95 to 100 per cent for the diagnosis of PCP (5).
Histology on lung biopsy demonstrates the formation of a foamy, eosinophilic alveolar exudate; severe cases are associated with oedema and interstitial fibrosis (5).
References:
1) Catherinot E, Lanternier F, Bougnoux ME, et al. Pneumocystis jirovecii Pneumonia. Infect Dis Clin North Am. 2010;24(1):107.
2) Miller RF, Huang L, and Walzer PD. Pneumocystis pneumonia associated with human immunodeficiency virus. Clin Chest Med. 2013;34(2):229.
3) Bossart S, Mühlethaler K, Garzoni C, et al. Is real time PCR preferable to the direct immunofluorescence in the diagnosis of Pneumocystis jirovecii pneumonia in HIV-infected patients?. BMC Res Notes. 2020;13(1):235.
4) Huang L, Cattamanchi A, Davis JL, et al. HIV-associated Pneumocystis pneumonia. Proc Am Thorac Soc. 2011;8(3):294.
5) Paul E Sax. Clinical presentation and diagnosis of Pneumocystis pulmonary infection in patients with HIV. © 2023 UpToDate (accessed on 18 February 2023).
Thank you for your reply
The picture is for PCP (Pneumocystis pneumonia caused by Pneumocystis Jirovecii) diagnosed by lung biopsy
What are the modalities used in the diagnosis of this disease?
The best way to make the diagnosis of PJP pneumonia is to perform a Gomori Methenamine Silver (GMS) Stain on Lung tissue or BAL Fluid,
The Cyst wall is stained and the organisms appear as crushed ping-pong balls, or crescent shapes, or folded spheres, or flattened beach balls, or deflated tennis balls as appeared in attached slide
PJP Diagnosis :
Identification of organisms at the site of infection:
– PCR of Respiratory Fluids
– Gomori Methenamine Stain for cysts
– Cytopathology for trophozoites /Cysts
What are their sensitivities and specificities?
– Induced Sputum: Sensitivity < 50-90%
– BAL : Sensitivity 90-99%
– Lung Biopsy : Sensitivity 95-100%
Non- Invasive Tests:
Beta- Glucan : Sensitivity 92.8%, Specifity : 75%, PPV 36.3%, NPV 60.0%
PCR : Infection vs Colonization ?
LDH : Non Specific , Prognostic ?
What is the place of lung biopsy in the diagnosis of PJP?
References:
•Lecture by Prof Gamal Saadi
Thank you for your reply
1- BAL fluid sample is of choice
2-induced sputum simple and cost effective and time saving and less invasive than bronchoscopy
3- oral washing or aspiration are highly sensitive
4- An immunofluorescence assay with anti- Pneumocystis antibodies is commercially available: it provides the best specificity and sensitivity of the general stains and some immunofluorescent monoclonal antibodies can reveal both trophic and asci forms. Before the development of PCR, this method represented the “gold standard” technique for the diagnosis of PCP.
5-real time PCR highly sensitive to diagnose missing infection and more specific than conventional PCR
6- Although microscopic methods are often sufficient to diagnose P. jirovecii in BAL fluids from AIDS patients, the sensitivity of these methods is often too low to diagnose PCP in non-HIV immunocompromised patients. In SOT recipients, molecular diagnostic methods are required to avoid missing a Pneumocystis infection in this population.
7-lung biopsy not used
references:
xavier Iriart, Marine Le Bouar, Nassim Kamar, Antoine Berry. Pneumocystis Pneumonia in Solid-Organ Transplant Recipients. J. Fungi 2015, 1(3), 293-331.
Thank you for your reply
What are the modalities used in the diagnosis of this disease?
Diagnosis of PCP include-
clinical suspicion
patient risk factors
laboratory evaluation- raised LDH, Elevation of serum beta-D-glucagon. An arterial blood gas analysis -show an elevated Alveolar-arterial (A-a) oxygen gradient in the setting of PCP
chest radiograph – diffuse bilateral peri-hilar interstitial infiltrates. These changes become increasingly homogenous as the disease course progresses.
Other radiographic findings include-
solitary or multiple nodules, which may progress to cavitary lesions
lobar infiltrates such as upper lobe lesions
pneumothorax
chest computed tomography (CT) – ground glass attenuation or cystic lesions with high sensitivity.
sputum studies, evaluation of bronchoalveolar lavage fluid- polymerase chain reaction assays of respiratory specimens, dye staining, or fluorescein antibody staining
lung biopsies.
What are their sensitivities and specificities?
Chest xray is less sensitive and specific.
LDH raised has a sensitivity of 70-90%
PCR in BAL has a sensitivity of 90%
Open lung biopsy has a sensitivity of 100%
What is the place of lung biopsy in the diagnosis of PJP?
Sometimes, a small sample of lung tissue (a biopsy) is used to diagnose PCP. The patient’s sample is sent to a laboratory, usually to be examined under a microscope. Polymerase chain reaction (PCR) can also be used to detect Pneumocystis DNA.
Pneumocystis Jirovecii Pneumonia
Justina Truong