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Departments of Infectious Diseases, Infection Control, and Employee Health, Bone Marrow Transplantation
Leukemia, The University of Texas M. D. Anderson Cancer Center, University of Houston College of Pharmacy
Spectral Genomics, Inc., Houston, Texas
National Cancer Institute, National Institutes of Health, Bethesda, Maryland
Background.
Anecdotal evidence suggests a rise in zygomycosis in association with voriconazole (VRC) use in immunosuppressed patients.
Methods.
We performed prospective surveillance of patients with zygomycosis (group A; n = 27) and compared them with contemporaneous patients with invasive aspergillosis (group B; n = 54) and with matched contemporaneous high-risk patients without fungal infection (group C; n = 54). We also performed molecular typing and in vitro susceptibility testing of Zygomycetes isolates.
Results.
Nearly all patients with zygomycosis either had leukemia (n = 14) or were allogeneic bone marrow transplant recipients (n = 13). The Zygomycetes isolates (74% of which were of the genus Rhizopus) had different molecular fingerprinting profiles, and all were VRC resistant. In multivariate analysis of groups A and C, VRC prophylaxis (odds ratio [OR], 10.37 [95% confidence interval {CI}], 2.7638.97]; P = .001), diabetes (OR, 8.39 [95% CI, 2.0434.35]; P = .003), and malnutrition (OR, 3.70 [95% CI, 1.0313.27]; P = .045) were found to be independent risk factors for zygomycosis. Between patients with zygomycosis (after excluding 6 patients with mixed mold infections) and patients with aspergillosis, VRC prophylaxis (OR, 20.30 [95% CI, 3.85108.15]; P = .0001) and sinusitis (OR, 76.72 [95% CI, 6.48908.15]; P = .001) were the only factors that favored the diagnosis of zygomycosis.
Conclusions.
Zygomycosis should be considered in immunosuppressed patients who develop sinusitis while receiving VRC prophylaxis, especially those with diabetes and malnutrition.
Recent anecdotal reports from geographically diverse bone marrow transplant (BMT) centers have suggested the emergence of zygomycosis in the setting of voriconazole (VRC) prophylaxis [14]. Increasing use of VRC as prophylaxis and as empirical [5], preemptive, and targeted treatment of invasive aspergillosis (IA) [69] has raised concerns about an increase in incidence of zygomycosis [4], an opportunistic mycosis that is resistant to VRC [10, 11].
Given the complex pathogenesis of zygomycosis, it is unclear whether selection pressure from VRC is the predominant factor accounting for the increasing incidence of zygomycosis. An increase in the incidence of zygomycosis has been noted in several cancer centers since the mid-1990s, predating the availability of VRC [4, 12, 13]. Trends in the reporting of zygomycosis infections can be further complicated by the fact that nosocomial transmission of zygomycosis, including outbreaks, have been rarely reported [14].
After the introduction of VRC in the M. D. Anderson Cancer Center's formulary (August 2001), we began to witness an increase in the number of invasive Zygomycetes infections in patients in our leukemia/BMT services. We therefore performed an observational matched case-control study comparing consecutive patients with a Zygomycetes infection (case group) with 2 contemporaneous control groups: (1) patients without an invasive mold infection (IMI), who were matched according to their underlying malignancy and duration of hospitalization at the time of diagnosis of zygomycosis, and (2) patients with IA, the most common IMI in our institution.
PATIENTS, MATERIALS, AND METHODS
Patients and setting.
We identified unselected consecutive patients with cancer at the M. D. Anderson Cancer Center who had either zygomycosis or IA (1 September 2002 to 31 March 2004), by active surveillance of weekly reviews of the microbiology laboratory records, autopsy reports, and premortem histopathology specimens. The corresponding medical records were reviewed by use of a standardized form. The clinical data analyzed included demographic characteristics; the type and status of the underlying malignancy at the time of diagnosis of infection; the type of BMT (if applicable); risk factors for IMIs present at or within 1 month before the diagnosis of infection, such as neutropenia, grade IIIIV graft-versus-host disease (GvHD), cytomegalovirus (CMV) infection, history of diabetes mellitus, hyperglycemia, acidosis, or malnutrition; HIV positivity; APACHE II score at the time of diagnosis of infection; the need for admission to the intensive care unit (ICU); previous antifungal use (drug, dose, and duration of treatment, given as prophylaxis or as empirical, preemptive, or targeted therapy) within 3 months of diagnosis of infection; the presence of intercurrent fungal or viral infections; and the clinical outcome. The duration of neutropenia (500 cells/mm3) and receipt of adrenal corticosteroids (cumulative dose [prednisone equivalent] and duration of treatment) and of tumor necrosis factor (TNF) inhibitors within the month before diagnosis of either zygomycosis or IA was also assessed.
