Progress in diagnosis and treatment of invasive aspergillosis after liver transplantation

PAN Sheng-hui, SUN Jiang-bo, XU Jian

Department of Organ Transplantation, The Second Affiliated Hospital of Hainan Medical University, Haikou 570105, China

Keywords:

ABSTRACT Invasive aspergillosis is an opportunistic fungal infection disease, and the risk factors of Invasive aspergillosis after liver transplantation are increasing, which seriously affects the quality of life of patients.Invasive Aspergillus has no specific clinical manifestations and occurs most frequently in the lungs.The diagnostic methods for invasive aspergillosis are continuously updated, including serological tests, polymerase chain reaction (PCR), nextgeneration sequencing, Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, Aspergillus GM lateral flow test, and some new markers under study have made the diagnosis of invasive aspergillosis infection more definitive.Voriconazole is the drug of choice for the treatment and prevention of invasive aspergillosis, and immunotherapy may become an adjuvant therapy or monotherapy for invasive aspergillosis with the emergence of more and more resistant strains.This article summarizes the progress in the diagnosis and treatment of invasive aspergillosis after liver transplantation, in order to provide a reference for clinical practice.

Invasive Fungal Disease (IFD), also known as invasive fungal infection (IFI), is a pathophysiological process in which fungi invade human tissues and blood and grow and multiply in them causing tissue damage, organ dysfunction and inflammatory responses.Liver transplantation (LT) recipients are at increased risk of IFI, with morbidity ranging from 4% to 40% of all LT recipients and mortality ranging from 25% to 67%[1].Invasive aspergillosis(IA) has long been recognised as one of the most significant opportunistic fungal infections in LT, with mortality rates of 80-90% in LT recipients with IA infection[2].Early diagnosis of IA and timely initiation of antifungal drug therapy are essential for the management of patients after LT[3], along with a comprehensive assessment of immunosuppressive regimens and the source of infection.In this paper, we review the following new developments in the clinical presentation, diagnosis and treatment of IA.

1.Epidemiology

Liver transplantation is one of the major advances in modern medicine and has become the best treatment option for end-stage liver disease.The increased intensity of immunosuppression in liver transplant patients to prevent or treat rejection increases the risk of IA.The chance of IA in patients after liver transplantation ranges from 1%-9%[4-6].Relevant national and international studies and guidelines state[1,7-11] that postoperative IA in LT recipients contains many risk factors and that the risk factors are increasing and currently include severe immune dysfunction, model end-stage liver disease score >30, diabetes mellitus, prolonged hospitalisation,hepatitis and extrahepatic failure, traumatic operations during hospitalisation, prolonged surgery, extensive blood product input,postoperative indwelling drains, central intravenous catheters,prolonged antibiotic therapy, rejection, acute kidney injury, renal failure, patients requiring continuous renal replacement therapy(CRRT), secondary surgery, re-transplantation, cytomegalovirus(CMV) infection, and bile-intestinal anastomosis.A number of agents that target immune cell populations and signalling pathways,including malignancies and autoimmune diseases, have also been identified as risk factors for IA.For example, tumour necrosis factor-α (TNF-α) inhibitors are widely used in autoimmune diseases and are associated with an increased risk of infection and cancer[12].The earliest studies found that LT recipients were susceptible to IA infections in the early stages, but more recent epidemiological studies have found a shift in IA infections towards the late postoperative period (>3 months)[10].Therefore, it is important to consider all relevant factors, including underlying disease, comorbidities (e.g.pre-existing lung disease) and the surrounding environment, when assessing the risk of IA in LT recipients.As invasive Aspergillosis is more difficult to diagnose,the identification of its risk needs to be enhanced and antifungal prophylaxis strategies need to be developed after LT[13].Targeted prophylaxis with antifungal agents in high-risk LT recipients can reduce the incidence of IA, particularly in liver transplant waiting patients at high risk of IA.

2.Clinical presentation

The identification of clinical signs of IA is challenging due to the varying doses and regimens of antifungal and immunosuppressive agents in post-LT patients, as well as the occurrence of other post-LT complications.Advances in imaging technology, combined with suspicious clinical presentations, have allowed clinicians to identify the onset of post-LT IA earlier.

