Paradoxal Trends in Azole-Resistant Aspergillus fumigatus in a National Multicenter Surveillance Program, the Netherlands, 2013-2018.

We investigated the prevalence of azole resistance of Aspergillus fumigatus isolates in the Netherlands by screening clinical A. fumigatus isolates for azole resistance during 2013-2018. We analyzed azole-resistant isolates phenotypically by in vitro susceptibility testing and for the presence of resistance mutations in the Cyp51A gene. Over the 6-year period, 508 (11%) of 4,496 culture-positive patients harbored an azole-resistant isolate. Resistance frequency increased from 7.6% (95% CI 5.9%-9.8%) in 2013 (58/760 patients) to 14.7% (95% CI 12.3%-17.4%) in 2018 (112/764 patients) (p = 0.0001). TR34/L98H (69%) and TR46/Y121F/T289A (17%) accounted for 86% of Cyp51A mutations. However, the mean voriconazole MIC of TR34/L98H isolates decreased from 8 mg/L (2013) to 2 mg/L (2018), and the voriconazole-resistance frequency was 34% lower in 2018 than in 2013 (p = 0.0001). Our survey showed changing azole phenotypes in TR34/L98H isolates, which hampers the use of current PCR-based resistance tests.

Lateral flow assays for diagnosing invasive pulmonary aspergillosis in adult hematology patients: A comparative multicenter study.

Fast diagnosis of invasive pulmonary aspergillosis (IPA) is essential as early adequate therapy improves survival. However, current microbiological methods suffer from a low sensitivity or a long turnaround time, often as a result of batching. Recently, two lateral flow assays for diagnosing IPA have been CE (Conformité Européenne)-marked and commercialized. These assays can be used for fast single sample testing. However, clinical validation and comparative studies are lacking. We therefore sought to evaluate and compare these assays in adult hematology patients. We retrospectively tested 235 bronchoalveolar lavage fluid (BALf) samples of adult hematology patients from four centers using the AspLFD (OLM Diagnostics) and the sona Aspergillus galactomannan LFA (IMMY). Both tests were read out independently by two researchers and by a digital reader. We included 11 patients with proven IPA, 64 with probable IPA, 43 with possible fungal disease, and 117 controls with no signs of IPA. In cases of proven IPA, the performance of both assays was similar. In cases of proven and probable IPA, we found an identical specificity for both assays, but a higher sensitivity (0.83 vs 0.69, P = .008) and a better negative predictive value (0.89 vs 0.82, P = .009) for the LFA. Digital readout improved the diagnostic performance of both tests. In conclusion, both assays showed a good performance for the diagnosis of IPA in BALf from adult hematology patients. Results were further improved by using a digital reader, especially for weakly positive results.

Mycovirus therapy for invasive pulmonary aspergillosis?

With the current revived interest in the use of bacteriophages for the treatment of bacterial infections, the study of mycoviruses as novel therapeutic solutions for invasive aspergillosis is the logical next step. Although ssRNA, dsRNA, and ssDNA mycoviruses have been identified, the majority of characterised mycoviruses have dsRNA genomes. Prevalence of dsRNA mycoviruses in Aspergillus spp. varies, and mycoviruses can have different effects on their fungal hosts: hypovirulence, hypervirulence, or a killer phenotype. Therapeutically, extracellular transmission of the mycovirus is essential. DsRNA mycoviruses lack an extracellular phase; however, a single ssDNA mycovirus with homologues in Aspergillus genomes has been described with an extracellular mode of transmission. Mycoviruses can induce hypovirulence or a killer phenotype, and both can be exploited therapeutically. Mycoviruses inducing hypovirulence have been used to control chestnut blight, however for aspergillosis no such mycovirus has been identified yet. Mycovirus encoded killer toxins or anti-idiotypic antibodies and killer peptides derived from these have been demonstrated to control fungal infections including aspergillosis in animals. This indicates that mycoviruses inducing both phenotypes could be exploited therapeutically as long as the right mycovirus has been identified.

Chitinase Induction Prior to Caspofungin Treatment of Experimental Invasive Aspergillosis in Neutropenic Rats Does Not Enhance Survival.

