Aims The purpose of this study was to investigate the performance of non-invasive diagnostic tests such as galactomannan enzyme immunoassay and quantitative PCR in the early diagnosis of invasive aspergillosis (IA), and how these tests are impacted upon by the use of different classes of antifungal agents in an in-vivo model of IA.
Methods A standardised rat inhalation model of IA was used to examine the effects of an azole, posaconazole, a polyene, amphotericin B and an echinocandin caspofungin. Daily blood samples were collected for subsequent analysis using a commercially available galactomannan assay and an inhouse qPCR assay.
Results No significant differences were observed in the CE/g of Aspergillus fumigatus in the lungs of each group. qPCR was statistically more sensitive than galactomannan for both the early detection of infected controls (p=0.045) and for overall detection (p=0.018). However, antifungal treatment significantly reduced the overall sensitivity of qPCR (p=0.020); these effects were due to posaconazole and caspofungin. In the latter stages of infection (days 4 and 5) there were no significant differences in the numbers of infections detected by galactomannan and qPCR; however, the antifungal class used caused significant qualitative differences (p=0.041). Galactomannan showed improved detection in posaconazole-treated animals.
Conclusions Previous exposure to antifungal therapy must be considered when interpreting either qPCR or galactomannan-based IA diagnostics as this study has shown that individual classes of antifungal agents impact upon the dynamics of antigen and DNA release into the circulation.
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Invasive aspergillosis (IA) remains a major concern in the management of patients undergoing haematopoietic stem cell transplantation. As a result of the acknowledged risk of IA a number of strategies have been developed for the use of antifungal agents ranging from prophylaxis,1 via preemptive therapy2 to empiric therapy.3 Despite the availability of a number of new mould active antifungal agents IA continues to have a high mortality rate1 and the diagnosis of IA remains problematical4 with many cases not diagnosed until after death.3
Symptoms such as fever, cough, or chest pain are non-specific and many patients may be asymptomatic. Some radiological findings, such as the presence of a halo sign or cavitating nodules in the lungs, may be strongly suggestive of aspergillosis but are not specific as these can also be found with other infections.5 Diagnostic tests such as galactomannan enzyme immunoassay and quantitative PCR have been widely employed, but it is not clear how the different antifungal treatment strategies influence these tests.
This study utilises an animal model of IA infection to examine the effect of antifungal treatment using the three main classes of drugs, azoles, echinocandins and polyenes, on two diagnostic tests, galactomannan and an optimised qPCR molecular detection assay.6
A standardised rat inhalation model of aspergillosis was used as previously described.6 Briefly, each animal was immunosuppressed using 75 mg/kg cyclophosphamide and 16 mg/kg depo-medrone. Prophylactic antibiotics (baytril 50 ppm) were added to the drinking water throughout the study. Two days after immunosuppression infection was induced by inhalation of 1×109 Aspergillus fumigatus spores/ml for 1 h. The animals were given a further dose of 75 mg/kg cyclophosphamide, with daily antifungal treatment commencing 5 h post-infection.
The animals were split into four groups, each comprising 12 animals. These included infected untreated controls and daily treatment groups receiving either caspofungin (5 mg/kg intraperitoneally), posaconazole (2.5 mg/kg by mouth) or amphotericin B (1 mg/kg intraperitoneally) and vehicle-treated saline (intraperitoneally). Posaconazole was chosen instead of other azoles because of its pharmacokinetic profile in a rat model, and it has good tissue penetration.7 Three animals from each group were humanely killed according to control and treatment groups daily for 2–6 days post-infection. Three uninfected animals were also included as negative controls. Terminal blood samples were taken by cardiac puncture following terminal anaesthesia, which were then separated into clot and serum samples by centrifugation, as previously described by our group.6 The serum was then used for the galactomannan enzyme immunoassay (Bio-Rad, Hertfordshire, UK), which was performed in duplicate as per the manufacturer's instructions, with the galactomannan index of 0.5 or greater defined as positive. The clot was processed and analysed using qPCR, as described below. Lungs were also removed from each animal, homogenised in phosphate-buffered saline and using an 18S real-time PCR as described previously8 colony forming equivalents of A fumigatus (CE/g) were determined.
