Invasive Aspergillosis

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ASPERGILLUS PCR - FORMIDABLE CHALLENGES AND PROGRESS Lena Klingspor, MD PhD Karolinska Institute, Department of Laboratory Medicine , Division of Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden. Juergen Loeffler, University of Wuerzburg, Medical Hospital II, Wuerzburg, Germany Wednesday, January 16, 2008, 11:50 AM – 12:15 PM Invasive aspergillosis (IA,) are important causes of morbidity and mortality in immunocompromised patients, with an incidence of 4-10%, and with a mortality rate of 80-90%, in allogeneic stem cell transplant recipients. Conventional diagnostic tests, such as blood culture, show poor sensitivity for the detection of Aspergillus spp. Non-culture based techniques that has been used in the past, has lacked sensitivity and specificity in immuno-compromised patients. New rapid methods which can detect IA early in the course of disease, with high sensitivity and specificity are needed since treating these infections at an early stage is often essential for a favourable outcome. Especially, the polymerase chain reaction (PCR) offers great promise for the rapid diagnosis of fungal infections, including detection of fungi that does not grow in blood cultures such as Aspergillus spp. At Karolinska University Hospital we have established an assay, using a combination of a manual extraction and a robot for automated extraction of Candida and Aspergillus DNA, in combination with real-time PCR. To asses its clinical applicability, a large number of patient samples, from patients with suspected invasive fungal infection have been analysed with real time PCR. Data will be presented with focus on Aspergillus R-T PCR results in immunocompromised patients However, a range of different PCR assays (conventional-, nested-, real-time- based) have been developed, targeting different gene regions (cytochrome p450, heat shock proteins, 18S, 5.8S, 28S, ITS) and including a variety of amplicon detection methods, such as gel electrophoresis, hybridization with specific probes, ELISA and restriction fragment length polymorphism (RFLP). These molecular assays provide high potential in terms of sensitivity and specificity, but vary widely in their feasibility and are up until now not standardized. Despite of this progress, there are certain questions to be addresses using those assays, such as the risk of contamination with spores and amplicons (which can be minimized by the use of cabinets and real-time PCR assays), the frequency of prospective sampling as well as the number of positive results of a PCR assay required to initiate antifungal therapy. Furthermore, only few commercially available, standardized assays are available. This particular challenge will be addressed by the Working Group “EAPCRI“(European Aspergillus PCR Initiative) under the auspices of ISHAM. Twenty-four centres have started to establish an European standard for Aspergillus -PCR. The principal goal of this initiative is to achieve a standard for PCR that can be incorporated into the next revision of the EORTC/MSG definitions for IA. Besides the use of PCR assays for the diagnosis of IFI in symptomatic patients, this highly sensitive technology can be also performed to preemptively monitor patients at risk to develop IFI Thus, they might help to reduce empirical antifungal therapy and might be valuable tools for early initiation and monitoring of preemptive antifungal therapy. Future, prospective studies evaluating the potential benefits of early therapy based on R-T PCR in patients at high risk for IA infections are needed.

+05/01/2008

Aspergillus PCR formidable challenges and progress

Lena Klingspor, Karolinska Institute, Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden. Juergen Loeffler, University of Wuerzburg, Medical Hospital II, Wuerzburg, Germany

Introduction Opportunistic invasive fungal infections are a major cause of morbidity and mortality in immuno-compromised patients .Patients at highest risk are those with prolonged periods of neutropenia , for example during treatment for acute leukaemia or after bone marrow / hematopoietic stem cell (HSCT) and solid organ transplantation (1, 2, 3, 4). Aspergillus fumigatus is a ubiquitous saprophytic mould that forms airborne spores. Humans inhale, on average, hundreds of these spores daily. Fungal spores range in sizes from 1–50 µm and are easily spread into the environment through many routes including air. In immune competent hosts, spores are killed and cleared by cells of the pulmonary immune system. However, immunocompromised patients may develop invasive pulmonary aspergillosis, a life-threatening infection, mainly caused by Aspergillus fumigatus. Early detection of invasive aspergillosis (IA) is crucial for the outcome of the patient. The increasing incidence of IA, in immunocompromised patients, emphasizes the need to improve the currently limited diagnostic tools. Conventional diagnostic tests, such as blood culture, show poor sensitivity for the detection of Aspergillus spp. (5, 6, 7)