Host chitinases, chitotriosidase and acidic mammalian chitinase (AMCase), improved the antifungal activity of caspofungin (CAS) against Aspergillus fumigatus in vitro These chitinases are not constitutively expressed in the lung. Here, we investigated whether chitosan derivatives were able to induce chitinase activity in the lungs of neutropenic rats and, if so, whether these chitinases were able to prolong survival of rats with invasive pulmonary aspergillosis (IPA) or of rats with IPA and treated with CAS. An oligosaccharide-lactate chitosan (OLC) derivative was instilled in the left lung of neutropenic rats to induce chitotriosidase and AMCase activities. Rats instilled with OLC or with phosphate-buffered saline (PBS) were subsequently infected with A. fumigatus and then treated with suboptimal doses of CAS. Survival, histopathology, and galactomannan indexes were determined. Instillation of OLC resulted in chitotriosidase and AMCase activities. However, instillation of OLC did not prolong rat survival when rats were subsequently challenged with A. fumigatus In 5 of 7 rats instilled with OLC, the fungal foci in the lungs were smaller than those in rats instilled with PBS. Instillation of OLC did not significantly enhance the survival of neutropenic rats challenged with A. fumigatus and treated with a suboptimal dosage of CAS. Chitotriosidase and AMCase activities can be induced with OLC, but the presence of active chitinases in the lung did not prevent the development of IPA or significantly enhance the therapeutic outcome of CAS treatment.

The diagnosis and treatment of invasive aspergillosis in Dutch haematology units facing a rapidly increasing prevalence of azole-resistance. A nationwide survey and rationale for the DB-MSG 002 study protocol.

Patients with haematological malignancies are at risk for invasive fungal diseases (IFD). A survey was conducted in all Dutch academic haematology centres on their current diagnostic, prophylactic and therapeutic approach towards IFD in the context of azole-resistance. In all 8 centres, a haematologist and microbiologist filled in the questionnaire that focused on different subgroups of haematology patients. Fungal prophylaxis during neutropaenia was directed against Candida and consisted of fluconazole and/or amphotericin B suspension. Mould-active prophylaxis was given to acute myeloid leukaemia patients during chemotherapy in 2 of 8 centres. All centres used azole prophylaxis in a subset of patients with graft-versus-host disease. A uniform approach towards the diagnosis and treatment of IFD and in particular azole-resistant Aspergillus fumigatus was lacking. In 2017, all centres agreed to implement a uniform diagnostic and treatment algorithm regarding invasive aspergillosis with a central role for comprehensive diagnostics and PCR-based detection of azole-resistance. This study (DB-MSG 002) will re-evaluate this algorithm when 280 patients have been treated. A heterogeneous approach towards antifungal prophylaxis, diagnosis and treatment was apparent in the Netherlands. Facing triazole-resistance, consensus was reached on the implementation of a uniform diagnostic approach in all 8 centres.

Echinocandin to fluconazole step-down therapy in critically ill patients with invasive, susceptible Candida albicans infections.

Invasive Candida spp. infections are increasingly diagnosed in critically ill patients. For initial treatment, an echinocandin is recommended with a possible step-down to fluconazole when the patients' condition is improving and the isolate appears susceptible, but there are no data to support such policy. We studied the safety and efficacy of step-down therapy in critically ill patients with culture proven deep seated or bloodstream infections by C. albicans susceptible to fluconazole. All patients admitted into the intensive care unit from January 2010 to December 2014, who had a culture proven invasive C. albicans infection and received initial treatment with an echinocandin for at least 4 days were included. Data on patient characteristics, treatment and vital outcomes were assessed. Of the 56 patients, 32 received step-down fluconazole therapy, at median day 5, whereas the echinocandin was continued in the other 24. No differences where seen in baseline characteristics or risk factors for invasive C. albicans infection between the two groups. Response rates were similar and no difference where seen in 28-day or 90-day mortality between the groups. Step-down therapy to fluconazole may be safe and effective in critically ill patients with invasive infections by C. albicans, susceptible to fluconazole, who have clinically improved as early as 4 days after start of treatment with an echinocandin.

Validation of a new Aspergillus real-time PCR assay for direct detection of Aspergillus and azole resistance of Aspergillus fumigatus on bronchoalveolar lavage fluid.