2.1 Invasive pulmonary aspergillosis (IPA)

As the most common mechanism of exposure is inhalation of Aspergillus spores, the lung becomes the most infested organ for IA in LT recipients, as IA is widely disseminated and can invade various parts of the body including the heart, eyes, muscles, thyroid and central nervous system (CNS)[14,15].Pulmonary invasive aspergillosis (IPA) is the most common type of IFI, often involving extra-pulmonary organ infections and developing Aspergillus sepsis[16].IPA has no specific clinical presentation and can present with fever, haemoptysis and chest pain, and through the blood route, can invade all organs of the body with serious consequences.patients with IA have no specific clinical symptoms and the likelihood of other pathogens infecting patients after LT is increased, making it difficult to make a definitive diagnosis.IPA can present with multiple patterns on CT: dense, well-defined lesions with or without the halo sign (attenuation of nodular surrounding tissue), air crescent sign (an advanced and non-characteristic image); cavity formation; wedgeshaped, segmental or lobar solid lesions; and bronchopneumonia may present with the inverse halo sign[17].The affinity of Aspergillus for blood vessels causes vascular damage in the lung and the formation of infarct foci; when coagulative necrosis occurs in the centre of the infarct foci, the density of the tissue surrounding the centre attenuates, forming the halo sign.necrotic resorption of lesions in late IA forms air-containing cavities, which are typically seen as crescentic air signs on CT [18].

2.2 Non-pulmonary invasive aspergillosis

Non-pulmonary IA infections in the rest of the lung are less common and non-specific in their clinical presentation.For example,in invasive fungal sinusitis, ulcers, nodules, pseudomembranes,plaques or crusts of the pulmonary trachea and bronchi (observed in bronchoscopic analysis), localised pain (which may radiate to the eyes) and nasal ulcers with black crusts that may extend to the orbit may be seen[19].The disease progresses rapidly and diagnosis usually requires histological or pathological examination.

In recent years, CNS IA has been progressively reported in postoperative patients with LT.The diagnosis was often previously confirmed by autopsy and surgical pathology.IA in CNS has no specific clinical signs and can lead to cerebral infarction, cerebral vasculitis, cerebrovascular accident (stroke), haemorrhage,multiple or isolated abscesses and meningitis.Extremely immunocompromised patients may experience multiple alterations of the cranial nerves, granulomas, brain abscesses and meningitis.A detailed neurological examination is required; laboratory evaluation includes serum Aspergillus antigens, blood, urine and sputum cultures, chest radiographs; magnetic resonance imaging of the brain with contrast, lumbar puncture, EEG for seizures,possible neurosurgical intervention and biopsy are key factors in the evaluation of these patients[20].Meningeal enhancement may be seen on CT in focal lesions and in enhanced images[11,13,21], and the presence of sinusitis, endocarditis and large vessel damage is noted.

3.Diagnosis

The 2019 EORTC/MSG IFI guidelines state that there are three levels of diagnosis for invasive fungal disease: 1.definitive (proven):fungal infection is suggested by microscopic examination (including microbioscopy, histopathology and cytopathology) or culture of a sterile site specimen; 2.clinical (probable): the patient needs to meet host factors, clinical criteria and microbiological criteria 1 each; 3.Possible: 1 each of host factors and clinical criteria are met, but not microbiological criteria[13].The diagnosis of probable infection is based on host factors, clinical manifestations and mycological evidence, with (1,3)-β-D glucan not being used as a single mycological evidence for invasive mycobacterial disease[22].different clinical signs and may be combined with other complications after LT, which can be difficult to identify.Clinical mycological evidence can be obtained from microscopy, specimen culture, serological examination, imaging and molecular biology.

The diagnosis of IA relies on a multilevel approach that takes into account risk factors and local epidemiology, as well as the performance and limitations of available diagnostic tools.early identification and treatment of IA is challenging, and delayed or missed diagnosis is associated with higher morbidity and patient mortality in IA[23-25].The lack of sensitivity and specificity of currently available diagnostic tests is a major cause of delayed diagnosis, and timely diagnosis of IA in LT recipients requires a high index of suspicion.When patients presenting with fever, respiratory symptoms or symptoms of disseminated infection should be assessed using advanced chest imaging such as computed tomography (CT);plain radiographs may produce false negative results and should not be used to rule out pulmonary infection.Other sites presenting with symptoms, such as the sinuses, should also have appropriate imaging modalities such as dedicated sinus CT.sinus imaging may reveal invasive local structures, but it is not possible to differentiate between the species causing invasive fungal sinusitis still requiring microbiological examination.Aspergillus brain abscesses present as hypointense images on CT with ring-like enhancement; on magnetic resonance imaging, T1 low signal and T2-weighted high signal.The characteristics of its enhancement are diverse, such as nodular,jagged and lace-like[26], and the degree of enhancement on MRIenhanced images increases with increasing disease.MRI is more recommended for CNS than CT.