Case definitions.
Patients with a diagnosis of definite or probable zygomycosis (without a prior history of zygomycosis) were designated as group A. Control patients with a diagnosis of definite or probable IA during the same period were designated as group B. Patients with concomitant zygomycosis and IA were excluded from group B. Finally, control patients without evidence of IMIs who were matched with the patients in group A according to their underlying malignancy and time and duration of hospitalization for the treatment of zygomycosis were designated as group C. Patients in this control group were identified by screening patient databases in the Department of Medical Informatics for underlying diseases, dates of admission, and ward of inpatient stay corresponding to those of the patients in group A. The ratio of the numbers of patients in group B and in group C to that in group A was 2 : 1.
To define zygomycosis and IA, we applied standardized diagnostic criteria [15]. We defined nosocomial IA and zygomycosis as corresponding infections occurring at least 14 days after admission [16]. Additionally, we defined breakthrough zygomycosis and IA as infections documented during antifungal prophylaxis. Prophylaxis was defined as the use of an antifungal with activity against Aspergillus, such as VRC, caspofungin (CAS), itraconazole (ITC), or amphotericin B (AMB), for >5 continuous days in the absence of signs and symptoms of infection. Preemptive therapy was defined as use of an antifungal in patients with signs or symptoms of a presumed IMI. Neutropenia was defined as a neutrophil count 500 cells/mm3, lymphopenia was defined as a lymphocyte count 500 cells/mm3, and monocytopenia was defined as a monocyte count 10 cells/mm3 at the time of diagnosis of infection. Hyperglycemia was defined as a glucose level 200 g/dL, malnutrition was defined as a serum albumin level 3 g/dL, and acidosis was defined as a pH value of <7.35 at the time of diagnosis of infection.
Fungal isolates.
Isolates of Zygomycetes and Aspergillus were identified according to standard morphological criteria [17, 18] and were stored in our research mycology laboratory in 20% glycerol at -80°C until they were subjected to susceptibility testing and molecular typing.
Antifungal susceptibility testing.
The in vitro susceptibility of Zygomycetes and Aspergillus isolates to antifungals was determined by use of the standard microdilution methods for filamentous fungi proposed by the NCCLS (guideline M38-A) [19, 20] and of Epsilometer (E-test) strips (AB Biodisk) [21]. We also determined the minimal fungicidal concentration (MFC) of AMB, ITC, and VRC for each isolate, by use of methods described elsewhere [22]. All susceptibility determinations were performed in triplicate.
DNA sequencing and analysis of Zygomycetes isolates.
The genus identification of Zygomycetes isolates was confirmed by sequencing the ITS1-5.8S-ITS2 region of fungal ribosomal RNA genes, as described elsewhere [23, 24]. Briefly, DNA fragments with an average size of 600 bp containing ITS regions were amplified. The amplicons were purified using the High Pure PCR Product Purification Kit (Roche Diagnostics), and bidirectional sequencing was performed using ITS4 (5-tcctccgcttattgatatgc-3) and ITS5 (5-ggaagtaaaagtcgtaacaagg-3) primers and the BigDye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems). The products were purified with PERFORMA DTR Gel Filtration Cartridges (Edge BioSystems) and were sequenced using an ABI-377 sequencer (Applied Biosystems). The results were analyzed using the Sequencing Analysis software program (version 3.3; Applied Biosystems). The contiguous sequences were constructed using SeqMan (DNASTAR) and were identified using the BLAST program on the National Center for Biotechnology Information Web site (http://www.ncbi.nlm.nih.gov/BLAST/).
Molecular typing of Zygomycetes isolates.
We used a polymerase chain reaction (PCR) method based on the amplification of repetitive genomic sequences, by use of the same procedure we recently described for Aspergillus typing [25]. Briefly, we extracted DNA from 10 L of hyphae from each Zygomycetes isolate by use of the UltraClean Microbial DNA Isolation Kit (Mo Bio Laboratories). We modified the manufacturer's instructions by extending the bead-beating period from 10 to 20 min and by decreasing the elution volume from 50 to 35 L. We standardized sample DNA to 25 ng/L and visualized the genomic integrity by use of agarose gel electrophoresis. Next, we amplified the extracted DNA with the DiversiLab Zygomycetes DNA Fingerprinting Kit (Spectral Genomics), in accordance with the manufacturer's instructions. We detected and analyzed repetitive PCR products by use of the DiversiLab System and software program (version 2.1.66); specifically, we separated and detected the amplified fragments with microfluidics chips and the Agilent 2100 Bioanalyzer (Agilent Technologies). The analysis was performed using DiversiLab software version 2.1.66, and the reports included a dendrogram, isolate information, and a virtual gel image of each sample.
