Aspergillus species can cause a variety of diseases. Among these, invasive pulmonary aspergillosis (IPA) is the most common opportunistic infection caused by moulds in immunocompromised patients and is characterized by acute invasion of hyphae into human tissue. Accordingly, the final proof of IPA is obtained histologically. The mere detection of Aspergillus on an external surface is not sufficient to make a diagnosis. If the presence of Aspergillus is detected in a sample, the correct classification of the findings will guide further diagnosis and treatment.
Aspergillus species can cause a variety of diseases. Among these, invasive pulmonary aspergillosis (IPA) is the most common opportunistic infection caused by moulds in immunocompromised patients and is characterized by the acute invasion of hyphae into human tissue. Accordingly, the final proof of IPA is obtained histologically. The mere detection of Aspergillus on an external surface is not sufficient to make a diagnosis, as Aspergillus is a ubiquitous organism that can be inhaled with the air we breathe and can also be swallowed. The lungs and intestines are considered external surfaces in the above sense. If the presence of Aspergillus is detected in a sample, the correct classification of the findings will guide further diagnosis and treatment.
Inhaled spores are either exhaled, removed mucociliary or destroyed by macrophages. If these mechanisms are prevented, the physiological defenses are bypassed and very different clinical pictures can develop. Chronic pulmonary aspergillosis (CPA) requires pre-existing lung structural changes, e.g. caverns. Spores cannot be exhaled from caverns because the air flow is chaotic. The situation is similar with impaired mucociliary clearance. In addition, pathologically dilated airways evade immune control. At the same time, such preformed cavities provide ideal temperature and humidity conditions for the growth of Aspergillus fumigatus. This is why this species is the most common cause of chronic aspergillosis.
Allergic bronchopulmonary aspergillosis (ABPA) is another chronic form of the disease. It is often associated with bronchiectasis. It is based on a pathological, ongoing inflammatory reaction that leads to tissue damage. There is no tissue invasion by Aspergillus hyphae, but a misdirected immune defense is the cause. Overall, the diagnosis and treatment of these rare diseases is complex, and registry studies can provide information on optimal strategies [1].
Epidemiology
IPA is an acute disease that occurs predominantly as a result of immunosuppression. Very rarely, however, people who have inhaled a particularly high inoculum may be affected.
Typical, however, would be a phase of pronounced immunosuppression, as is already present in acute myeloid leukemia at the time of diagnosis and is intensified by antileukemic therapy [2]. The rate of patients with invasive aspergillosis was up to 24% before the introduction of systemic prophylaxis [3]. Antifungal prophylaxis has significantly reduced the rate. The strongest predisposing factor is neutropenia, so that patients with myelodysplastic syndrome are also at high risk of aspergillosis. In adult patients with acute lymphoblastic leukemia, there is also a substantial risk with a rate of invasive pulmonary mycoses of 13%. However, not all intensive therapies for hematologic malignancies are associated with invasive aspergillosis [4]. Thus, autologous transplantation of hematopoietic stem cells does not predispose to multiple myeloma and lymphoma therapy and the rate is less than 1%. This is probably due to the easily controllable short neutropenia duration. In contrast, invasive aspergillosis is common in allogeneic stem cell transplantation. Although neutropenia may also be more short-lived than in other high-risk groups, drug immunosuppression comes into play, which is administered over weeks and months and sometimes years [2].
In patients treated in intensive care, viral pneumonia and viral tracheitis pave the way for aspergillosis. For example, severe COVID-19 leads to aspergillosis in up to 22% of cases [5]. For patients with influenza pneumonia requiring intensive care, this connection has long been known and has recently been confirmed in a Swiss cohort study. In this study, pre-existing bronchial asthma increased the risk of influenza-associated pulmonary aspergillosis (IAPA) to 17%. The extent to which pneumonia due to infection with other viruses, such as respiratory syncytial virus (RSV) or human metapneumovirus (hMPV), promotes aspergillosis is currently unknown. There is an association in immunosuppressed patients, but it has not yet been shown for non-immunosuppressed patients. However, infections caused by viruses for which no specific therapy is available are currently underdiagnosed. Aspergillus is also a potential pathogen for other patient groups. These include recipients of solid organ transplants, especially after lung transplantation (Table 1) .
