AN APPRAISAL OF THE EPIDEMIOLOGY OF MUCORMYCOSIS

ABSTRACT

Mucormycosis is an angio-invasive mycosis with high morbidity and mortality rates which mainly affects immunocompromised patients. It is no longer an uncommon disease due to the increased incidence of diabetes and use of immunosuppressive agents in the current era. Our objective was to review all cases of proven and probable mucormycosis — according to EORTC criteria — diagnosed from 2000 until 2007 at the University Hospitals Leuven, a 1900-bed tertiary care hospital, to assess the changing epidemiology of the disease. In 45 patients there was microbiological or histopathological evidence for the presence of a member of Mucorales during the hospital stay of which 12 cases fulfilled the criteria for proven mucormycosis and 6 for probable mucormycosis. The overall incidence was 0.042 cases per 10,000 patient days. A slight although not statistically significant increase in incidence was noticeable during the study period. The major site of infection was the lungs (78% of the cases), with haematological malignancy the most common underlying disorder and Rhizopus species the most often suspected etiologic agent. Overall mortality was 55% and co-infections with Aspergillus species, proven or probable, noted in 44% of cases. The highest survival rate was achieved with surgery combined with antifungal therapy.

epidemiology, mucormycosis, tertiary care hospital

Topic:

• lung
• epidemiology
• aspergillus
• mucorales
• mucormycosis
• rhizopus
• surgical procedures, operative
• infections
• mortality
• coinfection
• tertiary care hospitals

CHAPTER ONE INTRODUCTION

1.1 Background of the study

Mucormycosis is an infectious disease caused by fungi belonging to the order Mucorales (subphylum Mucormycotina) [1,2]. It has an angio-invasive pathology with high morbidity and mortality rates and mainly affects immuno-compromised patients [2,3]. Members of the family Mucoraceae are the most frequent cause of mucormycosis [3]. After inhalation of airborne spores, or in rare cases after percutaneous inoculation or ingestion, the fungi reach the lungs or the subcutaneous tissues [3]. Mononuclear and polynuclear phagocytes are able to kill the spores in healthy individuals [3]. Immunocompromised neutropenic patients and patients with impairment in phagocytosis (e.g., as a consequence of hyperglycaemia) are accordingly at higher risk for developing mucormycosis [3]. In addition, patients receiving corticosteroid therapy, or are transplant recipients and those with burn wounds or trauma [3],4] are at risk of infection. Deferoxamine treatment and neutropenia are risk factors for the development of disseminated disease. While the most common predisposing illness for gastrointestinal infection is severe malnutrition [5], rhinocerebral mucormycosis is often associated with diabetic keto-acidosis [3],6] and cerebral mucormycosis has been diagnosed in AIDS patients and drug addicts [4],6]. Mucorales are unique among filamentous fungi in their greater ability to cause infections in a broader, more heterogenous population of human hosts than other opportunistic moulds.

The fungal hyphae invade skin, mucosae, and tissues causing thrombosis in blood vessels, infarction, haemorrhage and necrosis [7]. According to Roden et al., mucormycosis is most frequently located in sinuses (39%), lungs (24%), skin (19%), brain (9%), gastro-intestinal tractus (7%), other organs (6%) and disseminates in 3% of the cases [3],6]. The diagnosis is often made at a late stage in the disease or even postmortem except for the rhinocerebral and cutaneous forms. Mucormycosis has become an increasingly common disease, representing the second most common infection caused by molds (after Aspergillus) among 109 cases of culture-proven cases in a large cancer center in which it represented 20% of all such cases [8]. The increased incidence of diabetes, use of immuno-compromising agents and the introduction of voriconazole prophylaxis or treatment among immunocompromised patients have been associated with the emergence of mucormycosis [2,9–13]. Several large centers have reported a steady increase in the incidence of disease during the last decades [2],14]. Our objective was to review all cases of proven and probable mucormycosis – according to European Organization for Research and Treatment of Cancer and Mycoses Study Group (EORTC) criteria – diagnosed from 2000 until 2007 at the University Hospitals Leuven, a 1900-bed tertiary care hospital facility. The incidence over the last seven years and possible risk factors for developing mucormycosis were assessed.