3.1 Serological tests

Serological tests can be used to increase the diagnostic basis for the diagnosis of invasive infections in combination with clinical symptoms and imaging.As a result, non-culture-based biomarkers for the detection of circulating fungal cell wall components have been developed over the last two decades.One of these diagnostic test markers is (1,3)-β-D glucan (BG), known as the G test.This fungal wall component can be found in fungi such as Candida and Aspergillus, but cannot be used to detect Cryptococcus and Trichoderma.the G test detects fungal infections, it does not identify the fungal species.False positives can occur in Gram-negative bacterial infections and with cephalosporins.It also does not indicate the absence of fungal infection when BG is negative and can be used as a screening test in patients at high risk of fungal infection who are not receiving antifungal infections.27 The value of the G test remains relatively low because it lacks the specificity of the Aspergillus fragment[28-29] and has limited sensitivity, particularly in liver transplant recipients, which ranges from 58% to 65%[25,30].

In addition, serum and bronchoalveolar lavage fluid (BALF)galactomannan assays (Galactomannan, GM) are recommended as markers for the diagnosis of IA[13].The sensitivity of serum GM is 61%-79%, the sensitivity of BALF-GM is 58%-90%, and the specificity of serum GM is 61%-79% and BALF-GM is 58%-90%[31].Serum GM is not recommended as a routine screening test in patients with LT, particularly in patients already receiving antifungal infections, which can significantly reduce the sensitivity of serum GM.Other non-specific inflammatory markers, such as C-reactive protein (CRP), interleukin 6 (IL-6) and calcitoninogen (PCT), have not been used to date in LT recipients for the assessment or diagnosis of IA.

3.2 Polymerase chain reaction (PCR)

With the continuous advancement of modern technology, molecular biology methods have also become one of the clinical tools for the diagnosis of IA, such as PCR techniques and gene sequencing,among others.PCR testing of different fluid samples (e.g.whole blood, plasma, cerebrospinal fluid, drainage fluid, etc.) and tissues is of great value for the clinical diagnosis of IA, especially for postoperative LT patients with a high chance of infection and a wide variety of infectious agents.a unique feature of PCR is its ability to simultaneously detect Aspergillus species and also to identify directly from clinical species certain mutations associated with triazole drug resistance The PCR is unique in its ability to simultaneously detect Aspergillus species and to identify certain mutations associated with triazole resistance directly from clinical species[32-33].However, at present, graded biology is not recommended as a routine test.

PCR can directly detect Aspergillus DNA in blood, serum or BALF with moderate accuracy for screening high-risk patients with suspected IA.a 2019 updated Cochrane review included 29 whole blood, serum or plasma PCR studies, showing a combined sensitivity of 79.2% and specificity of 79.6% for PCR testing.For two or more consecutive positive results, the sensitivity was even lower at 59.6%and the specificity improved to 95.1%[34].According to a 2012 systematic review, BALF-PCR reported a sensitivity and specificity of 77% and 94%, respectively[35].It has excellent negative predictive values (～95% for single or sequential assays), and sequential performance and or combination with other biomarkers can improve positive predictive values[34].PCR is more sensitive but slightly less specific than GM, whereas serial positive PCR is less sensitive but more specific.Unlike the GM and BG markers released by IA,Aspergillus DNA can be detected in the absence of active vascular invasive disease.Although the test does not differentiate between active disease and colonisation, it does offer a potential opportunity for early initiation of pre-emptive treatment in patients with high clinical suspicion but inconsistent imaging results, or for antifungal prophylaxis in high-risk individuals.

3.3 Next-Generation Sequencing (NGS)

Next-Generation Sequencing (NGS), also known as highthroughput/massively parallel sequencing, is an emerging diagnostic technology for identifying potential pathogens of CNS.In theory,with sufficiently long sequencing reads, a high enough hit rate and an all-inclusive reference database, almost any microorganism can be uniquely identified based on a specific nucleic acid sequence.There are many sequencing technologies and data analysis packages available[36].All human and microbial DNA is extracted from clinical samples (including blood, CSF, BAL fluid, etc.) without a priori knowledge of a specific target.sequencing is performed using the ‘birdshot’ method, removing human DNA and comparing the results with existing nucleotide sequences in a pre-formed database.If sequences are present in the reference database, rare pathogens can be identified as well as potential drug resistance mutations.The TAT (TURN-AROUND TIME; test turnaround time) is typically 12-24 h after the specimen is received by the reference laboratory.NGS technology enables evolutionary tracking and is used to identify outbreaks of various fungal infections.As costs decrease and databases expand, NGS can be routinely used as an adjunct to contraindication to invasive biopsy, so the clinical application and management of NGS sequencing technology for the diagnosis of infections still needs to be clarified.

