A Report of 2 Cases of Disseminated Invasive Aspergillosis with Myocarditis in Immunocompromised Patients

Invasive aspergillosis is a life-threatening opportunistic infection that occurs most often in immunocompromised patients, especially those with either hematological malignancies treated with chemotherapy or in organ transplant recipients. Here, we report 2 cases of disseminated invasive aspergillosis with myocarditis proven pathologically by autopsy. These 2 cases occurred in immunocompromised patients: the first patient received chemotherapy for acute myeloid leukemia with myelodysplasia-related changes (AML-MRC); the second patient received methotrexate therapy for rheumatoid arthritis (RA). Invasive aspergillosis with myocarditis has high morbidity and mortality rates, therefore it is necessary to make prompt diagnosis and to start intensive treatments quickly.


Introduction
Aspergillus species have the ability to cause aggressive invasive infections in a variety of major organs, including the lungs, heart, central nervous system, and gastrointestinal tract.These organisms most commonly affect a variety of immunocom promised patients with hematological malignancies, especially those who had acute leukemia under receiving chemotherapy, or organ-transplant patients [1].Here, we present 2 cases of disseminated invasive aspergillosis with myocarditis in immunocom promised patients who received either chemotherapy for acute myeloid leukemia (AML) or immunosuppressive therapy for rheumatoid arthritis (RA).
Anti-fungal therapy with liposomal amphotericin B (AMPH-B), as well as anti-micobacterial therapy was initiated.However, the patient developed severe neutropenia with a WBC count of 0.2 × 10 3 /mm −3 and he went into shock.An electrocardiogram (EKG) showed both an elevated ST-T segment in the broad leads and decreased wall motion, with a low echo region at the apex.The patient's clinical course strongly indicated a diagnosis of fungal myocarditis, therefore the therapy with methylpredonisoline (m-PSL; 0.5 mg/kg /day, 3 days) was initiated.Micafungin (MCFG) was also initiated in addition to liposomal AMPH-B, but no response was obtained and the patient died.The autopsy findings showed disseminated invasive aspergillosis involving the lungs, heart, liver and gastrointestinal tracts (Figures 1(a)-(f)).Leukemia cells were not detected in the extremely hypocellular bone marrow, indicating that leukemia progression had been denied.

Case Report 2
An 81-year-old woman was admitted to our hospital in October 2011 because of a worsening cough, sore throat, hemorrhagic stomatitis and melena.Progressive pancytopenia; WBC count, 0.23 × 10 3 /mm −3 with 20% granulocytes and 80% lymphocytes was also present.She had been diagnosed with Rhematoid artiritis (RA) at another clinic at age 76 and had started undergoing hemodialysis for chronic renal failure in May 2011.Ten days before her admission, a single dose of methotrexate (MTX; 2 mg) was administered to control the disease activity of RA.On her admission, she had high fever and Escherichia coli was detected in the blood culture, therefore anti-micobacterial therapy was immediately initiated.She was also treated with granulocyte-colony stimulating factor (G-CSF: 250 µg/day) intraveneously.During her hospitalization, the pancytopenia gradually improved.However, the patient suddenly lost her consciousness and went into shock.Chest imaging did not show any abnormal signs.Computed tomography scan of the head revealed that she had multiple cerebral infarctions.Since Candida tropicalis and Bacteroides fragilis were simultaneously detected in the blood culture, the therapy with MCFG and metronidazole was started.However, despite these supportive treatments, her respiratory symptoms worsened and she died.An EKG showed no definite change.The autopsy findings showed disseminated invasive aspergillosis affecting the lungs, heart, spleen, and kidneys (Figures 2(a)-(f)).The bone marrow showed almost normal cellularity.Although the cause of the cerebral infarctions could have been embolization caused by Aspergillus species, the patient's family did not consent to opening her skull for autopsy.

