Diagnosis of Invasive Septate Mold Infections A Correlation of Microbiological Culture and Histologic or Cytologic Examination

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1 Microbiology and Infectious Disease / DIAGNOSIS OF SEPTATE MOLD INFECTIONS Diagnosis of Invasive Septate Mold Infections A Correlation of Microbiological Culture and Histologic or Cytologic Examination Jeffrey J. Tarrand, MD, 1 Mathias Lichterfeld, MD, 2 Irfan Warraich, MD, 1 Mario Luna, MD, 1 Xiang Y. Han, MD, 1 Gregory S. May, PhD, 1 and Dimitrios P. Kontoyiannis, MD 2 Key Words: Invasive aspergillosis; Mold culture; Fungal recovery DOI: /EXBVYAUPENBM285Y Abstract We correlated results of microbiologic culture and histopathologic examination for 2,891 consecutive samples from autopsy tissue, surgical or biopsy tissue, and bronchoalveolar lavage (BAL) or bronchial washing (BW) specimens. For 23 autopsy cases with suspected invasive septate mold infections by histopathologic examination, culture yielded a mold in 12 cases (52%). For 1,683 surgical or biopsy samples, histopathologic evidence of invasive septate mold infection was present in 30 samples, 9 of which also grew mold by culture (30%); 20 additional samples grew mold in culture alone, possibly representing culture contamination. Of 1,185 BAL and BW samples, mold was evident in 28 by cytologic examination and culture, 20 by cytologic examination alone, and 68 by culture alone. These results suggest a positive concordance for culture and histologic-cytologic examination of 23%, although both methods were negative in 96% of surgical and biopsy tissue and BAL and BW samples. The septate molds cultured from these samples were Aspergillus fumigatus (19), Aspergillus flavus (15), Aspergillus terreus (13), Aspergillus niger (7), Fusarium species (3), and Scedosporium apiospermum (2). A flavus was isolated significantly more frequently from tissue than from BAL and BW samples. Invasive septate mold infections caused by Aspergillus, Scedosporium, and Fusarium organisms and other septate molds frequently present a diagnostic challenge. Although histologic examination of the infected tissue remains the gold standard for diagnosis, obtaining tissue during surgery or biopsy may be impractical in many cases owing to risks associated with the underlying disease. In many instances, when the tissue diagnosis is made, invasive septate mold infection already is in the advanced stage. Since most invasive septate mold infections have associated pulmonary disease in the early course of the infection, bronchoalveolar lavage (BAL) and bronchial wash (BW) frequently are performed and the resulting specimens subjected to cytologic examination and microbiologic culture. The results then are correlated with clinical and radiologic findings to determine an early diagnosis. Aspergillus, Fusarium, and Scedosporium organisms exhibit similar septate hyphae in tissue and BAL and BW specimens on microscopic examination, and examples of branching morphologic features frequently are missing. Correlation with culture results is necessary for the definitive identification of the organism. To date, most correlation studies have been from autopsy series, 1-3 whereas few data exist from surgical or biopsy tissue or BAL or BW specimens. We report the correlation for 2,891 consecutive specimens from autopsy tissue, surgical and biopsy tissue, and BAL and BW specimens from a cancer patient population. Materials and Methods We retrospectively reviewed all fungal culture records for autopsy tissue samples, surgical and biopsy tissue samples, BAL specimens, and BW specimens examined from January 854 Am J Clin Pathol 2003;119: DOI: /EXBVYAUPENBM285Y