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There has been some progress in the diagnosis of invasive aspergillosis (IA) in recent years, mainly due to use of high-resolution CT-scanning and other imaging procedures. Still, established IA is difficult to treat with a death rate of 80-90% (8, 9, 10). Therefore, it is mandatory to develop and evaluate non-culture-based methods for the detection of systemic fungal infections. The galactomannan assay (Platelia®, BioRad) is commercially available and approved by the FDA, as well as in Europe and has been included to the criteria of the European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC-MSG) for classification of IA. (11). A further antigenemia ELISA assay detects the polysaccharide ß-1-3-glucan. (12).. However, this assay is not able to distinguish between fungal species (13.). The assay is approved by the FDA and has been included in the EORTC/MSG criteria for possible IA. More recently, PCR protocols for diagnosing fungal infections have been described (14-20). A range of different PCR assays (conventional-, nested-, real-time- based) have been developed, targeting different gene regions (cytochrome p450, heat shock proteins, 18S, 5.8S, 28S, ITS) and including a variety of amplicon detection methods, such as gel electrophoresis, hybridization with specific probes, ELISA and restriction fragment length polymorphism (RFLP). These molecular assays provide high potential in terms of sensitivity and specificity, but vary widely in their feasibility and are up until now not standardized. Furthermore, only few commercially available, standardized assays are available. This highlights the problems when evaluating publications designed to compare PCR methodologies. Yet, a consensus concerning the type of specimen, the extraction method and the PCR format and platform still has to be reached.

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Types of sample As for the best blood fraction to test, a consensus has yet to be reached on the method of choice for nucleic acid extraction and purification, and of course, the choice of specimen has a great influence on the extraction methodology. Different types of specimens have been analyzed, most often blood or serum samples since they are easy to obtain and offer the possibility to analyze them on a prospective base. Testing serum specimens by PCR relies on the detection of free circulating Aspergillus DNA, while the use of EDTA–whole-blood samples allows the detection of spores, hyphal fragments or free circulating DNA. The pathogenesis of IA is poorly understood and there is a lack of knowledge regarding the source of the fungal nucleic acid in the blood. No consensus has been achieved on the source for fungal DNA in plasma/serum or whole white blood cell pellet. Some scientists recognize that, as blood culture samples rarely yield actively growing Aspergillus spp (21), it is unlikely that the source would be viable spores or fungal cells. On the other hand the spread of viable Aspergillus cells through the bloodstream to internal organs must occur since Aspergillus can be cultured from tissue biopsies such as heart and brain . Loeffler et al., found that whole blood was a better specimen for fungal DNA extraction than plasma (22). White and Barnes hypothesized that, depending on the patient population being studied, the fungal DNA associated with the white blood cell fraction could be conidia previously ingested and partly digested by macrophages, hyphae attacked by neutrophils, or free fungal fragments damaged through platelet attachment (23). We would therefore recommend the collection of whole-blood in EDTA tubes, in order to have access to both the free- and the cell-associated DNA. Moreover, EDTA is known to inhibit DNases that are naturally present in the blood without interfering with the PCR assay which is the case of citrate (24) (and heparin has to be avoided because of Taq-inhibition),

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making it the compound of choice when blood has to be stored for any length of time before being processed.

Extraction methods A consensus has yet to be reached on the method of choice for nucleic acid extraction and purification .DNA is generally considered the target of choice due to its relative stability, and easiness of extraction (compared to RNA). (24). Specimens such as plasma and serum, in which DNA would be free in solution, allow more rapid and less complex protocols (red cell and white cell lysis buffers are not needed) for nucleic acid extraction compared to whole blood (16). If hyphal elements or conida are present in the specimens, the protocols have to rely on longer and more complex processes. This is due to the fact that the fungal cell wall is difficult to break and DNA to be released. For whole blood (red cell and white cell lysis buffers are often used), enrichment of fungal DNA by decanting human DNA with the supernantant increases the sensitivity . Protocols currently used to break the fungal cell wall usually rely on enzymatic digestion, such as recombinant lyticase (25), or mechanical disruption by bead-beating(26). If enzymatic lysis is performed, recombinant lyticase has to be used instead of zymolyase which might be contaminated by fungal DNA (27). For DNA extraction, either spin columns or an automated method can be performed. Sources of contamination during fungal DNA extraction may also include buffers and spin columns (the silica membranes) (28).

Fungal nucleic acid target for the assay The advantage of selecting a multi-copy gene is, obviously, the increased chances to successfully amplify it to a detectable level. During the last years, the ribosomal DNA gene

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region has been shown to be a promising target, which is composed of the 18S (1800 bp) gene, the 5.8S (159 bp) gene and the 28S (3396 bp) gene. These are respectively separated by internal transcribed spacer 1 (ITS 1) (361 bp) and ITS 2 (231 bp). Up until now, numerous studies made use of its different components, 18S (18, 29), 28S (30, 31), ITS1 (32) and ITS 2 (20).

