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Indication - Pneumocystis pneumonia
Pneumocystis jirovecii nucleic acid test
Facility level:
2. Laboratory
Assay formats
NAT
Status history
First added in 2020
Purpose type
Aid to diagnosis
Purpose
To aid in the diagnosis of Pneumocystis pneumonia
Specimen types
Respiratory specimens (sputum, bronchoalveolar lavage fluid)
WHO prequalified or recommended products
N/A
Codes
ICD11 code: CA40.20

Summary of evidence evaluation

The guidelines related to the test used rigorous methods, and the recommendations were based on evidence varying from low to moderate quality. The meta-analysis suffered from high heterogeneity, but the confidence intervals were rather small. The primary studies selected children hospitalized with suspected PCP in a prospective manner and using valid reference standards. All patients were included in the analysis. The challenge is that PCR seems to detect more PCP cases than the current reference standard. In summary, the test was evaluated in various subgroups of patients on various specimens. No recommendations in guidelines were made specifically for children, and only two primary studies in children were added to the evidence portfolio. With the sensitivity of the test at more than 97%, it is safe to rule out patients to avoid inappropriate use of cotrimoxazole. But the test cannot distinguish infection from carriage, which might hamper clinical usefulness. The full evidence review for this test category is available online at: https://www.who.int/medical_devices/diagnostics/selection_in-vitro/selection_in-vitro-meetings/new-prod-categories_3

Summary of SAGE IVD deliberations

PCP is a significant public health problem, whose incidence in LMICs is closely linked with the burden of HIV disease and, more specifically, the proportion of patients who still present with advanced disease. P. jirovecii cannot be cultured and has historically been diagnosed by stains or antibody-based microscopy, with limited sensitivity. Molecular assays are valuable in confirming P. jirovecii as the causative agent of pneumonia, when combined with clinical signs and symptoms. In these cases, the assays are also reliable for excluding PCP and thus preventing inappropriate use of cotrimoxazole. But current tests are not inexpensive; and they are only appropriate for centralized testing. SAGE IVD also noted that while the submission broadly covers NAT formats, the evidence base provided is predominantly for PCR tests. Group members noted that LAMP tests tend not to be as sensitive as PCR, although LAMP tests do have advantages, particularly in LMICs. In considering the use of PCR tests, SAGE IVD raised some concerns about the potential for variability in results. Some procedures use conventional PCR, while others use real-time PCR. And the specimen type includes a broad range of respiratory tract specimens, from sputum to BAL to tracheal secretion. Diagnostic accuracies vary with both specimen and technology type. The group confirmed, however, that this potential for variability in results applies to many other conditions where different PCR types and specimens are used. SAGE-IVD also acknowledged that addressing quality assurance and standardization issues is beyond the scope of the EDL (although the group emphasized that this should be done as and when countries establish and implement their own national EDLs).

SAGE IVD recommendation

SAGE IVD recommended including the P. jirovecii test category in the third EDL: • as a disease-specific IVD for use in clinical laboratories (EDL 3, Section II.b); • using a nucleic acid test format; • to aid in the diagnosis of Pneumocystis pneumonia. The group requested the addition of a note to the test category entry in the EDL stating that it is particularly relevant in immunocompromised patients.