The clot was initially pretreated by mixing with an equal volume of G2 buffer and 10 μl proteinase K, which was incubated at 56°C until this became fully lysed (approximately 1 h). DNA was then extracted from 200 μl of the lysed sample using the automated EZ1 BioRobot (Qiagen, Crawley, UK) and eluted in 100 μl, which was then used for qPCR analysis. All qPCR assays were performed using A fumigatus-specific primers ASPCF–5′–CTC GGA ATG TAT CAC CTC TCG G–3′, ASPCR–5′–TCC TCG GTC CAG GCA GG–3′, and the probe 5′–FAM–TGT CTT ATA GCC GAG GGT GCA ATG CG-TAMRA–3′ targeting 28S gene.2 The 25 μl PCR mixture contained 1× QuantiTect PCR master mix (Qiagen), 300 nM of each primer, 150 nM of probe and 7.5 μl of DNA. DNA amplification and fluorescence detection was performed using an ABI Prism 7000 (Applied Biosystems, Warrington, UK) under the following conditions: 50.0°C for 2 min, 95.0°C for 15 min followed by 40 cycles, 95.0°C for 15 s (denaturation) and 58°C for 1 min (hybridisation and elongation) with fluorescence detected after each cycle. Automated analysis using ABI Prism software calculated the Ct values. PCR positivity was defined as a Ct value of less than 40, as previously reported by the European Aspergillus PCR Initiative.9
Statistical analysis was performed to analyse both diagnostic tests and the effect of the antifungal agent on these tests. Cross tabulations were performed using SPSS and analysis performed using Fisher's exact test. In addition, a two-way analysis of variance was performed to determine whether statistical differences in fungal burden were observed between each group.
Fungal burdens in the lungs of all infected animals, regardless of treatment group, ranged from 2.30 to 4.88 log10 (CE/g) (table 1). No statistical differences were observed between any groups on any of the days (2–5) with respect to fungal burden. No fungal growth was detected in the uninfected control animals.
Diagnostic analysis of the infected controls indicated that the overall qPCR was significantly more sensitive than galactomannan for early detection (p=0.045), showing 67% positivity at day 1 and 100% positivity thereafter until day 4 post-infection. In comparison, galactomannan only demonstrated 33% positivity after 2 days and 100% positivity on days 3 and 4 post-infection (table 2). For all samples tested qPCR was significantly more sensitive (73%) when compared with galactomannan (67%, p=0.018). However, when analysis was performed on the antifungal-treated groups compared with the vehicle-treated group the sensitivity of qPCR was significantly reduced to 67% (p=0.020), and galactomannan sensitivity increased marginally (70%).
Overall, antifungal treatment affected diagnostic detection; this effect was mainly due to posaconazole and caspofungin as amphotericin B treatment did not affect the detection by galactomannan and only slightly affected qPCR detection on day 2. While there were no significant differences in the number of positive infections detected by qPCR and galactomannan at the latter stages of infection (days 4 and 5) there were qualitative differences in detection depending on which antifungal agent had been used. Galactomannan was better at detecting infections treated with posaconazole (p=0.041). No significant difference was observed for caspofungin and amphotericin B.
Galactomannan analysis showed that amphotericin B closely matched the vehicle control, with 100% positivity at day 3 and thereafter. Caspofungin, likewise, was 100% positive at days 3 and 4, but dropped to 67% positivity at day 5. Posaconazole was negative by galactomannan at day 2 and only achieved a maximum 67% positivity after day 5 (figure 1).
qPCR analysis showed that amphotericin B was 100% positive 3 days post-infection and thereafter. Caspofungin showed a time-dependent increase in qPCR positivity, with 33% at days 2 and 3, 67% at day 4 and 100% at day 5 post-infection. In contrast, posaconazole qPCR positivity was only 33% throughout, except at day 4 when a transient 100% positivity was observed (figure 2).
Our previous work has shown the importance of using an optimal clinical sample and how the method of extraction improves the diagnostic yield from qPCR.6 In this follow-up study we have examined the effect of antifungal treatment on diagnostic sensitivity, which we have shown to play a significant role. It has previously been demonstrated that the performance of galactomannan and qPCR diagnosis of IA varies depending upon treatment with mould-active antifungal agents.10 11 However, to our knowledge this is the first study to model the effect of treatment simultaneously with the three most widely used classes of antifungal agent on diagnostic sensitivity of qPCR and galactomannan in a rat model of IA.