If IPA is suspected, differentiation from mucormycosis is necessary. The standard antimycotics against aspergillosis are only partially effective against the pathogens of mucormycosis. To make matters worse, mixed infections occur. This can be explained by the fact that mucormycosis pathogens are also inhaled. The upper and lower respiratory tract are also the target organs for these. The frequency of mixed infections varies from region to region and is reported to be up to 30%. This risk depends on environmental factors. It is determined by exposure to spores. From the occurrence of soil dust to the maintenance of air conditioning systems, many individual factors have been described [6].
Diagnostics
In immunosuppressed patients, the differential diagnosis of IPA should definitely be considered in an extended diagnostic work-up. The growing number of immunocompromised fungi and the increasing use of antimycotics in medicine, veterinary medicine and agriculture promote the growth of resistance and thus also the selection of more aggressive, multi-resistant mold species and azole-resistant aspergilli.
Appropriate diagnostics should be initiated particularly in patients with persistent or recurrent neutropenic fever >72 hours, which does not respond to antibiotics, or under other severe immunosuppression. Other clinical signs are non-specific, often mild respiratory symptoms such as productive or non-productive cough, pleuritic symptoms, mild shortness of breath and hemoptysis. The latter should already be considered a warning signal, as pulmonary bleeding due to invasive fungal growth is a frequent fatal complication of invasive mycoses; however, hemoptysis can also occur in the early stages of IPA or in sinusoidal or tracheal infestation. Rapid, interdisciplinary collaboration is essential in the diagnosis of IPA. Admission – if not already treated as an inpatient – should take place in the case of highly suspected cases of the above-mentioned syndromes.
Radiology
Low-dose computed tomography of the thorax should be performed as a primary diagnostic test – also to differentiate lobar pneumonia or atypical pneumonia in immunosuppression. This is also suitable for the more elective diagnosis of ABPA and CPA. A chest X-ray examination is not useful. In this case, infiltrates that indicate pulmonary aspergillosis cannot usually be clearly identified. The findings should be made by a radiologist experienced in invasive mycoses in order to achieve the greatest possible certainty in the diagnosis; a high degree of examiner dependency was found in the diagnosis of IPA despite precise examination techniques. In severely immunosuppressed patients with persistent febrile neutropenia, a PET-CT scan may be performed to exclude or detect further organ involvement and other infectious foci, if available. Further infectious differential diagnoses of pulmonary CT findings and CT-graphic signs of CPA and ABPA will not be discussed in detail here.
According to the EORTC/MSG definition, various signs can indicate invasive mycosis on CT chest [7]. If a CT with angiography is performed, indications of angio-invasive growth (“vessel occlusion sign”) can already be detected here, which is associated with a high risk of fatal intrapulmonary bleeding in patients with IPA. Nonspecific signs are frosted glass infiltrates and roundish infiltrates. Nodular lesions with surrounding frosted glass infiltrate (=”halo”) are considered more specific, Fig. 1A). The latter may be an incipient sign of invasive growth and is a morphological correlate of hemorrhage surrounding the infiltrate, so that the halo is more pronounced in hematology, particularly in thrombocytopenic patients. However, there are many other possible differential diagnoses such as other infectious agents of the bacterial and parasitic spectrum as well as malignancies, lymphomas or metastases. Another more specific CT-graphic sign is a cavity caused by invasive growth and displacement of vital tissue by fungal hyphae (Fig. 1B) . The presence of pulmonary tuberculosis in particular could be considered in the differential diagnosis, so that an unconditional clinical correlation must also be made here. In the further course of invasive aspergillosis and especially under effective antifungal therapy, the so-called “air crescent sign” often appears, which is often a crescent-shaped formation within a cavity (Fig. 1C). Morphologically, this is usually a receding infiltrate, which now leaves behind a cavity after previous invasiveness. In addition, wedge-shaped or segmental consolidation may occur. It is important to understand that the absence of such infiltrates or non-specific infiltrates in no way excludes IPA [7]. Other fungal pathogens sometimes have other pulmonary manifestations and can therefore already confirm a suspected diagnosis on CT; these include in particular the aforementioned mucormycosis (“inverse halo sign”) or abscessing metastases as manifestations of bloodstream infections caused by yeasts such as Candida spp. which, however, do not usually cause pneumonia themselves. If invasive pulmonary mycosis is still suspected, a targeted bronchoalveolar lavage (BAL) of the area identified on CT should be performed with preservation of multiple samples for sending to microbiology, molecular biology, serology and pathology. A pulmonary biopsy should be performed if the bronchial system is conspicuous in the BAL or if mold is already macroscopically visible. In hematology, this is often not possible due to severe thrombocytopenia, but should be evaluated in any case.