Details of submission from 2020

Background

PCP is a life-threatening illness of largely immunosuppressed patients, such as those with HIV/AIDS (1). When diagnosed rapidly and treated, survival rates are high. The etiologic agent of PCP is P. jirovecii, a human-only, commensal fungus that does not grow on microbiological agar. Other mammals have their own Pneumocystis species. AIDS-related PCP has a variable incidence: 5.9–55% in adults and 5–49% in children (1). In the 1980s, during the HIV pandemic, PCP was one of the most prevalent AIDS-defining diseases in HICs; this remains an issue in cases of undiagnosed HIV, particularly in LMICs (2). Untreated, the mortality is 100%. In HICs, the mortality is about 10% among AIDS patients, but it is about 30% in LMICs (1). Overall, the day 30 all-cause mortality is 28%, ranging from 10% to 62%; it is higher in HIV-negative patients due to fulminant disease (3). In addition to high-dose cotrimoxazole, adjunctive corticosteroids reduce mortality in AIDS in moderate and severe cases, but are unhelpful in non-AIDS cases. Second-line therapy requires clindamycin and primaquine. Does the test meet a medical need? The historical difficulties in diagnosing PCP (lack of culture and poor sensitivity of microscopic investigation) have resulted in many cases being diagnosed on clinical suspicion (3). Clinically, signs are non-specific. Typically, bilateral chest infection presents with respiratory signs of distress, which can be mild in the HIV positive but are typically more severe in HIV-negative. Radiological signs can be absent in the early stages; when present, although typical of PCP, they are generally non-specific (bilateral ground-glass opacification, leading to consolidation) and could also represent an underlying condition (4, 5). With the advent of modern diagnostic techniques – real-time PCR and (1-3)-β-D-glucan (BDG) – reliable laboratory-based diagnosis can now be achieved. Cotrimoxazole (trimethoprim-sulfamethoxazole) is the most effective agent for both preventing and treating PCP. In the absence of laboratory diagnosis, cotrimoxazole is instituted empirically and a 3-week course prescribed. If a precise laboratory diagnosis is made, empirical and possibly inappropriate use of cotrimoxazole could potentially be prevented. How the test is used The current gold standard for confirming a diagnosis remains histological and/or microscopic identification of cysts and trophic forms of P. jirovecii in clinical specimens, usually respiratory samples using conventional or immunofluorescent (IF) antibody stains. Standard microscopic investigation is highly subjective; this can influence specificity and sensitivity. While IF microscopy improves sensitivity, it is still suboptimal (67%). PCP PCR is primarily a diagnostic test. It is used in conjunction with a lactate dehydrogenase (LDH) test, oxygen saturation and radiology. Detection of Pneumocystis DNA in blood is a poor prognostic feature.

Public health relevance

Prevalence There are an estimated 400 000 PCP cases in AIDS patients annually and more than 100 000 in patients with other immunosuppressive diseases (6). In addition to patients diagnosed with HIV, an ever increasingly susceptible HIV-negative population is at risk of PCP (3, 7–11). Those at increased risk include patients with solid tumours or suffering from haematological malignancy, solid organ transplant recipients, patients with autoimmune and inflammatory conditions receiving immunomodulating therapies (e.g. high-dose corticosteroids or anti-TNF therapy) and patients diagnosed with primary immune deficiencies (11, 12). Generally, the incidence of disease is low (8–10). But the epidemiology of PCP is changing, associated with more aggressive immunosuppressive and immunomodulatory approaches when managing auto-immune conditions (RA and vasculitis), pre-existing respiratory conditions (COPD), and other conditions (haematology and transplants, particularly renal transplants) (3, 7). Socioeconomic impact In a recent analysis in the USA, mean inpatient costs for PCP patients on various insurance plans were US$ 23 342–63 388 per visit; for outpatients they were US$526–1061 per visit (13).

WHO or other clinical guidelines relevant to the test

The following organizations all have guidelines relevant to the test: • British Society for Medical Mycology 2015 (14) • Infectious Diseases Working Party of the German Society of Hematology and Medical Oncology (DGHO) 2015 (15) • Fifth European Conference on Infections in Leukaemia (ECIL-5) 2016 (16) • American Society of Transplantation Infectious Diseases Community of Practice 2019 (17) • CDC, the National Institutes of Health and the HIV Medicine Association of IDSA 2019 (18). ECIL-5 is a joint venture of the European Group for Blood Marrow Transplantation, the European Organisation for Research and Treatment of Cancer, the Immunocompromised Host Society and the European LeukemiaNet.