qPCR has recently received considerable attention in a drive to improve diagnostics for IA. The European Aspergillus PCR Initiative has recently made advances by publishing validated extraction guidelines,9 but unlike galactomannan detection it is not yet included within the EORTC guidelines for the diagnosis of IA. In our study we have demonstrated that qPCR was statistically superior to galactomannan at the early detection of IA. qPCR was also shown to be highly sensitive in early detection in a guinea pig model of IA, with levels rapidly reaching a plateau before becoming undetectable on day 6.12 These are in contrast to a previous study that demonstrated that galactomannan (80%) was more sensitive than qPCR (63%) in the serum of untreated infected rats.13 However, in this study both the clinical specimen and the DNA extraction method were inferior to our methodology, which may account for their reduced sensitivity of qPCR. Morton and colleagues14 recently demonstrated experimentally that A fumigatus conidia inoculated into blood were preferentially detected by qPCR in a dose-dependent manner, but not by galactomannan. The authors suggest that this was because of the failure of the conidia to grow in blood and release galactomannan into the plasma. The kinetics of fungal growth and their response to antifungal action will therefore also impact the release of diagnostic target molecules.
The primary aim of this study was to investigate the effects of antifungal treatment upon qPCR detection and to compare this with galactomannan. Previous reports have suggested that molecular detection is influenced by previous antifungal exposure in animal models and in patients.10 12 Similarly, it has been reported that the highest accuracy achieved from galactomannan testing is in the absence of antifungal therapies.15 We have shown a significant reduction in qPCR sensitivity within antifungal-treated rats, whereas overall galactomannan sensitivity marginally increased following treatment. The class of antifungal agent played a role in determining the most appropriate diagnostic test, with posaconazole treatment shown to favour galactomannan detection in the later infections, whereas caspofungin treatment and amphotericin B treatment were shown to have little effect on either method. Interestingly, no statistical differences were observed between any of the treatment groups and the infected control with respect to lung fungal burden at each time point when evaluated by colony-forming equivalents, so it is unlikely that the diagnostic sensitivity of either test was a direct reflection of changes in cell numbers.
Previous studies have investigated a variety of models, including the treatment of a guinea pig model of IA treatment with amphotericin B in which it was shown that serum galactomannan levels were equivalent to the control until between day 3 and day 5.16 This was a similar trend to the data reported herein for galactomannan, but was overall less sensitive for early detection compared with qPCR. In a guinea pig model it was also shown that serum galactomannan remained lower in voriconazole-treated animals than control animals for the duration of the experiment.12 It has also been reported that circulating galactomannan levels are decreased during posaconazole therapy in a rabbit model of IA.17 Further studies have shown that despite a reduction in pulmonary infiltrates, lung weights and mortality, serum galactomannan levels were increased following prophylaxis and treatment with caspofungin.16
These findings and our data provide insights into how diagnostic testing strategies could be optimised. PCR became positive earlier in our control animals therefore the use of posaconazole or caspofungin in either prophylaxis or treatment may reduce the value of a negative PCR result early in the course of the infection. Amphotericin B had less effect, delaying a positive PCR result by 1 day. It may therefore be necessary to perform daily PCR for the first week after the diagnosis of invasive fungal disease is suspected to obtain the highest likelihood of a positive specimen.
Treatment with caspofungin and amphotericin B resulted in no delay in a positive galactomannan compared with control. In contrast, however, treatment with posaconazole causes a delay in positivity. Our results also suggest that there may be a ‘window of positivity’ for galactomannan testing during treatment with posaconazole and caspofungin as test positivity declined rapidly by day 5 in animals treated with these agents. This again suggests that early and frequent testing may be necessary to optimise the use of this test. New developments in antigen testing such as the novel lateral flow device deigned as a near patient testing kit may improve diagnostics, but again the impact of antifungal agents must be evaluated in this system.18 Overall, our results suggest that it is vital that the interpretation of these diagnostic tests must take into account both the type and duration of antifungal treatments.
Optimised and standardised qPCR reactions are statistically superior to galactamannan for the early detection of IA.
Antifungal treatments, particularly fungi static agents, negatively effect laboratory-based diagnostic tests.
When patients are receiving antifungal drugs the results of diagnostic tests should always be interpreted with this in mind.
Regardless of how well optimised a diagnostic test the appropriateness of the clinical samples taken and the stage of infection is key to meaningful diagnostic results.
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Funding The authors would like to acknowledge the financial support from Merck Sharp & Dohme and Schering Plough. This project was also supported in part with federal funds from the National Institute of Allergy and Infectious Diseases under contract no NOI-AI-30041.
Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed.
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