Polymerase chain reaction (PCR) Hundreds of manuscripts have been published dealing with the detection of fungal DNA. However, up to now, the standardization procedures and inter-laboratory validation remains insufficiently. Which PCR assay that is most suitable is dependent on the demands of the method. PCR assays can be used as (i) a diagnostic tool only, (ii) as a tool for the early diagnosis of IA, ideally prior to the onset of clinical symptoms and finally (iii) as a tool to monitor preemptive antifungal therapy. Dependent on this decision, different technical aspects have to be considered, including the types of samples, the extraction of the nucleic acids, the fungal DNA targets and the frequency of sampling. White and colleagues recently published a multi-centre study, comparing two primer sets (28S and 18S) and three machines (LightCycler (Roche), TaqMan (Applied Biosystem) and RotorGene (Corbett Research)), on a panel consisting of 8 negative and 8 positive samples (10 – 5000 conidia per ml) (23). The study revealed that the 28S assay was more specific than the 18S assay, particularly since the 18S was found to amplify a portion of the human rDNA gene in the absence of DNA from A. fumigatus. This interference was particularly strong with the LightCycler and was responsible for a decrease in sensitivity. Overall, the sensitivity, specificity, negative (NPV) and positive (PPV) predictive value were higher with the 28S than the 18s primer set, regardless of the platform used. The platforms themselves were found to

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have a major influence on the assay, as sensitivity and NPV were 100% on the TaqMan machine, whereas specificity and PPV were at 100% using the Rotor-Gene system (23). However, the sensitivity of PCR assays with blood samples from healthy donors spiked with Aspergillus conidia might not reflect the sensitivity of the assay when clinical blood samples are analyzed because we do not know if conida, hyphal fragments or free circulating DNA are detected.

Contamination: risks and prevention Molds, such as Aspergilli, may come from many sources in the cleanroom, such as bags, boxes, markers, intervention equipment, and cart wheels. All clinical specimens should be prepared in sterile benches. Before any anlysis are performed, cabinets, pipettes, racks, and microcentrifuges, including DNA extraction robots, rotors and adaptors, should be wiped down with agents like Microsol (Anachem, United Kingdom) and DNAzap (Ambion, United Kingdom). Each stage (DNA extraction, preparing mastermix, and PCR) should be

carried out in

separate laboratories that are independently equipped, including laboratory coats. The risk of contamination may occur also during fungal PCR. Using real-time PCR, the capillaries do not have to be reopened for post-PCR analysis, and the risk of carryover contaminations can therefore be minimized. As a precaution, the use of a sufficient number of negative controls for DNA extraction and PCR is mandatory.

Practical considerations in the clinical laboratory If a test has to be used routinely in a clinical laboratory, there are additional factors of great importance. The volume of blood to be processed has to be within the range that is usually

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taken from a patient. Again, a big discrepancy exists between studies, with blood sample volumes ranging from 200 µl (16) to 10 ml (17). As the fungal load in the circulation of a patient may be less than 1 genome per ml, we recommend to process between 3 and 5 ml, which corresponds to the volume of blood accommodated by most EDTA collection tubes in hospitals. (Larger blood volumes may increase the sensitivity of the assay due to the higher fungal yield) Another crucial consideration in the development of a diagnostic laboratory test for aspergillosis would be the potential for automation of the method. In our routine laboratory, we have developed a real-time LightCycler assay for the detection and identification of Candida and Aspergillus spp., using the MagNa Pure LC Instrument (Roche Diagnostics, Basel, Switzerland) for automated extraction of fungal DNA (18). The assay takes 5–6 h to perform. The oligonucleotide primers and probes used for species identification were derived from the DNA sequences of the 18S rRNA genes of various fungal pathogens. All samples are screened for Aspergillus and Candida to the genus level in the real-time PCR assay. If a sample is Candida-positive, typing to species level is performed using five speciesspecific probes. The assay detects and identifies most of the clinically relevant Aspergillus and Candida spp. with a sensitivity of 2 CFU ⁄mL blood. (EDTA-blood 3-5 ml) To assess clinical applicability, 1650 consecutive samples (1330 blood samples, 295 samples from other body fluids and 25 biopsy samples) from patients with suspected invasive fungal infections were analysed and compared to cultures, direct microscopy, serology or CT scan results. In total, 114 (6.9%) samples were PCR-positive, 5.3% for Candida and 1.7% for Aspergillus spp.