Microbiology
The microbiological detection of Aspergillus spp. is based on specific methods that are not identical to the test methods for the detection of bacteria. Therefore, the microbiology laboratory must be notified of suspected Aspergillus pneumonia so that the sample can be processed accordingly.
The detection of Aspergillus in patient samples that do not originate from a primary sterile compartment must always be interpreted with care and in conjunction with all available findings. Aspergillus spp. are ubiquitous environmental germs that are regularly detected even without clinical relevance. Instead, the detection may be an expression of transient colonization or environmental contamination. The latter must be taken into consideration, especially in the case of highly sensitive nucleic acid detection using PCR, as this method not only detects vital aspergilli, but also nucleic acid residues of dead pathogens.
Microscopy of primary samples: While routine Gram staining is well suited for the microscopic detection of bacteria or Candida spp. it is not recommended for molds. Fungal filaments (hyphae) of Aspergillus spp. and other molds can be better visualized with the help of optical brighteners (e.g. Calcofluor-White) (Fig. 2 ).Aspergillus hyphae are narrow (3-6 µm) and have a regular septation, branches are usually acute-angled. However, it must be emphasized that the reliable identification of moulds at the genus or species level is generally not possible with this method, as microscopic identification is dependent on the formation of characteristic fruit forms (sporulation) under standardized culture conditions. The sensitivity of microscopic examination is unsatisfactory and is at best around 50% in the case of invasive aspergillosis [8].
Fungal culture: The fungal culture must be explicitly requested in the laboratory, as special culture media (e.g. Sabouraud glucose agar, malt agar) are used and the incubation temperature and time differ from those of the standard bacterial culture. The culture rate is influenced by the quality and volume of the sample material, by the suitability of the culture media and incubation conditions used, and by sample pretreatment and antifungal therapy of the patient. Despite adapted cultivation conditions, cultural mold detection is difficult and less sensitive than bacterial culture. Nevertheless, it should not be dispensed with, as cultural cultivation enables the precise identification of Aspergillus spp. Traditionally, this is done on the basis of macroscopic and microscopic characteristics (Fig. 3 and 4) . With the sequence analysis of certain genes, such as the β-tubulin gene, strains that do not develop typical morphological characteristics can also be identified. In addition, it is possible to distinguish between very closely related species (“siblings”) or to discriminate within species complexes. The exact species diagnosis can influence the choice of antifungal therapy, as some Aspergillus speciesexhibit intrinsic resistance, e.g. A. lentulus, a close relative of A. fumigatus . Furthermore, susceptibility testing can be performed with cultured Aspergillus strains to detect acquired resistance. Phenotypic susceptibility testing using the reference method (bouillon microdilution) is complex and only established in specialized laboratories. However, it is possible to obtain an initial indication of resistance via screening procedures using selective nutrient media mixed with antimycotics [8].
Other detection methods: Several commercial PCR test systems are now available for the detection and identification of Aspergillus spp. directly from patient samples. Even if false-positive findings occur, the method is a useful addition to conventional mycological diagnostics. With the detection of cell wall components, further culture-independent examination methods are available. Galactomannan (“Aspergillus antigen”) can be determined from serum and bronchoalveolar lavage, the less specific 1,3-β-D-glucan only from serum. The significance of these biomarkers depends, among other things, on the patient population examined and the reproducibility of a positive measured value [8].
Pathology
If no definitive diagnosis can be made on the basis of the previous diagnosis and the material obtained, material should be obtained again by BAL and a biopsy of a suspicious focus should be performed.
In the histopathological, microscopic examination, fungal hyphae can already be seen in the HE stain; their invasive growth is evidence of the presence of invasive pulmonary aspergillosis – especially in immunocompromised patients – and excludes colonization. In extended histopathological diagnostics, Gomorri-Grocott staining (silver staining) should also be used to clearly identify mold hyphae and their branching, and the width of hyphae should be measured to rule out other mold species such as the aforementioned Mucorales, Fusarium spp. or rarer molds as the causative agent. Histology can also be used for subsequent molecular diagnostics via PCR. The National Reference Center in Jena can already be consulted for diagnosis. In addition, centers of excellence of the European Confederation of Medical Mycology (ECMM) advise treating colleagues on the selection of targeted diagnostics.