Azole resistance in Aspergillus fumigatus is increasingly reported. Here, we describe the validation of the AsperGenius, a new multiplex real-time PCR assay consisting of two multiplex real-time PCRs, one that identifies the clinically relevant Aspergillus species, and one that detects the TR34, L98H, T289A, and Y121F mutations in CYP51A and differentiates susceptible from resistant A. fumigatus strains. The diagnostic performance of the AsperGenius assay was tested on 37 bronchoalveolar lavage (BAL) fluid samples from hematology patients and 40 BAL fluid samples from intensive care unit (ICU) patients using a BAL fluid galactomannan level of ≥1.0 or positive culture as the gold standard for detecting the presence of Aspergillus. In the hematology and ICU groups combined, there were 22 BAL fluid samples from patients with invasive aspergillosis (IA) (2 proven, 9 probable, and 11 nonclassifiable). Nineteen of the 22 BAL fluid samples were positive, according to the gold standard. The optimal cycle threshold value for the presence of Aspergillus was <36. Sixteen of the 19 BAL fluid samples had a positive PCR (2 Aspergillus species and 14 A. fumigatus samples). This resulted in a sensitivity, specificity, and positive and negative predictive values of 88.9%, 89.3%, 72.7%, and 96.2%, respectively, for the hematology group and 80.0%, 93.3%, 80.0%, and 93.3%, respectively, in the ICU group. The CYP51A real-time PCR confirmed 12 wild-type and 2 resistant strains (1 TR34-L98H and 1 TR46-Y121F-T289A mutant). Voriconazole therapy failed for both patients. The AsperGenius multiplex real-time PCR assay allows for sensitive and fast detection of Aspergillus species directly from BAL fluid samples. More importantly, this assay detects and differentiates wild-type from resistant strains, even if BAL fluid cultures remain negative.

An elevated pro-inflammatory cytokine response is linked to development of amphotericin B-induced nephrotoxicity.

OBJECTIVES: The underlying mechanism for amphotericin B-induced acute kidney injury (AKI) remains poorly understood and may be immunologically mediated. We assessed whether the development of nephrotoxicity is linked to a distinct cytokine profile in patients receiving amphotericin B deoxycholate (AmBD). PATIENTS AND METHODS: In 58 patients who received AmBD, circulating serum interleukin (IL)-6, IL-8 and IL-10 were measured at baseline, week 1 and week 2 of antifungal treatment and correlated to the development of renal impairment. The Cox proportional hazards model approach was adopted for analysis. RESULTS: The P value was 0.026 for the overall effect of IL-6 on time to development of AKI. An increasing or non-receding IL-6 trend by week 1 of AmBD treatment (followed by a decreasing or non-receding IL-6 trend from week 1 to week 2) correlated with an increased likelihood of nephrotoxicity [hazard ratio (HR) 6.93, P value 0.005 and HR 3.46, P value 0.035, respectively]. Similarly, persistently increasing IL-8 levels were linked to a 3.84-fold increased likelihood of AKI. CONCLUSIONS: In patients receiving AmBD, persistence of an elevated pro-inflammatory cytokine milieu is associated with a predisposition to drug-related kidney injury.

Unrecognized norovirus infections in health care institutions and their clinical impact.

Noroviruses (NoVs) have emerged as the leading cause of acute viral gastroenteritis (GE) in humans. Although diagnostic facilities have greatly improved, significant underdiagnosis of NoV in hospitals may still occur, thereby increasing clinical burden and nosocomial spread. We evaluated the underdiagnosis of sporadic NoV infections in a tertiary care hospital and estimated its clinical impact. From December 2008 until July 2009, fecal samples specifically referred for bacterial but not viral examination were retrospectively tested for NoV by real-time PCR. The clinical and virological data from patients with undiagnosed NoV infection (missed patients) were evaluated and compared with those from patients with recognized NoV. During the study period, 45 patients with undiagnosed NoV were detected, whereas 50 patients were regularly diagnosed. The missed NoV cases were more frequently adults than children (80% versus 46%; P < 0.001). The viral load levels did not differ between the diagnosed and missed patients, but missed patients more frequently presented without diarrhea (20% versus 4%; P < 0.07). The newly admitted missed NoV cases with GE underwent more diagnostic imaging (24% versus 4%; P < 0.01) and tended to be hospitalized longer. When missed-NoV patients were included, the number of nosocomial clusters doubled and missed patients were index cases in 5 of the 6 clusters. These data indicate that NoV infections are frequently missed despite routine laboratory testing and demonstrate that underdiagnosis of NoV patients is associated with costly abdominal imaging and nosocomial clustering. Awareness of NoV infection in adult patients and education about the importance of viral GE should be increased.

Early serum galactomannan trend as a predictor of outcome of invasive aspergillosis.