Discussion
Invasive aspergillosis is a serious opportunistic infection that occurs in a variety of immunocompromised patients, such as those receiving chemotherapy for hematological malignancies or organ-transplant patients and it has high morbidity and mortality rates.Its incidence has been estimated at around 10% -14%, and the mortality rates may be as high as 27% -60% in spite of antifungal therapy [2,3].In order to reduce mortality rates, to make a diagnosis quickly and to initiate intensive treatments are necessary.However, premortem diagnosis of invasive aspergillosis is difficult, because cultures were unreliable and to assess to the sites was limited [2,3].
Autopsy results in Japan have shown that the prevalence of visceral mycoses markedly increased from 3.7% in 1993 to a peak of 4.6% in 2009.Aspergillus became the predominant causative pathogen and the rate of aspergillosis exceeded that of candidiasis in 1994; it has continued to increase conspicuously and was even higher in 2001 (46.0%) [4,5].On the other hand, since 2001, there has been a slight decrease in the frequency of aspergillosis because of the development of novel, effective antifungal agents and advancements in non-invasive diagnostic tools.In addition, an increasing number of cases with collagen diseases as the underlying cause of visceral mycosis and a decreasing number of cases with leukemia as the underlying disorders were noted.However, overall, invasive aspergillosis are still one of the most serious infections not only in patients with malignant diseases but also in those with recieving steroids or immunosuppressive therapy for a variety of diseases [4,5].
The lung is the most common primary site of invasive aspergillosis with secondary hematogenous dissemination.Once Aspergillus forms abscesses in the lung, it can invade blood vessels and disseminate to the entire body.Cases of cardiac invasive fungal infection is difficult to prove premortem and has an especially high mortality rate.In the case of Aspergillus endocarditis, the mortality rate reaches 96% if the patient is treated by medication alone, and 68% even if surgical resection is performed [6][7][8][9].It is difficult to make a diagnosis of aspergillus myocarditis, because it requires histopathological evidence of characteristic fungal hyphae or positive culture results, yielding aspergillus species from myocardial specimens.Our patients discussed here showed disseminated invasive aspergillosis with myocarditis that was proven by autopsy.In Case 1, hemorrhagic necrosis was shown in the left ventricle and the presence of fungal hyphae in the myocardium was demonstrated.In Case 2, Abscess lesion observed in the myocardium and fungal hyphae in the myocardium was also detected.Both cases had several common risk factors for developing disseminated invasive aspergillosis.These factors include the underlying disease, long-term immunosuppressive therapies including steroids, prolonged neutropenia or neutrophil dysfunction and the use of broad-spectrum antibiotics which affect mucosal barriers [2,3].Leukemia and MDS have been the major underlying diseases to develop invasive aspergillosis, followed by solid cancers and bacterial infections and only a few reports have been published in which invasive aspergillosis with myocarditis was proven pathologically [4,[6][7][8]10].In Case 2, pancytopenia due to severe bone marrow suppression occurred after the patient received MTX.It was considered to be an adverse effect of MTX due to renal insufficiency and it may became the risk factor to develop invasive aspergillosis with myocarditis in Case 2. In most reported cases, it is difficult to determine what to extent the myocarditis contributed to the patient' death.The clinical course of aspergillus myocarditis is extremely short and results in fatality, so EKG changes consistent with severe symptoms was not necessarily revealed.Williams reported only 6 cases of 37 cases of secondary aspergillosis in which the myocardium was involved had electrocardiographic changes consistent with ischemia of myocardial infarction [11].In our case, Case 1 showed positive electrocardiographic and echographic changes, correlating with ischemia.We believe that our cases certainly died of asperugillus myocarditis and subsequent heart failure, by its extremely quick clinical course, laboratory and necropsy findings and the absence of other alternative etiologocal findings.

Conclusion
In conclusion, we reported 2 cases of disseminated invasive aspergillosis with myocarditis, which were all confirmed by autopsy.These 2 cases involved immunocompromised patients receiving chemotherapy or immunosuppressive therapies.Invasive aspergillosis, especially in cardiac infection including myocarditis, has high morbidity and mortality rates.It is therefore necessary to provide early diagnosis and intensive treatments, including novel antifungal therapies.

Figure 1 .
Figure 1.Autopsy results for Case 1, with evidence of invasive fungal infection (hyphae of Aspergillus).(a) Macroscopic image of the right upper lobe of the lung showing cavity lesions with hemorrhagic necrosis (arrow); (b) Diffuse hemorrhagic necrosis was observed in the lung lesion (hematoxylin and eosin [HE] stain; original magnification, ×100); (c) Grocott stain for Y-angle branching hyphae demonstrating massive lung tissue invasion (original magnification, ×400); (d) Macroscopic image of the heart, showing hemorrhagic necrosis in the left ventricle (arrow); (e) Inflammatory infiltration in the necrotic myocardium (HE stain; original magnification, ×100); (f) Grocott staining for the presence of fungi in the myocardium (Grocott stain; original magnification, ×400).

Figure 2 .
Figure 2. Autopsy results for Case 2, with evidence of invasive fungal infection (hyphae of Aspergillus); (a) Macroscopic image of the left upper lobe of the lung, showing a 10-mm nodule (arrow); (b) Fungal hyphae are seen proliferating radially from the lung wall (HE staining; original magnification, ×100); (c) Grocott staining for Y-angle branching fungal hyphae in the lung tissue (original magnification; ×400); (d) Macroscopic image of the heart; (e) Abscess lesion observed in the myocardium (HE stain; original magnification, ×100); (f) Grocott staining for the presence of fungal hyphae in the myocardium (original magnification, ×400).