Therapy
Treatment of IPA is complex and requires prolonged administration of antimycotics, close monitoring of response and toxicity, and close consultation with thoracic surgery. Various therapeutic approaches have been established.
Prophylaxis: Preventive measures should be taken to avoid mold infections in the home environment of severely immunocompromised patients. Potential exposure exists through houseplants, compost heaps, gardening, poorly maintained ventilation systems, air conditioning units and sanitary facilities. These sources should be avoided; if this is not possible, personal protective equipment with gloves and mouth and nose protection should be worn. For the prophylaxis of superficial colonization with pathogens from the mycological spectrum, in particular of the mucous membranes, which may exhibit dysbiosis due to exposure to antibiotics and medication, local mouth rinsing solutions (amphotericin B-based) and nourishing mucous membrane creams/solutions can be used. However, there is no evidence for the reduction of invasive mycoses. A so-called “low-germ” diet is now not considered beneficial for the prophylaxis of infections from the mycotic spectrum, even in cases of severe immunosuppression
In certain risk populations, primary antifungal prophylaxis with medication is indicated to reduce invasive mycoses, especially candidemia and IPA. These include, in particular, patients with acute leukemia, especially AML, as well as patients after allogeneic stem cell transplantation and lung transplantation. Drug prophylaxis should then be carried out with a mold-active triazole (e.g. posaconazole), which has even been shown to reduce overall mortality in patients with AML [9].
Due to potential drug interactions caused by inhibition of the cytochrome p450 (CYP3A4) enzyme apparatus by triazoles, echinocandins or triazoles with less potent CYP3A4 inhibition (fluconazole, not effective against molds) are also used here with simultaneous administration of calcineurin inhibitors. In patients at high risk who cannot receive systemic prophylaxis with a triazole for this reason (e.g. ALL when administering vinca alkaloids), regular (2-3×/week) monitoring of serum galactomannan can be carried out to detect an increase at an early stage. Another, growing group are patients with hematologic diseases, such as AML, undergoing oral therapy with tyrosine kinase inhibitors or similar molecularly targeted substances. Here, too, drug prophylaxis is sometimes indicated; attention should also be paid to potential drug interactions and side effects of oncological medication in the GP setting [10].
Preemptive and empirical therapy: In addition to the primary prophylactic administration of antimycotics, this can also be carried out empirically or preemptively. In immunocompromised patients with or without neutropenia and antibiotic-refractory fever lasting several days, it is not uncommon in clinical practice to administer a purely empirical antifungal drug, often an echinocandin or azole – without microbiological evidence of invasive mycosis. If empirical therapy is given to patients on antifungal prophylaxis, a change of class is recommended, often to liposomal amphotericin B. All three antifungal classes are approved for the empirical treatment of febrile neutropenia. It should be noted here that this approach is associated with a higher rate of side effects and higher costs than, for example, the use of a drug for the treatment of cancer. a preemptive approach. This refers to the administration of antimycotics to high-risk patients with corresponding clinical symptoms and clear indications of invasive mycosis. These may include typical fungal infiltrates in the CT thorax or positive biomarker monitoring (e.g. galactomannan). In a prospective study, this therapeutic approach was evaluated as non-inferior to empirical antifungal therapy with regard to the endpoint of survival [12].
Targeted therapy: As soon as invasive pulmonary aspergillosis is proven, targeted antifungal therapy should be given. Due to the disease burden of the patient population and the high morbidity and mortality rates, initial treatment is usually carried out by IPA in an inpatient setting.
Three subtype classes are available for the treatment of invasive aspergillosis. Standard therapy consists of oral or intravenous administration of a triazole such as voriconazole, posaconazole or isavuconazole. Fluconazole is not effective against molds. Liposomal amphotericin B is also approved for the treatment of IPA. Echinocandins (anidulafungin, caspofungin, micafungin) can be administered as second-line therapy in the event of non-response or intolerance to previous therapy or as a combination partner [2].