The monitoring and prediction of treatment responses to invasive aspergillosis (IA) are difficult. We determined whether serum galactomannan index (GMI) trends early in the course of disease may be useful in predicting eventual clinical outcomes. For the subjects recruited into the multicenter Global Aspergillosis Study, serial GMIs were measured at baseline and at weeks 1, 2, and 4 following antifungal treatment. Clinical response and survival at 12 weeks were the outcome measures. GMI trends were analyzed by using the generalized estimation equation approach. GMI cutoffs were evaluated by using receiver-operating curve analyses incorporating pre- and posttest probabilities. Of the 202 study patients diagnosed with IA, 71 (35.1%) had a baseline GMI of ≥ 0.5. Week 1 GMI was significantly lower for the eventual responders to treatment at week 12 than for the nonresponders (GMIs of 0.62 ± 0.12 and 1.15 ± 0.22, respectively; P = 0.035). A GMI reduction of >35% between baseline and week 1 predicted a probability of a satisfactory clinical response. For IA patients with pretreatment GMIs of <0.5 (n = 131; 64.9%), GMI ought to remain low during treatment, and a rising absolute GMI to >0.5 at week 2 despite antifungal treatment heralded a poor clinical outcome. Here, every 0.1-unit increase in the GMI between baseline and week 2 increased the likelihood of an unsatisfactory clinical response by 21.6% (P = 0.018). In summary, clinical outcomes may be anticipated by charting early GMI trends during the first 2 weeks of antifungal therapy. These findings have significant implications for the management of IA.

The Y238X stop codon polymorphism in the human ß-glucan receptor dectin-1 and susceptibility to invasive aspergillosis.

BACKGROUND: Dectin-1 is the major receptor for fungal ß-glucans on myeloid cells. We investigated whether defective Dectin-1 receptor function, because of the early stop codon polymorphism Y238X, enhances susceptibility to invasive aspergillosis (IA) in at-risk patients. METHODS: Association of Dectin-1 Y238X polymorphism with occurrence and clinical course of IA was evaluated in 71 patients who developed IA post hematopoietic stem cell transplantation (HSCT) and in another 21 non-HSCT patients with IA. The control group consisted of 108 patients who underwent HSCT. Functional studies were performed to investigate consequences of the Y238X Dectin-1 polymorphism. RESULTS: The Y238X allele frequency was higher in non-HSCT patients with IA (19.0% vs 6.9%-7.7%; P < .05). Heterozygosity for Y238X polymorphism in HSCT recipients showed a trend toward IA susceptibility (odds ratio, 1.79; 95% CI, .77-4.19; P = .17) but did not influence clinical course of IA. Functional assays revealed that although peripheral blood mononuclear cells with defective Dectin-1 function due to Y238X responded less efficiently to Aspergillus, corresponding macrophages showed adequate response to Aspergillus. CONCLUSIONS: Dectin-1 Y238X heterozygosity has a limited influence on susceptibility to IA and may be important in susceptible non-HSCT patients. This is partly attributable to redundancy inherent in the innate immune system. Larger studies are needed to confirm these findings.

Early proinflammatory cytokines and C­reactive protein trends as predictors of outcome in invasive Aspergillosis.

BACKGROUND: Monitoring treatment response in invasive aspergillosis is challenging, because an immunocompromised host may not exhibit reliable symptoms and clinical signs. Cytokines play a pivotal role in mediating host immune response to infection; therefore, the profiling of biomarkers may be an appropriate surrogate for disease status. METHODS: We studied, in a cohort of 119 patients with invasive aspergillosis who were recruited in a multicenter clinical trial, serum interleukin (IL)-6, IL­8, IL­10, interferon­Î³, and C­reactive protein (CRP) trends over the first 4 weeks of therapy and correlated these trends to clinical outcome parameters. RESULTS: Circulating IL­6 and CRP levels were high at initiation of therapy and generally showed a downward trend with antifungal treatment. However, subjects with adverse outcomes exhibited a distinct lack of decline in IL­6 and CRP levels at week 1, compared with responders (P = .02, for both IL­6 and CRP). Nonresponders also had significantly elevated IL­8 levels (P = .001). CONCLUSIONS: High initial IL­8 and persistently elevated IL­6, IL­8, and CRP levels after initiation of treatment may be early predictors of adverse outcome in invasive aspergillosis. Cytokine and CRP profiles could be used for early identification of patients with a poor response to antifungal treatment who may benefit from more­aggressive antimicrobial regimens.

Anti-Aspergillus human host defence relies on type 1 T helper (Th1), rather than type 17 T helper (Th17), cellular immunity.