1.2 Materials and methods

This retrospective study included patients hospitalized at the tertiary care University Hospitals Leuven from January 2000 to December 2007. Patients’ culture data were retrieved from the Sirscan 2000 system (i2a, Montpelier, France). Queries were run to obtain the protocols from histological examinations of tissue specimens. We used the criteria of the EORTC for proven, probable or possible invasive fungal infections [15]. As such, a proven case was based on a histopathologic examination of a needle aspiration or a biopsy specimen which revealed hyphae and evidence of associated tissue damage. Alternatively, a case could be supported by a positive culture of a sample obtained by sterile procedure from a normally sterile and clinically or radiologically abnormal site consistent with infection [15]. A probable case was based on at least one host factor and one microbiological criterion and one major or two minor clinical criteria from abnormal site consistent with infection as defined by the EORTC [15]. The myco-logical evidence for a probable case is based on direct examination of a sample or culture of specimens collected from sites that may be colonized (e.g., sputum, bronchoalveolar lavage) [15]. Mucorales have typically wide, irregular non-septate hyphae with right-angled branching [5]. The identification of the Mucorales was based on macroscopic and microscopic evaluation of the cultures growing on Sabouraud agar with 0.2% glucose (Takashio medium), growth temperature and/or internal transcribed spacer (ITS) sequence analysis on ABI Prism 310 automatic sequencer (Applied Biosystems, Foster City, California, USA). The following information was extracted from the electronic medical records of all patients with proven or probable invasive fungal infections, i.e., demographic characteristics, underlying disease, co-infection with other fungi, site of infection, symptoms and signs of infection, radiological findings, laboratory findings, therapy, and outcome.

1.3 Statistics (Microsoft Excel software)

The correlation between the incidence and the year was determined by means of the Spearman rank correlation coefficient. A P-value < 0.05 was considered signifcant.

Results

Incidence

We found 18 cases with mucormycosis, i.e., 12 patients with proven mucormycosis and 6 with probable disease. No patients were classified in the category of possible mucormycosis. While members of the mucorales were recovered from specimens of 27 other patients, they were considered to be contaminants.

When analyzing the incidence over the years 2000 to 2007, there was a slight increase during the last three years of the study (Fig. 1 ). The range fluctuated between 0 and 0.056/10,000 patient days except for a peak incidence of 0.15 cases/10,000 patient days in the year 2006. There was a positive correlation between incidence and year (Spearman correlation coefficient = 0.59), but this correlation was not statistically significant (P = 0.12).

Fig. 1

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Incidence of mucormycosis cases per year.

1.4 Demographic characteristics and underlying disease

The median age of the patients with proven and probable mucormycosis was 60 years (range, 42–69 years), of which 56% (10/18 cases) occurred in men.

Demographic characteristics, site of infection, underlying disease, EORTC criteria, co-infection with Aspergillus species, therapy, and outcome of the 18 mucormycosis patients is given in Table 1 and summarized in Table 2.

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There was no seasonal variation in the Mucorales infections, 4/18 cases were diagnosed in spring, four in summer, four in autumn and six in winter.

Patients with mucormycosis were hospitalized in the medical intensive care unit (n = 8), haematology ward (n = 7), and surgical intensive care unit (n = 3). The most common underlying condition was a haematological disorder/malignancy (50%) with or without haematopoietic stem cell transplantation (HSCT). The second largest group of patients were solid organ transplant recipients (17%). Two patients (11%) did not have obvious risk factors and were thought to have been infected secondary to trauma.

1.5 Pattern of infection — anatomic location

The lungs were the major sites of infection (14/18, 78%), with disseminated mucormycosis evident in two of the pulmonary patients. In one of the two the infection extended to the ethmoidal septum and the lamina cribrosa, while in the second, the patient suffered from heart and lung involvement. Subcutaneous infection was detected in one patient who had a motor-cycle accident with polytrauma. A Mucor species was isolated from femoral bone biopsies (deep extension).

One HIV-positive patient suffered from a cervical/vaginal mycetoma caused by a Rhizopus species after having undergone an incomplete abortus arte provocatus.

One patient suffered from a craniocerebral injury due to a car accident. One month after undergoing decompressive craniotomy in the fossa posterior, a cerebral infection developed due to a Mucor species which had been isolated from the submucosal tissues of the neck.

Sinus involvement was evident in only one case.

1.6 Underlying host factors and clinical risk conditions

Half of the mucormycosis patients (9/18) had a haematological disorder (44% of these patients underwent HSCT) which took the form of acute leukaemia (acute myeloid leukaemia, n = 3; acute lymphatic leukaemia, n = 1), lymphoma (small cell B-cell Non-Hodgkin lymphoma, n = 1), myeloprolipherative disorders (chronic myeloid leukaemia, n = 1), and stem cell disorders (myelodysplastic syndrome, n = 2; aplastic anaemia, n = 1). Neutropenia was present in all but two of the patients with haematological malignancies. Ten of the 18 patients received methylprednisolone or dexamethasone for more than three weeks in the three months prior to diagnosis.

