Pneumocystis jirovecii pneumonia (PcP) remains a major cause of respiratory illness among immunosuppressed patients. PcP is difficult to diagnose, in particular in non-HIV-infected patients due to the lack of specific clinical data associated. Since P. jirovecii could not be cultivated for many years, microscopic visualization of cysts or trophic forms in respiratory specimens based on cytochemical or immunofluorescence stainings are the standard procedures to identify this fungus. Polymerase chain reaction (PCR)-based methodologies have been developed to overcome the low sensitivity of microscopy in respiratory specimens, especially those with low fungal load, and in non-HIV-infected patients. Real-time quantitative PCR is the only format suitable for diagnosis since the risk of contamination is minimal and quantification is possible. Quantitative results have been used to differentiate PcP (high fungal load) from carriage/colonisation (low fungal load); however, this is inconclusive and has limited results in intermediate fungal loads. New strategies based on the measurement of blood biomarkers could be used to perform PcP diagnosis noninvasively. Several studies explored the usefulness of candidate serum biomarkers, such as (1–3)-β-d-Glucan (BG), Krebs von den Lungen-6 antigen (KL-6), lactate dehydrogenase (LDH) or S-adenosylmethionine (SAM), with the former presenting the most promising results. After approximately three decades of intense research, PcP diagnosis (detection processes and interpretation) remains a challenge for both microbiologists and clinicians.
Chapter 13
Laboratory diagnosis of Pneumocystis jirovecii pneumonia
O. Matos*
F. Esteves**
* Medical Parasitology Unit, Group of Opportunistic Protozoa/HIV and Other Protozoa, Global Health and Tropical Medicine, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Portugal
** Department of Genetics, Toxicogenomics & Human Health (ToxOmics), NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Portugal
Abstract
Keywords
Pneumocystis jirovecii
pneumonia
diagnosis
current methods
new alternatives
1. Introduction
Pneumocystis jirovecii (formerly Pneumocystis carinii f. sp. hominis) is an atypical fungus exhibiting pulmonary tropism and highly defined host specificity. The story of Pneumocystis began in Brazil in 1909 where Carlos Chagas, a young physician working at Oswaldo Cruz Institute, in Rio de Janeiro, mistakenly identified Pneumocystis as a stage of the life cycle of Trypanosoma cruzi, in the lungs of guinea pigs.1 In 1910, Antonio Carini, an Italian scientist living in the city of São Paulo also in Brazil, found similar cysts in the lungs of Rattus norvegicus, but doubted that the cysts were a part of the life cycle of T. cruzi.2 Because of this doubt, he sent his data and specimens to the Pasteur Institute in Paris, where in 1912, the researcher duo of Pierre and Marie Delanoë described the organism identified as a new biological entity, and suggested naming it Pneumocystis carinii because cysts were found only in the lungs of hosts and Carini had provided the specimens for study.3
In the early 20th century, this pathogen was considered an enigmatic lung pathogen, but the year 1981 became a turning point where P. carinii, was no longer seen as a relatively obscure human pathogen but became one of the leading causes of death in the global epidemic of acquired immunodeficiency syndrome (AIDS).4
Classified at first as a protozoan, it was latter reclassified as a fungus based on greater DNA sequence homology with fungal organisms.5,6 Pneumocystis infects a variety of mammalian hosts. The human form, because of its host specificity, was renamed Pneumocystis jirovecii in honor of Otto Jirovec, which linked it to epidemics of interstitial plasma cell pneumonia in neonates in Europe,7 while P. carinii is now reserved for the rat form of Pneumocystis.
Pneumocystis pneumonia (PcP or pneumocystosis) is an opportunistic disease with airborne transmission predominantly reported in patients with impaired immunity, mainly among human immunodeficiency virus (HIV)-infected persons.8,9 In developed countries, widespread use of PcP chemoprophylaxis and potent combination antiretroviral therapy (cART) have reduced the incidence of this pathology.10 Despite these advances, in the 21st century, PcP continues to be a serious problem for HIV-infected patients, especially for those who are undiagnosed or who are noncompliant with preventive medication.11 Recently, PcP has been reported as a serious emerging problem in non-HIV-infected persons who are undergoing immunosuppressive treatments related to malignancies, connective tissue diseases, or organ transplantation. Reports on pulmonary colonisation with P. jirovecii in patients presenting diverse levels of immunodeficiency, primary respiratory disorders, or even in the immunocompetent general population, is also an important epidemiological issue, especially in terms of transmission.9,12–14 In addition, in developing countries, where most HIV-infected persons reside, PcP is an emerging disease with high prevalence and is poorly controlled since the access to cART and PcP prophylaxis is still limited; and possibly also due to lack of PcP diagnostic resources and expertise.15–18
Therefore, prevention of PcP and control of existing cases through early diagnosis remains a very important objective from a public health point of view in all countries, and especially in the developing countries.