3.4 Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS)

To guide the selection of antifungal drugs, species level identification of moulds growing in culture is often required.MALDI-TOF MS has the advantage of being able to identify a broad spectrum of species from commercial and in-house databases.The use of MALDI-TOF MS for filamentous fungi has evolved over the last decade, but time-consuming sample preparation techniques(which can vary between producers) and limitations of spectral databases and available isolation challenge sets have delayed its widespread use.The mechanism of culturing mycorrhizal isolates and the stage of fungal growth may influence identification, as different levels of mycelium and spores are present in liquid versus solid media with different proteomic fingerprints[37].Several studies have reported identification rates of filamentous fungi ranging from 15% to 97%, depending on the platform and database used, and have been widely used for fungal isolates[38].There are significant differences between many commercially available platforms and the databases they manage, including the range of species included and the nomenclature used for species identification, which can make generalisation challenging.

MALDI-TOF MS is a reasonable alternative to conventional microbiological and molecular methods for the identification of strains from positive cultures, although the lack of standardised processing techniques and incomplete database spectra remain limiting factors.In cases where identification is not possible,additional molecular diagnostic techniques are required.

3.5 Aspergillus GM Lateral Flow Assay (GM-LFA)

GM-LFA is an immunochromatographic test for the qualitative and quantitative detection of Aspergillus GM in serum and BAL samples.The performance of the GM-LFA assay for BALF in Solid Organ Transplant (SOT) recipients varies, with lower sensitivity and specificity compared to patients with haematological malignancies[39].There are limited data on the performance of GM-LFA for the diagnosis of IPA in SOT recipients.This trial is currently undergoing further research.

3.6 Other test techniques

In the diagnosis of IPA, a combination of radioactive tracers, such as 18-fluorodeoxyglucose (a marker of metabolic activity), can be useful in CT and positron emission tomography (PET) to locate areas of abnormality, but cannot distinguish between malignancy,infection or inflammation[40].Additional radioactive tracers are being investigated to better image IPA.another non-invasive test uses exhaled breath to detect organic compounds released by exhalation from the IA environment, which may be the simplest and cheapest biomarker for diagnosing IA, but its efficacy is yet to be further certified.In addition, in a study by Kieren et al[41], a flowmetric test paper test optimised using a galactofuranose specific monoclonal antibody (mAb476) that recognises urinary antigens following Aspergillus fumigatus infection is currently dedicated to relevant clinical studies.

4.Treatment and prevention

An important principle in the treatment of severe infections after LT is to reduce or stop the use of immunosuppressive drugs.The choice of drugs for the treatment of IA is limited and requires considerable care due to the condition of the post-LT recipient, including underlying disease, complications and potential organ dysfunction.In addition, in LT recipients, drug-drug interactions significantly influence the choice of antifungal agents[8,42].The unique toxicity of different antifungal agents also adds to the complexity of treating IA in LT recipients[43], for example, there are important drug interactions between voriconazole (VCZ) and tacrolimus and sirolimus, so when administering antifungal therapy for IA, the immunosuppressive regimen needs to be adjusted according to the patient’s blood levels and reduced or discontinued if necessary.In particular, corticosteroid doses should be reduced.

Current national and international guidelines for the management of IA state[8,13,44] that voriconazole (VCZ) is the drug of choice for the treatment and prevention of IA, and that drug concentrations should be monitored.In some LT recipients who are voriconazole resistant or unsatisfied with treatment, amphotericin B and its remedial treatment or a combination of different antifungal drugs may be used, but drugs with the same toxicity should be avoided as far as possible.A comparative study of the efficacy of posaconazole versus voriconazole published by Johan A Maertens et al in The Lancet[45]suggested that posaconazole intravenous and tablet formulations with improved systemic absorption may be an effective alternative to voriconazole and that posaconazole was well tolerated in the study,with fewer treatment-related adverse events in subjects than in the voriconazole group, supporting posaconazole as IA as a first-line agent.The clinical efficacy of other antifungal agents alone in the treatment of IA needs to be investigated.