Statistical analysis.
Treatment groups were compared by 2 or Fisher's exact test, where appropriate. Forward stepwise logistic-regression analysis was performed for factors identified in univariate analysis with P .2. Factors identified in logistic-regression modeling were considered to be significant only if they had a P value of .05 without adjustment for multiple comparisons. Exploratory variables identified by logistic regression were tested for association by correlation matrix analysis. Odds ratios (ORs) and 95% confidence intervals (CIs) were reported for all significant variables. All analyses were performed using the Stata software program (version 8.0; Stata Corporation).
RESULTS
We identified 27 patients with zygomycosis (15 definite cases and 12 probable cases). Twelve cases were documented only by culture, 9 were confirmed by both histopathologic assessment and culture, and 6 were documented only by histopathologic assessment. An underlying hematological malignancy was seen in all but 1 patient. Thirteen patients (48%) were allogeneic BMT recipients. Pulmonary involvement (16 patients [59%]; pneumonia alone in 12 patients and pneumonia with concomitant sinusitis in 4 patients) and sinusitis (11 patients [41%]; isolated sinusitis in 4 patients, sinusitis with concomitant pneumonia in 4 patients, and sinusitis with concomitant orbitocerebral infection in 3 patients) were the most common clinical presentations of zygomycosis. Soft tissue and disseminated zygomycosis were seen in 2 patients each. Most of the patients (19 [70%]) had significant exposure to systemic corticosteroids, and a minority of the patients had neutropenia (6 [22%]) and CMV reactivation (4 [15%]).
Zygomycosis was a common IMI during the study period. Among the 109 cases of IMI (definite or probable) that were culture proven (Aspergillus, Fusarium, or Zygomycetes) during the study, zygomycosis was the second most common IMI (22 cases; 20% of IMIs; incidence, 0.095/1000 patient-days), compared with IA (70 cases; 64% of IMIs; incidence, 0.302/1000 patient-days) and fusariosis (17 cases; 16% of IMIs; incidence, 0.073/1000 patient-days). This incidence of zygomycosis was higher than that in our institution in 1999 (0.0079/1000 patient-days; P = .001). Additionally, the incidence of zygomycosis in our BMT service (13/834 [1.55%]) was higher than that during the period 19891998 (10/4020 [0.25%]) [13] (OR, 7.34 [CI, 3.2816.4]; P = .0001). In contrast, there was a trend toward decreasing incidence of IA during the study period (incidence, 0.499/1000 patient-days in 2001 vs. 0.359/1000 patient-days in 2003; P = .044) (figure 1A). No temporal or spatial clustering of Zygomycetes infections were observed. The majority of patients (17 [63%]) were receiving VRC (alone in 13 patients and in combination with CAS in 4 patients) at the time of diagnosis of zygomycosis. In 13 of these patients, VRC was given as prophylaxis. All of these patients received VRC for >5 days before the onset of zygomycosis. Nine patients were receiving CAS (alone in 5 patients and in combination with VRC in 4 patients) at the time of diagnosis. Finally, none of the patients with zygomycosis had a history of IA, but 6 patients (22%) had mixed infections with another mold (Aspergillus species in 5 patients and Scedosporium apiospermum in 1 patient).
Table 1 lists the characteristics of the 21 patients with zygomycosis (excluding the 6 patients with mixed mold infections) and of the 54 patients with IA (14 definite cases and 40 probable cases; group B). These 2 groups were comparable in terms of the patients' underlying disease, APACHE II score, and comorbidities. There was a trend toward more-frequent neutropenia at the time of diagnosis in the IA group (P = .06), but the duration of neutropenia before diagnosis in both groups was similar. In univariate analysis, VRC prophylaxis (OR, 13.07 [95% CI, 3.6946.19]; P < 0001) and sinusitis at presentation (OR, 48.18 [95% CI, 5.57416.0]; P < .001) were significantly more common in patients with zygomycosis, whereas the only factor associated with IA was active malignancy at the time of diagnosis (OR, 3.38 [95% CI, 1.2112.46]; P = .03). Four factors were retained in the final model for logistic-regression analysis: VRC prophylaxis, sinusitis, diabetes, and preemptive CAS therapy. Of these factors, only VRC prophylaxis (OR, 76.72 [95% CI, 6.48908.15]; P = .001) and sinusitis (OR, 76.72 [95% CI, 6.48908.15]; P = .001) were found to be independent factors favoring the diagnosis of zygomycosis over aspergillosis. The exposure to VRC (median duration and total dose of prophylaxis) was similar between the patients with zygomycosis and the patients with IA (table 1). Of interest, overall AMB use remained stable, whereas VRC use almost doubled in leukemia/BMT services during the study period (figure 1B).