Triazoles offer the possibility of early oral therapy after initial intravenous therapy initiation. All three triazoles mentioned require a loading dose on days 1 and 2 of therapy, after which voriconazole is administered twice daily and isavuconazole and posaconazole once daily. In addition to the tablet form, posaconazole is also available as an oral suspension to be administered 3 times a day, although the pharmacokinetics vary considerably.
With regard to adverse drug reactions, the triazoles are associated with gastrointestinal effects (nausea, vomiting, diarrhea), in particular hepatotoxicity and QTc time prolongation. However, a shortening of the QTc interval has also been described for isavuconazole in studies. Cutaneous (exanthema) and neurological (paresthesias, neuropathies, dizziness) side effects have also been described for posaconazole in rare cases. In addition to specific neuro-psychiatric side effects such as changes in color vision, hallucinations and encephalopathy, voriconazole has also shown an increased risk of developing squamous cell carcinoma with prolonged administration.
Liposomal amphotericin B is available intravenously and should be administered once daily at a dose of 3 mg/kg body weight. Due to hypokalemia, infusion reactions and nephrotoxicity, administration should only be carried out in an inpatient setting. Non-liposomal forms of amphotericin B should no longer be administered today due to unacceptable toxicity.
Echinocandins (anidulafungin, caspofungin, micafungin) can also currently only be administered intravenously. They should rather be used as a combination partner with an azole in severe infections or in the event of a non-response to IPA. It is administered once a day. A new echinocandin, Rezafungin, which has been approved for once-weekly administration in the USA since 2023, could simplify long-term therapy. Other new, orally available substances such as ibrexafungerp and olorofim represent promising future treatment options.
Therapeutic drug monitoring (TDM) with target levels of 1.0-5.5 mg/dl is recommended by guidelines with a high level of evidence for voriconazole due to its pronounced adverse effects that correlate with plasma levels.
The duration of IPA therapy depends on the clinical and radiological response as well as the immune status of the treated patient. As a rule, this should be given for at least four to six weeks; in patients with ongoing immunosuppression, the duration of therapy can be extended by months and switched to secondary prophylaxis in the course of treatment.
The clinical and radiological response should be closely monitored. The clinical symptoms should subside after just a few days. In the case of pronounced infestation with angioinvasion, however, these can last longer and become clinically apparent as hemoptysis, especially when the infiltrates decrease under antifungal therapy. CT thoraces to assess the response of the infiltrates should be performed on days 7, 14 and 28 after diagnosis, and beyond if necessary [2].
Surgical resection of the persistent focus of infection should be evaluated for infections that are refractory and localized (e.g. one lung lobe) under ongoing immunosuppression. Centers of Excellence of the European Confederation of Medical Mycology (ECMM) are certified in the diagnosis and treatment of mycoses, advise treating colleagues in the selection of targeted diagnostics, selection and management of therapy and are available for the evaluation of inclusion in a clinical trial. ECMM experts have developed “EQUAL scores” for various entities of invasive mycoses, which weight current guideline recommendations for diagnostics and therapy on the basis of a point value and thus make them measurable. These recommendations have been validated in prospective studies [13].
EQUAL Score Cards are available as handy pocket formats and can be downloaded free of charge in many languages at www.ecmm.info/equal-scores. A summary of the EQUAL Aspergillus score is shown in Table 2 [13]. In summary, IPA is a serious infectious disease in immunocompromised patients with a high mortality rate, which requires rapid, targeted and multidisciplinary collaboration and a high level of expertise in diagnosis and treatment. Guidelines and simplified pocket cards (EQUAL scores) from the microbiological-infectiological societies are available and can provide support.
Take-Home Messages
- In immunosuppressed patients with unclear fever and/or respiratory syndromes, invasive mycosis, especially pulmonary aspergillosis as the most common entity, should be considered at an early stage when selecting diagnostic tests.
- Diagnostics are interdisciplinary and include CT thorax, which can reveal evidence of IPA; serology with Aspergillus antigen (=galactomannan) and broncho-alveolar lavage with shipment for culture, pan-fungal and Aspergillus-specific PCR and galactomannan.
- An early start to treatment for invasive pulmonary aspergillosis is
is associated with better survival and should therefore be carried out with a mold-active triazole or liposomal amphotericin B if highly suspected and at the latest if a positive diagnosis is available.
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InFo PNEUMOLOGY & ALLERGOLOGY 2023; 5(4): 8-15