Both interferon-gamma-producing type 1 T helper (Th1)- and interleukin-17 (IL-17)-producing Th17 cells have been proposed to be involved in anti-fungal host defence. Although invasive aspergillosis is one of the most severe human fungal infections, little is known regarding the relative importance of the Th1 versus Th17 cellular immune pathways for the human anti-Aspergillus host defence. Using human peripheral blood mononuclear cells and a system consisting of monocyte-derived macrophages with lymphocytes, we found that Aspergillus fumigatus is a weak inducer of human IL-17 but induces a strong Th1 response. These data were validated by the very low IL-17 levels in bronchoalveolar lavage fluid and serum of patients with invasive aspergillosis. Surprisingly, live A. fumigatus reduced IL-17 production induced by mitogenic stimuli. This effect was mediated through the propensity of A. fumigatus to metabolize tryptophan and release kynurenine, which modulates the inflammatory response through inhibition of IL-17 production. In conclusion, A. fumigatus does not stimulate production of IL-17 and human host defence against aspergillosis may not rely on potent Th17 responses.

Modulation of Toll-like receptor 2 (TLR2) and TLR4 responses by Aspergillus fumigatus.

Toll-like receptor (TLR)-based signaling pathways in the host may be modulated by pathogens during the course of infection. We describe a novel immunomodulatory mechanism in which Aspergillus fumigatus conidia induce attenuation of TLR2- and TLR4-mediated interleukin (IL)-6 and IL-1beta proinflammatory responses in human mononuclear cells with suppression of IL-1beta mRNA transcription. Background TLR2 and TLR4 mRNA transcription was not influenced. A. fumigatus conidia induced TLR2 internalization and uptake into the phagosome with a resultant decrease in surface receptor expression. A. fumigatus hyphae, on the other hand, selectively downregulated the TLR4-mediated response. These novel immunosuppressive effects may facilitate the invasiveness of A. fumigatus.

Fungal strategies for overcoming host innate immune response.

A successful pathogen is one that is able to effectively survive and evade detection by the host innate immune defense. Fungal pathogens have adopted strategies which evade host defense and eventually cause disease in at-risk patients. Shielding of stimulatory surface recognition molecules, shedding of decoy components, induction of anti-inflammatory signals, complement evasion and resilient survival capacity are successful evasion mechanisms employed by fungal pathogens. Understanding these complex pathways of immune evasion can potentially contribute to development of novel therapeutic strategies against fungal infections.

Invasive aspergillosis in patients admitted to the intensive care unit with severe influenza: a retrospective cohort study.

BACKGROUND: Invasive pulmonary aspergillosis typically occurs in an immunocompromised host. For almost a century, influenza has been known to set up for bacterial superinfections, but recently patients with severe influenza were also reported to develop invasive pulmonary aspergillosis. We aimed to measure the incidence of invasive pulmonary aspergillosis over several seasons in patients with influenza pneumonia in the intensive care unit (ICU) and to assess whether influenza was an independent risk factor for invasive pulmonary aspergillosis. METHODS: We did a retrospective multicentre cohort study. Data were collected from adult patients with severe influenza admitted to seven ICUs across Belgium and The Netherlands during seven influenza seasons. Patients were older than 18 years, were admitted to the ICU for more than 24 h with acute respiratory failure, had pulmonary infiltrates on imaging, and a confirmed influenza infection based on a positive airway PCR test (influenza cohort). We used logistic regression analyses to determine if influenza was independently associated with invasive pulmonary aspergillosis in non-immunocompromised (ie, no European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group [EORTC/MSG] host factor) influenza-positive patients (influenza case group) compared with non-immunocompromised patients with severe community-acquired pneumonia who had a negative airway influenza PCR test (control group). FINDINGS: Data were collected from patients admitted to the ICU between Jan 1, 2009, and June 30, 2016. Invasive pulmonary aspergillosis was diagnosed in 83 (19%) of 432 patients admitted with influenza (influenza cohort), a median of 3 days after admission to the ICU. The incidence was similar for influenza A and B. For patients with influenza who were immunocompromised, incidence of invasive pulmonary aspergillosis was as high as 32% (38 of 117 patients), whereas in the non-immunocompromised influenza case group, incidence was 14% (45 of 315 patients). Conversely, only 16 (5%) of 315 patients in the control group developed invasive pulmonary aspergillosis. The 90-day mortality was 51% in patients in the influenza cohort with invasive pulmonary aspergillosis and 28% in the influenza cohort without invasive pulmonary aspergillosis (p=0·0001). In this study, influenza was found to be independently associated with invasive pulmonary aspergillosis (adjusted odds ratio 5·19; 95% CI 2·63-10·26; p<0·0001), along with a higher APACHE II score, male sex, and use of corticosteroids. INTERPRETATION: Influenza was identified as an independent risk factor for invasive pulmonary aspergillosis and is associated with high mortality. Future studies should assess whether a faster diagnosis or antifungal prophylaxis could improve the outcome of influenza-associated aspergillosis. FUNDING: None.