Table 2 lists the characteristics of all 27 patients with zygomycosis (group A) and of 54 matched contemporaneous control patients without any IFI (group C). The univariate analysis showed that patients with zygomycosis were more likely to have significant corticosteroid exposure (P < .0001), a prior allogeneic BMT (P < .0001), grade IIIIV GvHD (P = .005), neutropenia for >10 days at the time of diagnosis (P = .05), a history of diabetes mellitus (P = .04), hyperglycemia (P = .03), the need for ICU admission (P = .02), VRC prophylaxis (P = .0005), and, possibly, malnutrition at the time of diagnosis (P = .09). In stepwise logistic-regression analysis, VRC prophylaxis (OR, 10.37 [95% CI, 2.7638.97]; P = .001), a history of diabetes mellitus (OR, 8.39 [95% CI, 2.0434.5]; P = .003), and malnutrition (OR, 3.70 [95% CI, 1.0313.27]; P = .045) were the only factors identified in the final model as risk factors for zygomycosis in the comparison between groups A and C.
Finally, a comparison between the patients in groups B and C revealed that grade IIIIV GvHD (P = .024), receipt of >600 mg of corticosteroids (prednisone equivalent during the prior month), and, possibly, the presence of lymphopenia (P = .062), CMV reactivation (P = .07), and neutropenia for >10 days at the time of diagnosis (P = .12) were risk factors seen more frequently in association with IA. There were no statistically significant differences in the prophylactic use of AMB, CAS, or VRC between groups B and C. In multivariate analysis, grade IIIIV GvHD (OR, 1.33 [95% CI, 1.011.74]; P = .043) and the use of corticosteroids (OR, 1.39 [95% CI, 1.151.68]; P = .001) were independently associated with the development of IA.
Susceptibility testing.
Of the 21 Zygomycetes isolates, 20 were available for susceptibility testing, sequencing, and molecular fingerprinting analysis. Zygomycetes isolates had uniformly high MICs/MFCs of VRC and ITC and high minimal effective concentrations (MECs) of CAS, whereas all Aspergillus isolates had low MICs/MFCs of VRC and ITC and low MECs of CAS (table 3).
DISCUSSION
Here we report on what is, to our knowledge, the largest single-institution study of the risk factors for zygomycosis, an uncommon yet emerging opportunistic mycosis in patients with cancer. The patient population in our study was typical of those affected with this devastating infection. Sinopulmonary involvement was predominant among those with infection, implying host-specific differences in presentation, since rhinoorbital and rhinocerebral zygomycosis is more common in diabetic individuals [26, 27]. Not surprisingly, zygomycosis was seen almost exclusively in patients with leukemia and in recipients of allogeneic BMTs in our institution, in accordance with results of a previous study [12]. Leukemia, prolonged and profound neutropenia, GvHD, malnutrition, corticosteroids, and BMT have classically been associated with zygomycosis in patients with cancer [1214, 28].
What clinically differentiates zygomycosis from IA in patients at risk for infection In our study, the patients with zygomycosis had risk factors that overlapped with those for IA. These risk factors, which include significant corticosteroid exposure, GvHD, allogeneic BMT, CMV reactivation, and prolonged neutropenia, have been extensively described in studies of IA [29]. In addition, 43% and 21% of patients with zygomycosis and IA, respectively, in our study had a history of diabetes mellitus. Functional impairment of neutrophils in the setting of hyperglycemia identifies a subgroup of patients at high risk for opportunistic mold infections [30]. In addition to the overlapping risk factors, there were several clinical similarities between zygomycosis and IA. In particular, both infections were typically community acquired, had comparably late onset after allogeneic BMT, and often featured pulmonary involvement (in 54% of zygomycosis cases and in 91% of IA cases). However, 2 risk factors were the differentiating features that favored zygomycosis over IA: (1) initial presentation with sinusitis and (2) occurrence as a breakthrough infection in the setting of prior VRC prophylaxis.
Is there a causal link between VRC and excess cases of zygomycosis, or did VRC simply make a preexisting trend more apparent In the latter scenario, VRC use could simply be a marker for changing immunosupression practices that pose a greater risk for infection by rare resistant molds. However, the approach to the treatment of GvHD has not changed in our institution during the study period, and very few patients in our series received TNF inhibitors (tables 1 and 2). It is tempting to speculate that the trends in frequency of zygomycosis might reflect the increasing and prolonged use of oral VRC versus parenteral agents with activity against Zygomycetes, such as AMB. Large prospective studies would be required to dissect the multiple and complex host, pharmacological, and microbiological risk factors that predispose patients to infection by resistant molds [31] such as the Zygomycetes.