Seven mucormycosis patients had concomitant diabetes, all of whom were known to have the disease prior to the diagnosis of mucormycosis. Four of them had corticosteroid-induced diabetes, two had diabetes mellitus type 2 and one had diabetes mellitus type 1.

Three patients with mucormycosis had lung transplants and finally, one patient suffered from Wegener’s granulomatosis.

Clinical presentation

All but one (pulmonary mucormycosis) patient exhibited (acute) symptoms within 30 days of detection of the fungus in clinical samples.

Pulmonary mucormycosis (n = 14)

Eight patients presented with fever refractory to broad-spectrum antibiotics, 6 had a non-productive cough and 11 had dyspnea. Other symptoms included chest pain (4 patients) and haemoptysis (3 patients). Almost all patients needed mechanical ventilation. One patient had concomitant pansinusitis, which pointed the clinician towards mucormycosis instead of aspergillosis as the cause of the infection.

Other than pulmonary presentations (n = 4)

The patient with cutaneous mucormycosis initially presented after a motor vehicle accident. On day 10 of his ICU stay, he had evidence of muscle necrosis of the left leg which was not accompanied by fever because he was on hemofiltration. The leg had to be amputated and a culture inoculated with portions of the lesion and a bone fragment revealed Mucor species.

Another patient with well-controlled HIV infection presented with renal insufficiency and dehydration. Because she complained of long-existing lower abdominal pain, a CT scan was performed, revealing a large mass in the lower pelvis which required surgery. Preoperative cultures revealed the presence of a Rhizopus species and histological examination of pre- and peroperative biopsies showed hyphae of the Mucorales type resulting in the diagnosis of a mycetoma.

The patient with cerebral mucormycosis suffered from a polytrauma with associated craniocerebral trauma. On day 17, there was meningeal irritation and fever. Lumbar puncture revealed a raised white cell count of 284.4/μl.

Microbiologic and histopathologic assessment

From Table 3 , it is obvious that most isolates belonged to the genus Rhizopus species (8/14). One of the Rhizopus species was isolated from the cervical/vaginal mycetoma, all the other were recovered from respiratory specimens. The two Mucor species were isolated in 2007 and caused cutaneous and cerebral mucormycosis which were traumatic in origin. Rhizomucor pusillus and Mycocladus corymbiferus were identified in a total of four patients with pulmonary mucormycosis. In the remaining four patients, the diagnosis was established through observation of irregular and wide branched hyphae observed in histopatho-logic preparations.

*In four patients, no Mucorales were cultured. The diagnosis of mucormycosis was based on histopathological examination.

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Co-infection with Aspergillus (probable or proven invasive aspergillosis) was a feature in 50% (4/8) of the patients with proven pulmonary mucormycosis. Two of these patients had a positive culture result and three had positive galactomannan tests. Additionally, histopathological examination of a broncho-alveolar fluid sample revealed the presence of Aspergillus type hyphae in one patient. Of the six patients with probable pulmonary mucormycosis, four (67%) had concomitant aspergillosis (probable or proven). In three there was histopathological evidence for Aspergillus type hyphae, whereas in two cultures grew Aspergillus species. All patients had positive galactomannan test results.

There were 6 patients with both positive cultures and histopathological assessments suggestive for mucormycosis. Eight patients were diagnosed by means of positive culture results, while the histopathological examination either was not performed (n = 4) or showed no (n = 1) or Aspergillus-type (n = 3) hyphae. Four patients were diagnosed only by histopathological detection of Mucorales-like hyphae.

In four cases, an upgrade from possible/probable to proven mucormycosis could be made by means of the autopsy examination.

Radiologic assessment

Most of the patients with pulmonary involvement (13/14) had consolidations on chest radiography. Pleural effusion was described in 8/14 (57%) patients and five (36%) exhibited cavities on chest radiography. The finding of an air-fluid level (n = 1), a tree-in-bud sign (n = 1), an air-crescent sign (n = 1), or a halo-sign (n = 3), was uncommon. All patients with a halo sign were co-infected with Aspergillus spp. Nodules were seen in 6 patients five of whom had probable or proven co-infections with Aspergillus spp.

The sinus CT scan of the patient with disseminated mucormycosis of lungs and sinuses showed demineralization of the ethmoidal septum and the lamina cribrosa. Transthoracic echocardiography of the patient with disseminated mucormycosis of lungs and heart showed mobile vegetation in the left ventricle.

Soft tissue swelling was found in sinus mucormycosis, while fluid collections were described in the (sub)cutaneous and cerebral mucormycosis cases.