Evidence for diagnostic accuracy

PCP PCR is a semi-quantitative or quantitative test, validated by various manufacturers against sputum, BAL and other respiratory specimens. Measures of diagnostic accuracy considered therefore include accuracy of various thresholds to differentiate infection versus colonization. Meta-analytical reviews of PCP PCR on respiratory samples generate excellent sensitivity of ≥ 97%; the subsequent NPV of ≥ 99% is sufficient to rule out PCP when PCR is negative (19–21). Despite the detection of possible Pneumocystis colonization, positivity in respiratory samples readily confirms disease as shown by positive likelihood ratios of ≥ 10. PCR testing has been applied to a range of specimen types (BAL fluid, blood). PCR testing of BAL fluid is preferred, but positivity in upper airway samples (sputum, induced sputum, oral washes and nasopharyngeal aspirates), once thought to represent detection of transient colonization, likely reflects a significant burden lower in the respiratory tract and is specific for PCP (16). Expectorated and induced sputum samples have been studied in Africa and have good sensitivity (22, 23). In Namibia, 475 samples were analysed and 25 (5.3%) samples were positive for P. jirovecii; 17 (3.6%) using both real-time PCR and Grocott’s methenamine silver (GMS) staining and eight (1.7%) using real-time PCR only (22). P. jirovecii was present in 8/150 (5.3%) HIV-positive and TB smear-negative patients, and in 12/227 (5.3%) TB smear-negative patients with an unknown HIV status. In South Africa, P. jirovecii was identified in 51% (156/305) and 67% (204/305) specimens using immunofluorescence and real-time PCR, respectively (23). The cut-off value for the real-time PCR that best predicted the reference IFA results was 78 copies/5 μL (area under ROC curve 0.92). The sensitivity and specificity of real-time PCR using this cut-off was 94.6% and 89.1%, respectively, compared with the IFA. In children, PCP PCR is invaluable for diagnosis. Morrow et al. (24) studied 202 children (median age 3.2 months) in South Africa, including 124 (61.4%) who were infected with HIV. They identified PCP in 109 (54%) children using PCR, compared with 43 (21%) using IFA and GMS (P < 0.0001). Most PCP cases (88, 81%) occurred in HIV-infected children. All 21 cases (19%) occurring in HIV-negative children had another risk factor for PCP. Das et al. (25) investigated 94 immunocompromised children with pneumonia for PCP in India. PCR detected P. jirovecii in 14 children. The occurrence of PCP in HIV-infected children was 43% (6/14), renal disease on immunosuppressants 45% (4/9), primary immune deficiency 19% (2/11) and malignancies on chemotherapy 4% (2/57). Wang et al. (26) suggest that the presence of Pneumocystis DNA in blood samples is a poor prognostic marker. Detection of DNA in the plasma of HIV-positive patients was significantly higher in deceased patients (79%) compared with survivors (14%), as was the burden of disease (deceased: 54 610 copies/mL vs survivors: 935 copies/mL. Choi et al. (27) use PCR to determine prognosis. In their study of 81 HIV-negative PCP patients with respiratory failure that were initially PCR positive, PCP PCR-negative conversion was associated with a good prognosis, generating a hazard ratio of 0.433 (95% CI: 0.203–0.928, P = 0.031). Performance of PCR may vary between HIV-positive and HIV-negative patients. The lower burden encountered in HIV-negative patients may lower sensitivity, but PCR specificity remains high (16). Nevertheless, PCR negativity when testing BAL fluid can be used to exclude disease irrespective of the underlying condition (ECIL guidelines level of evidence: II; grade of recommendation: A).

Evidence for clinical usefulness and impact

Almost no real-life studies of the role of PCP PCR in LMICs have been done because the test is not generally available in these countries. Oladele et al. (28) reviewed the topic but did not do a systematic review, as this is not possible with the current state of knowledge.

Evidence for economic impact and/or cost–effectiveness

No reviews or primary studies of the economic impact or cost–effectiveness of the PCP PCR test are available. The price per test (excluding control testing, human resource and processing costs) varies. The development of the first freeze-dried assay using loop-mediated isothermal amplification (LAMP) technology is expected to greatly reduce shipping cost and enable transport to centres without a major airport. While the price per assay or the equipment cost cannot be determined, it is notable that no extraction system is required for this test format; the result is available in 25 minutes.

Ethical issues, equity and human rights issues

The test is for identifying and excluding PCP in vulnerable immunocompromised and HIV-infected individuals; it raises no concern for equity or breach of human rights.
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