Diagnosis of aspergillosis by PCR: Clinical considerations and technical tips

Medical Mycology, 2018, 56, S60–S72 doi: 10.1093/mmy/myx091 Review Article Review Article Diagnosis of aspergillosis by PCR: Clinical considerations and technical tips 1 Cardiff University School of Medicine, Cardiff UK, 2 Public Health Wales Microbiology Cardiff, Cardiff, UK, 3 Western Sydney University, Sydney, Australia, 4 Clinical Microbiology, Trinity College Dublin, Ireland, 5 Center of Community Medicine and Infectious Diseases Service, ULSS 20 Verona, 6 Wuerzburg University, Wuerzburg, Germany and 7 Radboud University Medical Centre, Nijmegen, The Netherlands ∗ To whom correspondence should be addressed. Dr P. Lewis White. Public Health Wales, Microbiology Cardiff, UHW, Heath Park, Cardiff. UK. CF37 1EN. Tel: +44 (0)29 2074 6581; E-mail: Received 2 May 2017; Revised 19 July 2017; Accepted 29 August 2017; Editorial Decision 22 August 2017 Abstract Standardization of Aspergillus polymerase chain reaction (PCR) protocols has progressed, and analytical validity of blood-based assays has been formally established. It remains necessary to consider how the tests can be used in practice to maximize clinical utility. To determine the optimal diagnostic strategies and influence on patient management, several factors require consideration, including the patient population, incidence of invasive aspergillosis (and other fungal disease), and the local antifungal prescribing policy. Technical issues such as specimen type, ease of sampling, frequency of testing, access to testing centers, and time to reporting will also influence the use of PCR in clinical practice. Interpretation of all diagnostic tests is dependent on the clinical context and molecular assays are no exception, but with the proposal to incorporate Aspergillus PCR into the second revision of the consensus guidelines for defining invasive fungal disease the acceptance and understanding of molecular tests should improve. Key words: Aspergillus PCR, EAPCRI, Invasive aspergillosis. Introduction The European Aspergillus PCR initiative (EAPCRI) was formed with the aim of standardising Aspergillus polymerase chain reaction (PCR) methodology in order to determine accurate analytical performance and clinical validity (www.eapcri.eu). In doing so, it has permitted the incorporation of the standardized methodology into revised guidelines for defining invasive fungal disease (IFD), with the ultimate goal of improving the diagnosis and subsequent management of patients at risk S60 of IFD. The EAPCRI has made significant advances in standardising Aspergillus PCR testing of ethylenediamine tetra-acetic acid (EDTA)-whole blood, serum, and plasma, determining that nucleic acid extraction procedures were the rate-limiting step governing optimal PCR performance.1–5 A range of technical recommendations that depends on sample type have been published, but there is limited information on how best to use these and interpret results in clinical practice. This review will explore the implementation of molecular diagnostic  C The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: Rosemary A. Barnes1 , P. Lewis White2,∗ , C. Oliver Morton3 , Thomas R Rogers4 , Mario Cruciani5 , Juergen Loeffler6 and J. Peter Donnelly7 Barnes et al. strategies and interpretation of results in different clinical contexts. Clinical considerations The patient population based studies may overestimate disease as the denominator is already weighted towards disease.7,8 Broadly, patients can be divided into low, medium, and high risk (Table 1). It must be recognized that individual patients may move from one risk category to another, depending on the aggressiveness of chemotherapeutic interventions and response to treatment. The use of mould active prophylaxis, such as with posaconazole, may be expected to markedly reduce the risk in some patient groups. The strategy PCR can be used in two main ways: first, to rule out aspergillosis and second, to rule in a diagnosis of IA. Ruling out IA utilizes the high negative predictive value of the test.9 This can be refined further according to whether the test is used as a screening test in asymptomatic patients or as part of a fever-driven approach during febrile neutropenia that can markedly reduce empirical use of antifungal agents during refractory fever. For both approaches, frequent testing is required. When testing blood, specificity of both galactomannan and β-D-glucan is higher than for PCR, while the sensitivity of PCR is higher.3 This sensitivity confers the high negative predictive value (NPV) such that a negative test may allow the diagnosis to be excluded. Positives show good specificity, but the low prevalence of disease leads to a low positive predictive value in diagnosis of IA. It is increasingly recognized that PCR Table 1. Underlying clinical conditions and the associated risk of invasive aspergillosis. Condition (age limits) Hematological malignancy (>16 years) Acute myeloid leukemia and myelodysplastic syndrome Acute lymphatic leukemia Chronic myeloid leukemia Chronic lymphatic leukemia Lymphoma Hodgkin’s disease Multiple myeloma Aplastic Anaemia (1–75 years) Stem cell transplantation (NS) Autologous Allogeneic Solid organ transplantation (NS) Kidney/pancreas Liver Heart/lung Small bowel Critical Illness (NS) NS, not specified. Approximate incidence of disease % Risk category Reference 8–12 4–6 2.5 0.5 1 0.3 0.3 15 High Moderate Low Low Low Low Low High 47 47 47 47 47 47 47 48 2–6 5–26 Low High 49 49 0–4 1–7 1–15 0–10 0.3–6 Low Low/moderate Moderate/high Limited data Low 49 49 49 59 50 The performance of any test will be heavily influenced by the prevalence of disease in a population and for opportunist infections, such as invasive aspergillosis (IA), is largely determined by the presence of several well-established risk factors (Table 1). These include neutropenia, high-dose corticosteroid treatment, graft versus host disease, and genetic predisposition. Most studies have focused on adult patients with hematological malignancies and those undergoing hematopoietic stem cell transplantation at risk of invasive disease. Other patient groups include those with other malignancies, undergoing solid organ transplantation, or with critical illness requiring intensive care treatment, but data from these groups are more limited, with less performance data available in pediatric populations. Preliminary data suggest performance is comparable but that the incidence of disease tends to be lower in children than in adults. Overall, IA is an uncommon infectious disease and incidence is low, reportedly less than 5% in hematological malignancy.6 However, there is a wide range of reported prevalence determined by the presence of risk factors and study design. Cohort studies may underestimate prevalence due to difficulties with accurate diagnosis, whereas (...truncated)