Diagnosis

The premortem diagnosis was often based on culture and histopathologic examination of bronchoscopic/endoscopic material (8 cases). Further, biopsy material obtained at surgery was diagnostic in five cases. Samples from non-sterile sites (sputum, bronchial aspirate, and swab) contributed to the diagnosis of mucormycosis in five patients.

The definitive diagnosis ‘proven mucormycosis’ was established at the time of postmortem histopathologic and/or microbiological examination in four patients (three localized and one disseminated pulmonary mucormycosis).

Treatment and outcome

Two patients did not receive specific therapy for mucormycosis due to the fact that the diagnosis was made just a few days prior to their death.

In the 16 other patients, amphotericin B formulations were employed in therapy.

Among the three patients receiving liposomal amphotericin B (LAMB), two survived and one died. Neither of the two patients treated with amphotericin B lipid complex (ABLC) survived. Of the three patients treated with amphotericin B deoxycholate, two survived. One patient was treated with LAMB followed by ABLC but this was not successful as the patient died. In seven patients, therapy was switched during treatment. In five, therapy was altered from amphotericin B (either LAMB, ABLC or LAMB/ABLC) to posaconazole (survival rate was 20%). One patient received amphotericin B deoxycholate and afterwards caspofungin but the individual died. Another patient with pulmonary mucormycosis caused by Mycocladus corymbiferus received posaconazole, but because of unsatisfactory response the therapy was switched to LAMB and the patient survived. In four of the 16 patients, amphotericin B therapy was combined with surgery with successful results.

Overall, six (33%) of the 18 patients diagnosed with probable or proven mucormycosis survived. Eight (44%) died from the infection, and 4 (28%) did not survive due to their underlying condition.

Mortality according to the site infection was as follows:

Of the 14 patients with localized and disseminated pulmonary mucormycosis, 3 (21.4%) survived. The deaths of eight of the 11 remaining patients were attributable to the fungal infections, but three died of uncertain causes. In these pulmonary mucormycosis patients, the eight patients with a haematological disorder with or without neutropenia all died. Of the three lung transplant patients, two died and one was cured.

Relative to the four patients with extrapulmonary mucormycosis, three survived and one died. The latter patient had exclusive sinus involvement, and the mucormycosis infection was not considered the cause of death.

When looking at the mortality according to the species, the mortality rate was 2/2 in cases caused by Rhizomucor pusillus , 7/8 due to Rhizopus species, 1 of 2 patients with infections caused by Mycocladus corymbiferus , and 0/2 for Mucor species infection.

Discussion

The objective of this study was to review all cases of probable and proven mucormycosis infections which occurred in a 1900-bed tertiary care university hospital between 2000 and 2007. We noticed a slight but not significant increase in the rate of mucormycosis infections during this period. The disease most often (78%) affected the lungs and haematological disorders were the most important risk factor. Rhizopus species were the most frequently recovered etiologic agents (44%). There was a high rate (44%) of co-infections with Aspergillus species. The overall mortality rate of proven and probable mucormycosis was 67%.

The incidence of mucormycosis over the eight study years was 0.042/10,000 patient days with a peak incidence of 0.15/10,000 patient days in 2006. The incidence of mucormycosis in our center is somewhat lower than that reported from the M.D. Anderson Cancer Center of Texas (0.079/10,000 patient days in 1999 and 0.95/10,000 patient days in 2002–2004) [8]. These and several other authors reported an increase in the incidence of mucormycosis in their hospitals’ population during the past decade [2,14,16,17]. Although not statistically significant, we also registered an increase in our institution beginning in 2005. According to Kauffman this increase in infections caused by members of the Mucorales is mainly seen in allogeneic HSCT recipients and in HSCT patients treated with high-dose corticosteroids for severe graft-versus-host-disease [14]. The four allogeneic HSCT patients in this study were all treated with corticosteroids for chronic graft-versus-host-disease. Several authors believe that the increased incidence of mucormycosis could be due to the increased prophylactic and empiric use of voriconazole, which is effective in treatment of Aspergillus species and other filamentous fungi, but has no activity against Mucorales [8,10,11,13,14,18]. The influence of prophylactic or empiric treatment with voriconazole on the incidence of mucormycosis could not be evaluated in this study as voriconazole is not used in these settings in our institution. Most studies, including this, registered a higher incidence of mucormycosis among men than women [4,6,17,19–22]. The median age of 60 years for patients in our studies is somewhat higher as compared to previous reports (range, 40–52 years) [6,8,20]. In contrast with the data in the review of Roden et al . (929 cases starting from the year 1940 up to 2003) and Chakrabarti et al. (129 cases over the years 1990–1999), diabetes was not the most common underlying disorder [6,19]. Instead, haematological disorders were the most frequent underlying conditions in our study. Similar results were found in the investigations of Petrikkos et al . in 2003 (24 cases, from 1993 to 2002) [21]. This is probably related to the HSCT activity (∼ 120 HSCT/year) in our hospital [23]. In the studies of Chakrabarti et al . and Roden et al ., rhino-orbito-cerebral mucormycosis was reported as the most frequent clinical manifestation [6,19]. In our study, the lungs were the most common (78%) site of infection. This is not unexpected because of the well known relationship between clinical presentation of mucormycosis and the underlying disease. The most frequent predisposing underlying condition of pulmonary mucormycosis has been described as haematological malignancy with neutropenia [2,6,9,24–26]. In the present investigation, most of the patients with pulmonary mucormycosis had acute leukaemia as underlying disorder, which also was the case in the study of Pagano et al. [24]. Pulmonary mucormycosis was noted in 64% of the patients, and 78% of the patients had acute leukaemia [24].