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In patients with positive PCR results for Candida and Aspergillus, verification with conventional methods, was possible in 83% and 50% of cases, respectively. Since conventional methods such as blood cultures have poor sensitivity for Aspergillus spp., we could only verify the Aspergillus PCR positive results in 50% of the patients The feasibility of PCR testing to detect Candida and Aspergillus spp. was further demonstrated not only in blood samples, but also in bronchoalveolar lavages (BAL) and other body fluids (18). We experienced that the DNA extraction method for fungi is crucial since DNA extracted from different body fluids contains not only human DNA but may also contain fungal, bacterial, viral and parasite DNA. The total amount of DNA may vary a lot from sample to sample. Using a probe system in the LightCycler, we experienced that the total amount of DNA is of importance to know. If the total amount of DNA exceeds 500 ng / sample, inhibition may occur in our assay and give a false negative result. Thus, we routinely measure the DNA concentration in all samples using the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., Rockland, DE, USA). If the concentration is too high, we dilute the sample before running the PCR. We started to use this assay in daily routine in June 2002. Samples for PCR have been analysed in patients, at risk for fungal infection, on clinical suspicion of fungal infection, and for confirmation of fungal infection. Samples have been taken before, during and after treatment. Between the period June 2002-September 2006, (52 months) 5408 various samples have been analysed for both Candida and Aspergillus spp., and 702 out of 5408 (13%) were PCR positive, 475 (8.8%) for Candida spp. and 227 (4.2%) for Aspergillus spp.. The total number of Aspergillus positive PCR blood samples was 127 / 4565 (2.8%). In 78 patients (108 Aspergillus PCR positive blood samples) the underlying disease is known; .49

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patients underwent allogeneic HSCT (with 81 PCR positive samples), 2 patients underwent solid organ transplantation, 19 were leukaemic patients and 8 patients were in ECMO (Extracorporal Membrane Oxygenation) treatment. In the immunocompromised patient, fungal spores and ⁄ or hyphae as well as naked DNA may be released into the bloodstream, with clinical signs, but also without real evidence of disease, which is not equal to the absence of disease. Some authors claim that at least 2 consecutive positive PCR results are required to initiate antifungal therapy (33-36) How to interpret a single positive PCR result is unclear. A transient presence of fungal DNA in clinical specimens might be possible but an infection cannot be excluded. The interpretation of a single Aspergillus PCR positive test has to be seen in relation to host factors, risk factors and the clinical signs of the patient. During the same period, 120 BAL samples were analysed by PCR. Thirty-one samples (26%) from 23 patients were Aspergillus-PCR positive, in 6 / 16 patients (38%), the PCR result could be confirmed by culture. In 7 patients the result of cultures is unknown. At present, only positive results from conventional cultures and / or histological examination provide definitive proof of invasive aspergillosis (37). However, there are data supporting the considerable clinical value of this PCR assay for confirming and improving diagnosis of pulmonary aspergillosis in high-risk patients (38). Recently, the greater sensitivity of the galactomannan EIA or PCR compared to culture in the detection of Aspergillus spp in BAL fluid has been confirmed (39, 40). However, BAL is difficult to perform or to repeat, but testing BAL samples may help in diagnosing invasive pulmonary aspergillosis.

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During the 52 months period, 31 biopsies were analysed by PCR in our lab and 12 were positive for Aspergilus. These results could be confirmed by culture in 100%, as well as by direct microscopy which showed hyphae. The high sensitivity of a PCR assay in the detection of Aspergillus spp has been demonstrated in patients with cerebral aspergillosis (41). Also tissue biopsy (42) has been employed. However, obtaining such samples requires invasive procedures which might not be performed in critically ill patients. Despite of this progress, there are certain questions to be addresses using those assays, such as the risk of contamination with spores and amplicons (which can be minimized by the use of cabinets and real-time PCR assays). The frequency of prospective sampling as well as the number of positive results of a PCR assay required to initiate antifungal therapy is not known and need to addressed. Is frequent PCR testing (2-3 times per week) in high risk patients necessary to detect IA early enough to achieve reduction of mortality by immediate start of treatment? Besides the use of PCR assays for the diagnosis of IFI in symptomatic patients, this highly sensitive technology can be also performed to preemptively monitor patients at risk to develop IFI, a strategy successfully used to monitor opportunistic viral infections. The aim of this strategy is to clearly define negative (no therapy) and positive (initiation or continuation of therapy, additional screening tests) samples. In these patients, maximum sensitivities and high negative predictive values are mandatory to minimize the risk of false negative results. Taken together, those new non-culture-based diagnostic assays are appropriate as simple and rapid screening tests with high sensitivities and quick turnaround times. Thus, they might help to reduce empirical antifungal therapy and might be valuable tools for early initiation and monitoring of preemptive antifungal therapy.

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However, the lack of standardisation of the techniques is a main reason why the EORTC/ MSG have not yet included PCR in the recently published list of criteria for the diagnosis of IA. This particular challenge will be addressed by the Working Group “EAPCRI“(European Aspergillus PCR Initiative) under the auspices of ISHAM. Twenty-four centres have started to establish a European standard for Aspergillus -PCR. The principal goal of this initiative is to achieve a standard for PCR that can be incorporated into the next revision of the EORTC/MSG definitions for IA. Future prospective studies evaluating the potential benefits of early therapy based on real-time PCR in patients at high risk for IA infections are needed. To be able to validate Aspergillus PCR, we need to perform multi-center studies in order to include enough patients with proven and probable infection according to the EORTC/MSG criteria.

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