Addition of 17-(allylamino)-17-demethoxygeldanamycin to a suboptimal caspofungin treatment regimen in neutropenic rats with invasive pulmonary aspergillosis delays the time to death but does not enhance the overall therapeutic efficacy.

Caspofungin (CAS) which is used as salvage therapy in patients with invasive pulmonary aspergillosis (IPA) inhibits the 1,3-ß-D-glucan synthesis in Aspergillus fumigatus. Inhibiting 1,3-ß-D-glucan synthesis induces a stress response and in an invertebrate model it was demonstrated that inhibiting this response with geldamycin enhanced the therapeutic efficacy of CAS. Since geldamycin itself is toxic to mammalians, the therapeutic efficacy of combining geldamycin with CAS was not studied in rodent models. Therefore in this study we investigated if the geldamycin derivate 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) was able to enhance the therapeutic efficacy of CAS in vitro and in our IPA model in transiently neutropenic rats. In vitro we confirmed the earlier demonstrated synergy between 17-AAG and CAS in ten A. fumigatus isolates. In vivo we treated A. fumigatus infected neutropenic rats with a sub-optimal dose of 0.75 mg/kg/day CAS and 1 mg/kg/day 17-AAG for ten days. Survival was monitored for 21 days after fungal inoculation. It appeared that the addition 17-AAG delayed death but did not improve overall survival of rats with IPA. Increasing the doses of 17-AAG was not possible due to hepatic toxicity. This study underlines the need to develop less toxic and more fungal specific geldamycin derivatives and the need to test such drugs not only in invertebrate models but also in mammalian models.

Phagocytosis and intracellular killing of Candida albicans blastoconidia by neutrophils and macrophages: a comparison of different microbiological test systems.

Polymorphonuclear neutrophils (PMN) and mononuclear phagocytes represent an important first line and effector function in the control of Candida infections. Their relative contribution to host defence is frequently assessed by means of microbiological assays. However, reported results are divergent and might well be associated with study design-related issues. In the present study, we compared frequently used microbiological candidacidal assays, with the purpose of determining the most adequate method for assessment of phagocytosis and intracellular killing. We concluded that microbiological assays using yeast-phagocyte suspensions are inappropriate for the assessment of intracellular killing of Candida blastoconidia by murine macrophages, due to adherence or clumping of cells. In contrast, an adherent monolayer of phagocytes can be applied as a single microbiological assay to independently study the process of phagocytosis and intracellular killing, by exudate peritoneal macrophages as well as exudate peritoneal PMN.

Voriconazole or amphotericin B as primary therapy yields distinct early serum galactomannan trends related to outcomes in invasive aspergillosis.

An improved number of anti-fungal drugs are currently available for the treatment of invasive aspergillosis (IA). While serial galactomannan index (GMI) measurement can be used to monitor response to treatment, the extent to which different anti-fungal regimens can affect galactomannan levels is unknown. In 147 IA patients receiving either voriconazole (VCZ) or conventional amphotericin B (CAB) in a multicentre clinical trial, we performed post-hoc analyses of GMI trends in relation to outcomes. The generalized estimation equations approach was used to estimate changes in the effect size for GMI over time within patients. Patients who received VCZ primary therapy and had good treatment response 12 weeks later showed earlier decreases in GMI values at Week 1 and Week 2 (p = 0.001 and 0.046 respectively) as compared to patients who only received CAB. At end-of-randomized therapy (EORT), which was a pre-set secondary assessment point for all patients who switched from randomized primary (CAB or VCZ) to an alternative anti-fungal drug, treatment failure was associated with increasing GMI at Weeks 1 and 2 in CAB-primary treated patients (p = 0.022 and 0.046 respectively). These distinct trends highlight the variations in GMI kinetics with the use of different anti-fungal drugs and their implications in relation to IA treatment response.