The fact that pulmonary mucormycosis was the most common site of infection corresponds with the high rate of recovery of Rhizopus species as the fungus is most frequently associated with the lungs [25].

Striking in this study is the high rate of co-infections by Aspergillus species. Concomitant pulmonary Aspergillus infection (probable or proven) was present in 57% of the cases of patients with pulmonary mucormycosis. This is in accord with the results of the study of Kontoyiannis et al. among 13 patients with histopathologically proven pulmonary mucormycosis of whom 6 (46%) had concomitant pneumonia caused by Aspergillus or Fusarium species [28]. Radiological signs associated with Aspergillus co-infection were the presence of a halo sign (all three patients with a halo sign were co-infected with Aspergillus) and the presence of noduli (five of six patients with noduli were co-infected with Aspergillus ). Our data suggest that when noduli or a halo sign are detected radiographically in a patient with mucormycosis, additional diagnostic testing should be undertaken in order to check for a co-infection with Aspergillus . In principle, invasive aspergillosis can be detected with high specificity by means of the Platelia Aspergillus antigen immunoassay (Bio-Rad Laboratories, Marnes-la-Coquette, France) for the detection of galactomannan. A few Mucorales have been tested for cross-reactivity with galactomannan immunoassays of which negative results were obtained with Mucor plumbeus, Rhizopus oryzae, Rhizopus species, Syncephalastrum racemosum, Cunninghamella bertholletiae, and Mycocladus corymbiferus [29,30]. However, we cannot exclude that there are possible members of the Mucorales which also express these galactomannans and could cross-react in the Aspergillus antigen tests. Further studies are necessary to increase our insight in this problem of possible cross-reactivity and co-infections. The treatment of choice for mucormycosis is surgery combined with antifungal therapy [3]. Just four patients were treated in this way in this study. The most obvious explanation is that the majority of the patients suffered from pulmonary mucormycosis with serious underlying conditions (haematological, transplant patients) limiting the possibility for surgery. It is generally believed that surgical resection in combination with antifungal therapy leads to better survival rates than antifungal therapy alone [2,3,6,9,19,21–25,31]. This is also supported by our study. There is however a bias for the better survival results after surgery. Surgical intervention is often precluded in the severely ill patient. The poor clinical condition of the patients accounts for a higher mortality risk as compared to those in better condition. In addition, surgical intervention is only feasible in patients with localized disease.

Pulmonary mucormycosis was associated with a high mortality rate in a study of Roden et al . (76%) and of Chayakulkeeree et al. (76%) [2,6,31]. In our study, patients with proven or probable pulmonary mucormycosis likewise had a high mortality rate of 79%. Just one out of six patients with pulmonary mucormycosis treated with ABLC, alone or in combination with other antimycotica, survived. The other two survivors of pulmonary mucormycosis were treated with LAMB with or without posaconazole.

Conclusion

In conclusion, this review study of 18 cases of proven and probable mucormycosis confirms the importance of mucormycosis in an era of HSCT and organ transplantation. Clinicians, microbiologists and pathologists should have a low threshold for suspicion of a fungal infection in immunocompromised patients. Further studies examining co-infections of Mucorales with Aspergillus species are certainly needed. The existence of cross-reactivity from non- Aspergillus species with the galactomannan test should be examined in depth. The application of molecular detection techniques on tissue specimens may also lead to new insights regarding the occurrence of infections caused by more than one fungal pathogen.