Nevertheless, our study design had several advantages. We compiled a sizeable study group in an attempt to prevent the publication biases associated with small case series, and we used 2 contemporaneous control groups in an attempt to avoid selection bias. The use of a control group of high-risk patients without any IMIs might be important for adjustment for confounding factors. Case-control studies of risk factors for infection by resistant bacteria have shown that comparing patients with infection caused by a resistant pathogen and patients with infection caused by a sensitive pathogen frequently results in an overestimation of the contribution of a drug to the selection of a resistant pathogen [32].
We also documented the genotypic diversity of the causative Zygomycetes species, as shown by rep-PCR, a new molecular-typing approach that has been proposed as having many advantages over existing methods [33]. The lack of temporal-spatial clustering of the zygomycosis cases and the apparent lack of clonality of the Zygomycetes isolates make it extremely unlikely that these infections were transmitted from a provider or other common source to other patients. Further rep-PCR protocol optimization may be required for more-extensive strain discrimination among Rhizopus isolates.
To ensure accurate species characterization, sequencing-based identification was used to characterize the relative distribution of the various Zygomycetes genera among our clinical isolates. Thus, in view of the imprecision of the morphological identification of Zygomycetes at the genus level (e.g., Mucor, Rhizopus, Rhizomucor, and Cunninghamella), we used ITS gene sequencing to identify the genus in almost all of our isolates [23, 24]. We showfor the first time, to our knowledgethat the results of ITS sequencing are >20% discordant with those of morphological identification and that most of the morphologically misidentified Zygomycetes isolates were actually Rhizopus species.
Finally, we show that the Zygomycetes isolates were resistant to several antifungals, including VRC, in vitro, whereas Aspergillus isolates were not (table 3), which is in accordance with results of previous in vitro susceptibility studies [10, 11, 34]. Because of the small number of isolates, however, we were not able to demonstrate species-specific differences in breakthrough rates of zygomycosis on the basis of degrees of in vitro susceptibility to VRC.
Our study had several limitations, chief among which are its relatively small sample size and lack of concomitant environmental and colonizing Zygomycetes isolates. Studying the genus distribution of such isolates and the risk factors for acquisition of Zygomycetes not causing invasive infection might have allowed us to identify additional, occult risk factors not appreciated in our analysis. Because of the noninterventional nature of the study and lack of these data for most patients, we were not able to assess iron overload [35] as a risk factor for zygomycosis in comparison with patients in the IA and control groups. Also, we cannot exclude the possibility of undiagnosed zygomycosis in control patients, in view of the low autopsy rate in our control groups. Finally, we cannot rule out the possibility that other differences among the groups could have influenced the validity of our findings. For example, most (56%) of the Zygomycetes infection cases in our study were definite, in contrast to only 26% of the IA cases; however, this discrepancy was largely due to the predominance of sinusitis and the frequent performance of sinus biopsy in the former group.
In conclusion, the spectrum of IMIs in immunosuppressed patients is evolving [36], and these trends in the frequency of IMIs might have implications for the index of suspicion of certain mycoses as well as for the choice of empirical and preemptive antifungal therapy. Proper identification of molds is more critical than ever for effective early antifungal therapy. A particular concern is that there is not a reliable method of early zygomycosis diagnosis based on antigen detection or PCR [37, 38]. Therefore, we believe that indiscriminate prophylaxis with antifungals that lack activity against non-Aspergillus molds might result in the selection of resistant molds. In view of the lack of a method for early diagnosis of zygomycosis, risk-based approaches and AMB-based preemptive regimens [39]or, possibly, posaconazole, a triazole with activity against the Zygomycetes [40]should be considered in high-risk patients with possible IMIs who are receiving VRC, especially those with initial presentation of sinusitis.
Acknowledgments
We thank Dr. Anthony Harris for useful comments; Susan Wallace, from the Department of Medical Informatics, for assistance in identifying patients in the 3 groups; and Frank Hung, from the Department of Pharmaceutical Policy and Outcomes Research, Division of Pharmacy, for providing information about total amphotericin B and voriconazole use in leukemia and bone marrow transplant services. We also thank Nathaniel D. Albert, Kristy Reece, and Dobbie Walton, for technical assistance. D.P.K. dedicates this work to Gerald P. Bodey, a pioneer in the study of fungal infections in patients with cancer, in honor of his 70th birthday.
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