2. Laboratory diagnosis of PCP
Pneumocystis jirovecii is an opportunistic pathogen that is usually found in the lungs of humans, but which has also been found in extrapulmonary sites.19 In general, laboratory findings are less severe in HIV-infected patients than in non-HIV immunosuppressed patients.20
The presumptive diagnosis of PcP is based on: clinical manifestations, pulmonary function testing, arterial blood gas testing (ABG) at rest and after exercise, and nonspecific radiological and laboratory tests. The history and physical examination are part of the evaluation of the patient with pulmonary symptoms. Over the years it was noticed that the presentation of PcP depends on the underlying disease. Thus, in HIV-infected patients the duration of the clinical manifestations is longer and the diagnosis is more difficult than in patients with other immune deficiencies.21. Pulmonary manifestations are the most frequent in the natural history of PcP. Symptoms and signs typical of a patient with PcP are fever between 38 and 40°C in more than 80% of patients, nonproductive cough in more than 50% of patients which may be accompanied by sputum in 20–30% of the cases, progressively worsening dyspnea in more than 60% of patients, and sometimes discrete crackling rales. This condition occurs over a period of one to 3 weeks. In addition, tachypnea, tachycardia, and occasionally cyanosis can also be observed, but the auscultation may also be normal.22,23 The chest X-ray characteristics of a patient with PcP can, in 40–80% of cases, show a classic image of diffuse interstitial bilateral infiltrates. However, in 6–20% of the cases of PcP, the radiological patterns may be atypical or even normal.22,23 Taking into account that PcP is an interstitial disease, and as such, causes serious difficulties in gas exchange occurring in the lungs, another parameter used in the presumptive diagnosis of PcP is ABG at rest and after exercise. A partial pressure of oxygen (PaO2) in peripheral blood ≤ 9.3 kPa (70 mmHg) is indicative of PcP inspite of the fact that 10–20% of cases of PcP have normal levels of PaO2.23 The measurement of lactate dehydrogenase (LDH), which increases in the beginning phase of the infection, is a nonspecific laboratory test for PcP diagnosis that can be used as a prognostic tool, and to assess response to PcP therapy.24 In addition, the evaluation of the immune status of the patient is important to determine the risk of developing PcP. This disease occurs most frequently in patients with CD4+ T cells count ≤ 200/μL blood. This information applies to HIV-infected patients and to non-HIV-infected patients with other immunodeficiency, such as cancer patients receiving chemotherapy.
All these elements are useful but nonspecific, neither confirming or nor denying the diagnosis of PcP. For many years P. jirovecii could not be cultured—a culture system to propagate P. jirovecii in vitro was only developed in 2014. Since this culture system still needs to be validated, disseminated, and shown to be cost-effective for diagnostic purposes,25 microscopic visualization of cysts or trophic forms in respiratory specimens with cytochemical staining or immunofluorescent staining with monoclonal antibodies (IF/Mab) are the standard procedures to identify this microorganism and diagnose the disease.
2.1. Current methods for diagnosis of PCP
A comparative analysis of the most applied/studied laboratory diagnostic method for PcP diagnosis is summarized in Table 13.1. This table briefly shows the most important variables, such as sensitivity and specificity, operational and time costs, useful specimens, benefits, and disadvantages implicated in the laboratory diagnostic procedures available for P. jirovecii identification/detection and PcP diagnosis in a clinical laboratory.