The recommended dose of VCZ for the treatment of IA in LT recipients is 6 mg/kg intravenously for q12h on the first day and a maintenance dose of 4 mg/kg intravenously for q12h or 200-300 mg orally twice daily[1].2018 pharmacology in China recommends controlling VCZ concentrations at 0.5-5 μg/mL[44], with too low or too high concentrations having a prognostic and therapeutic impact on patients causing a significant impact, too low for insufficient intensity of infection control and too high leading to hepatic impairment confused with postoperative complications of LT and other drug toxicity leading to hepatic impairment.It has also been noted that voriconazole concentrations in Asian populations should be controlled to 3 μg/mL, and that the incidence of postoperative hepatotoxicity in LT patients increases if this target is exceeded[46].The duration of treatment for IA process is generally 6-12 weeks,with specific treatment varying from individual to individual, and termination of treatment is indicated by the absence of evidence of IA on imaging, fungal and pathogenic examinations and clinical symptoms[8,47].

Due to the inhibitory effect of azoles on cytochrome P450, calciumregulated phosphatase therapy is initiated at a 50% dose and the appropriate tacrolimus concentration needs to be re-established.After completion of 90 days of voriconazole prophylaxis, an increase in calcium-regulated phosphatase dose is required to achieve appropriate blood levels due to the increase in cytochrome P450 activity when voriconazole is discontinued[16].If patients develop neurological deficits, cyclosporine may be administered in place of tacrolimus.

Recent studies have now found that the emergence of VCZresistant strains puts the preferred regimen of VCZ at risk[48], such as genetic alterations in the CYP51A gene of Aspergillus fumigatus.It has also been found that triazole-resistant strains without CYP51A mutations have also been reported in more than 50% of strains in some collections[49].Recently, mutations in the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase(hmg1) have been identified and associated with triazole resistance in many clinical Aspergillus fumigatus isolates[50-51].The prevalence of hmg1 gene mutations and their role in triazole resistance in Aspergillus fumigatus is unclear.Further studies are needed to unravel the mechanistic role and clinical significance of hmg1 mutations.Neofytos et al.found[1] that multiple triazole resistance in Aspergillus fumigatus is increasingly being reported worldwide, in most cases due to TR34/L98H mutations.

Patients with IA face numerous challenges in the treatment of standard antifungal drugs.No new class of antifungal drugs has been invented for over a decade, the number of fungal isolates resistant to azoles has steadily increased, and conventional antifungal drugs have serious drug toxicity.Immunotherapeutic approaches hold the promise of improving antifungal treatment to reduce high mortality rates.In immunocompromised patients, this usually involves therapeutic immune boosting, including cellular therapeutic approaches such as cellular infusion of innate (granulocytes,dendritic cells, natural killer cells) and adaptive immune systems (T cells) and the administration of different cytokines, chemokines and antibodies[52].However, the use of cell therapy in transplant patients carries the risk of inducing fatal rejection, inflammatory factor outbreaks, etc.How to circumvent these risks has become the current focus of cell therapy and is still under continuous research.These approaches may be used in the future as adjuvant therapy or even as stand-alone treatment.

Prophylaxis for IA is necessary in LT patients who are immunocompromised and whose disease progresses rapidly after IA infection, which not only affects the function of the transplanted liver and the quality of patient survival, but also greatly increases the cost of treatment for LT patients after IA.Prophylactic regimens of voriconazole are administered via the enteral route because of high bioavailability (>83%) (74%-94%), ease of administration,and low cost relative to intravenous administration.In addition,the pharmacokinetics (PK) of oral voriconazole is not significantly affected by renal impairment according to Johnson et al.Therefore,no adjustments are required for oral administration or CRRT use in patients with mild to severe renal impairment.LT recipients generally receive tapered doses of tacrolimus as well as prednisone and morte-macrolimus as immunoprophylaxis.

5.Summary

The causes of post-operative IA in LT are multifaceted and it is difficult to complete the diagnosis of IA with independent tests alone.New markers and more advanced tests show some potential.It is crucial to optimise post-operative anti-rejection drugs and IA-related tests.For high-risk patients, dynamic detection of IA infection indicators should be performed in parallel with fungal tests, active treatment should be given in case of infection, and discontinuation or reduction of immunosuppressive drugs is an important principle.Among the therapeutic options for the treatment of IA after LT, voriconazole is the drug of choice, but alternative drugs or alternative treatments may emerge in the course of ongoing research.In conclusion, the occurrence of postoperative IA after LT seriously affects the prognosis of patients and early diagnosis and treatment should be achieved to improve the quality of survival and survival cycle of LT recipients.

Authors’ contribution and author conflicts of interest.

First author: Shenghui Pan: collection of relevant literature and writing of the paper; corresponding author: Jian Xu: conception and review of the article project; the rest of the authors participated in the collection and collation of literature.All authors have no conflict of interest.