Table 13.1
Laboratory Methods for PcP Clinical Diagnosis. Includes Worldwide Microscopic, Molecular and Serologic Standard Technical Procedures for P. jirovecii Identification/Detection
| Method | Technique | Sensitivity | Specificity | Estimated Cost/Sample USD (€) | Approximate Time Load (h) | Most Suitable Specimens (Also Available Specimens) | Observations | References |
| Microscopy | GMS | 79% (BALF) | 99% (BALF) | 112.1 (102.9) (BALF) | 3 | BALF or biopsy | Needs experienced/qualified microscopist; needs invasive and expensive samples; cumbersome protocol; identification of cysts; recommended combination with Giemsa or Giemsa-like stains; allows semi-quantification methods; optical microscope | [26] |
| TBO | 68% (BALF) | 100% (BALF) | 103.2 (94.7) (BALF) | 2 | BALF or biopsy | Needs experienced/qualified microscopist; needs invasive and expensive samples; identification of cysts; recommended combination with Giemsa or Giemsa-like stains; allows semi-quantification methods; optical microscope | [27] | |
| Giemsa (or DQ) | 68% (BALF) | 88% (BALF) | 104.2 (95.7) (BALF) | 1 | BALF or biopsy | Needs experienced/qualified microscopist (very difficult to read); needs invasive and expensive samples; rapid/easy protocol; identification of trophic forms and spores; recommended combination with GMS or TBO; allows semi-quantification methods; optical microscope | [28] | |
| IF | 97% (BALF) | 100% (BALF) | 110.7 (101.6) (BALF) | 2 | BALF, IS or biopsy | Excellent sensitivity/specificity (robustness); most accurate/robust microscopic method; easy to read; needs invasive and expensive samples; identification of cysts and/or trophic forms; allows semi-quantification methods; needs expansive/specific equipment (fluorescence microscope) | [29] | |
| Molecular | nPCR | 76–100% (BALF) | 53–86% (BALF) | 114.1 (104.7) (BALF) | 8 | BALF, IS or biopsy (OW, SS, NA) | Needs experienced/qualified staff; needs invasive and expensive samples; alternative non-invasive samples may be used; detection of low fungal burdens (eg, colonised patients); possible false positives; allows further genotyping; needs expensive/specific equipment (thermocycler) | [30] |
| RT-qPCR | 94–99% (BALF) | 89–96% (BALF) | 116.6 (107.0) (BALF) | 4 | BAL, IS or biopsy (OW, SS, NA) | Highthroughtput format; needs experienced/qualified staff; needs invasive and expensive samples; alternative non-invasive samples may be used; possible false positives; detection of low fungal burdens (eg, colonised patients); allows quantification; needs expensive/specific equipment (real-time apparatus) | [31] | |
| Serologic | BG | 91% (serum) | 77% (serum) | 26.1 (24.0) (serum) | 3 | Serum | Highthroughtput format; minimally invasive or inexpensive samples; suitable for screening; positive results in other fungal infections (false positives); recommended confirmation of results with GMS/Giemsa, TBO/Giemsa or IF; not quantitative; allows indirect quantification; needs expensive/specific equipment (microplate reader) | [32,33] |
| KL-6 | 72% (serum) | 79% (serum) | 26.6 (24.4) (serum) | 2.5 | Serum | Highthroughtput format; minimally invasive or inexpensive samples; positive results in other interstitial lung diseases (false positives); needs combination with GMS/Giemsa, TBO/Giemsa or IF; not quantitative; needs expensive/specific equipment (microplate reader) | ||
| LDH | 80% (serum) | 52% (serum) | 4.8 (4.4) (serum) | 2 | Serum | Highthroughtput format; minimally invasive or inexpensive samples; positive results in organ damage cases (false positives); very low specificity; needs combination with GMS/Giemsa, TBO/Giemsa or IF; not quantitative; low cost but needs expensive/specific equipment (microplate reader) | ||
| SAM | 68% (serum) | 52% (serum) | 14.3 (13.1) (serum) | 2 | Serum (plasma) | Highthroughtput format; minimally invasive or inexpensive samples; very low robustness/accuracy; needs combination with GMS/Giemsa, TBO/Giemsa or IF; not quantitative; needs expensive/specific equipment (microplate reader) | ||
| BG/KL-6 | 94% (serum) | 90% (serum) | 49.4 (45.4) (serum) | 5.5 | Serum | Highthroughtput format; minimally invasive or expensive samples; most accurate serologic method; suitable for screening; not quantitative; needs combination with GMS/Giemsa, TBO/Giemsa or IF; not quantitative; needs expensive/specific equipment (microplate reader) | ||
| BG/LDH | 97% (serum) | 72% (serum) | 30.9 (28.4) (serum) | 5 | Serum | Highthroughtput format; minimally invasive or expensive samples; needs combination with GMS/Giemsa, TBO/Giemsa or IF; not quantitative; needs expensive/specific equipment (microplate reader) | ||
| LDH/KL-6 | 89% (serum) | 74% (serum) | 31.4 (28.8) (serum) | 4.5 | Serum | Highthroughtput format; minimally invasive or expensive samples; low specificity; needs combination with GMS/Giemsa, TBO/Giemsa or IF; not quantitative; needs expensive/specific equipment (microplate reader) |
GMS, Grocott’s Methenamine Silver stain; TBO, Toluidine Blue O; DC, Diff-Quick; IF, immunofluorescence staining; RT-qPCR, real-time quantitative PCR; BG, (1-3)-β-d-Glucan quantification assay; KL-6, Krebs von den Lungen-6 antigen quantification assay; LDH, lactate dehydrogenase quantification assay; SAM, S-adenosylmethionine quantification assay; BG/KL-6, combination test using BG and KL-6 quantification assays; BG/LDH, combination test using BG and LDH quantification assays; LDH/KL-6 combination test using LDH and KL-6 quantification assays; BALF, bronchoalveolar lavage fluid; IS, induced sputum; OW, oropharyngeal washing; SS, spontaneous sputum; NA, nasopharyngeal aspirate.
Serologic combination tests (BG/KL-6, BG/LDH, LDH/KL-6) are considered positive when both biomarkers levels are indicative of PcP, negative when both biomarkers levels are below the cutoff level for PcP, and undetermined when either one of the two biomarker assays yields contradictory results. Estimated costs per sample include sample collection (BALF or serum) and laboratorial technical procedure. Approximate time load was estimated based on previous data.33
2.1.1. Biological specimens
The diagnosis of PcP is determined by cyto-histopathological examination of respiratory specimens obtained by invasive techniques, such as open lung biopsy (LB), transbronchial biopsy (TBB), bronchoalveolar lavage fluid (BALF), and bronchial secretions (BS), and specimens obtained by less invasive techniques such as spontaneous sputum (SS), nasopharyngeal aspirate (NA), and oropharyngeal washing (OW). Currently, BALF and IS are the most widely used clinical specimens for the diagnosis of PcP. In general, noninvasive tests should be tried in order to make the initial diagnosis, and invasive techniques should be used only when necessary and clinically feasible.34 Invasive diagnosis should be considered when dealing with non-HIV immunosuppressed patients with suspicion of PcP.
2.1.1.1. Specimens obtained by invasive methods
LB represents the gold standard laboratory diagnostic method in the assessment of lung inflammatory processes in immunosuppressed hosts.34 The diagnosis of lung tissue fragments allows the observation of the microorganism in more than 95% of cases of infection.35 Transbronchial biopsy (TBB) also allows the identification of P. jirovecii in 95% of cases, and should be considered mainly in non-HIV immunosuppressed patients with strong suspicion of PcP and negative BALF.36,37 Bronchoalveolar lavage (BAL) is performed by fiberoptic bronchoscopy and instillation of 150 mL of physiologic saline solution preheated at 37°C, divided into three syringes of 50 mL each, into the middle lobe and then slowly aspirating.38 According to some studies, the BALF analysis allows diagnosis in more than 80% of all patients with PcP, and in more than 95% of patients with HIV co-infection.37,39 It is, however, an expensive and invasive procedure, which involves secondary hazards, such as pneumothorax.40 Given its high sensitivity, this is the most frequently used technique in the diagnosis of fungal infections, particularly of PcP.41–44 The BS are obtained by the aspiration of secretions from the main bronchi of the bronchial tree during bronchoscopy. However, in some cases, there may be a need to instill sterile saline solution in order to assist in the removal of secretions and to stimulate coughing.45 This is especially performed in very young children.46
Sputum is a biological sample which may be obtained spontaneously by a noninvasive and inexpensive method that carries a low risk of complications, which, if any, are mostly transient and devoid of severity.47 The SS collected routinely for culturing bacterial and fungal agents, is rarely diagnostic in cases of PcP.48 In turn, induced sputum (IS) is based on the concept that changes in the microenvironment of the airways such as pH and osmolarity, as well as the activation of inflammatory mediators, can acutely increase secretions and make it possible to obtain specimens in patients who originally had unproductive cough.49 The IS is obtained by inhaling 1.8% saline with the aid of an ultrasonic nebulizer for 10–15 min, which promotes transudation and tracheobronchial exfoliation. Patients should have taken nothing by mouth for 6–8 h prior to induction.50 This is a less invasive method with less discomfort and risk for the patient than BALF collection, but it should be obtained only in infectious diseases clinics that carefully control sputum induction (having a single room with a ventilation system that allows for the total exhausting of air from the room to the external environment to avoid infectious droplets, if present, produced by coughing to be expelled into the room air).51 There are also contraindications for sputum induction, such as, recent history of pneumothorax, extreme asthenia, or active tuberculosis. Sputum induction for the diagnosis of PcP is widely used for patients with AIDS, but its utility for patients with other forms of immunosuppression is less defined. Non-HIV immunosuppressed patients with PcP have lower burden of organisms, and sputum induction may consequently have lower diagnostic yield in these patients.52 This technique allows Pneumocystis detection in 30–55% of cases of infection, after staining with nonspecific dyes, sometimes with difficult interpretation.34,50,53 The problem of low sensitivity can be overcome with IS liquefaction with dithiothreitol, followed by cell sedimentation and staining. Mucus liquefaction allows the concentration and better visualization of clusters of cysts and trophic forms of P. jirovecii existent in the specimen observed under the microscope.54 The application of immunofluorescence with monoclonal antibodies (MAb-IF) anti-P. jirovecii or PCR methodologies to liquefied IS, increases the sensitivity of detection from 60 to 97%.50,52,55 In addition to the risk of associated complications, bronchoscopy as well as sputum induction are expensive and they require specialized personnel, rooms, and equipment.
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