2 Microbiology and Infectious Disease / ORIGINAL ARTICLE 1998 to December 1999 at the microbiology laboratory of the University of Texas M.D. Anderson Cancer Center, Houston. A total of 3,220 such cultures were performed, including 23 autopsy samples, 1,913 surgical or biopsy tissue samples, and 1,284 BAL or BW specimens. Corresponding histologic or cytologic examinations within 2 weeks of these cultures were matched as follows: all 23 autopsy cases, 1,683 tissue samples (87.98%), and 1,185 BAL or BW specimens (92.29%). Correlation analysis was performed in these 2,891 samples. Microbiologic Culture All specimens from autopsy tissue, surgical or biopsy tissue, and BAL and BW submitted or fungal culture were plated routinely on Sabouraud dextrose plates, Mycosel agar (BBL, Cockeysville, MD), Sabouraud dextrose slants, and brain-heart infusion agar supplemented with 10% sheep blood, chloramphenicol, and gentamicin. The media were incubated at 25 C without carbon dioxide for a total of 4 weeks and read twice per week. Molds were identified by using standard morphologic criteria. 4 Molds isolated from routine bacteriologic culture were transferred to fungus culture for definitive identification. Histologic Criteria Tissue sections for histologic examinations were stained routinely with H&E and, when indicated, with Gomori methenamine silver. For antemortem tissue, we included only specimens from potentially sterile sources biopsies, aspirates, surgical wounds, and surgical tissue fragments. Nonsterile specimens, such as sputum, stool, and urine, and sources prone to contamination, such as drains, skin, and nonsurgical wounds, were excluded. The diagnosis of invasive septate mold infection was made when septate hyphae with a pertinent background of inflammation, necrosis, or granuloma and demonstrating acute 45 branching were observed histologically. In addition to these microscopic criteria, autopsy cases also had macroscopic lesions compatible with invasive septate mold infections. Specimens showing Zygomycetes or yeast infections were not included in the study. Similar morphologic criteria were used for the cytologic examination of BAL and BW specimens. Statistics When appropriate, the χ 2 test was used for statistical analysis. 5 Results Even in autopsy-confirmed cases of invasive septate mold infection, the culture yield of mold was low. Autopsy tissue samples typically have a heavy load of organisms and a large inoculum size for culture. For the 23 autopsy cases with invasive septate mold infections detected by histopathologic examination, 12 cases (52%) had mold growth: Aspergillus terreus (4 cases), Aspergillus flavus (3 cases), Aspergillus fumigatus (1 case), Fusarium species (2 cases), and Scedosporium apiospermum (2 cases) Table 1. The size of culture inoculum for surgical and biopsy tissue samples was more variable depending on the size of tissue. In these specimens (n = 1,683), histologic examination revealed that 30 were consistent with invasive septate mold infections, of which culture yielded a mold in 9 (30%) (Table 1). The 9 samples included 8 from nasal or sinus biopsies, and the molds recovered were all Aspergillus, including A flavus (4 samples), A fumigatus (3 samples), and A niger (1 sample), and 1 sample from a bone abscess that grew Fusarium species. The 30 culture-negative tissue types were mostly abscesses, lung mass lesions, and soft tissue lesions. In addition, 17 samples showed mold growth but had no histologic evidence of invasive septate mold infection, likely representing culture contamination by mold. The rate of mold isolation, including 17 possible contaminants, was 1.54% (26/1,683). Autopsy cases showed a trend toward higher culture yield compared with surgical and biopsy specimens (12/23 vs 9/30; P =.15 [not significant]). The results of correlation of the culture and cytologic examination results for BAL and BW samples were different Table 1 Presence of Septate Mold in Tissue or BAL or BW Specimens by Histologic and Cytologic Examination and by Culture Positive Positive Culture and Positive Culture, Negative Specimen Type No. of Specimens Histocytologic Findings Histocytologic Findings Histocytologic Findings Autopsy tissue Surgical or biopsy tissue 1, BM, FL, LN, and NL Soft tissue lesion Lung mass lesion Abscess All other, excluding skin BAL or BW 1, BAL, bronchoalveolar lavage; BM, bone marrow; BW, bronchial wash; FL, sterile fluid aspirate; LN, lymph node; NL, nonlung fine-needle aspirate. Am J Clin Pathol 2003;119: DOI: /EXBVYAUPENBM285Y 855

3 Tarrand et al / DIAGNOSIS OF SEPTATE MOLD INFECTIONS from those for surgical and biopsy tissue. Cytologic evidence of a septate mold was seen in 48 BAL and BW samples, 28 of which (58%) grew the organism by culture (Table 1), which is significantly higher than the culture yield from tissue samples (9/30 [30%]) (χ 2 = 4.86; P <.03). In addition, 68 samples grew septate mold alone, making a total of 96 (8.10%) of 1,185 samples positive by culture. This rate also was significantly higher than that for surgical and biopsy tissue samples (26/1,683 [1.54%]) (χ 2 = 71.9; P <.01). The 28 samples that were positive cytologically and by culture all grew Aspergillus species, including A fumigatus (15 samples), A terreus (9 samples), A niger (6 samples), and A flavus (4 samples) (3 cases had multiple species). Because these samples were positive cytologically and by culture, they probably had higher numbers of organisms, and for many of them (15/28 [54%]), there were multiple isolations of the same mold during 6-month periods before and after the index sample was obtained. In contrast, for the samples positive by culture alone, there were fewer multiple isolations of the same mold (11/68 [16%]) (χ 2 = 12.2; P <.01). The distribution of septate mold recovered from tissue (combined autopsy and surgical and biopsy) vs BAL and BW specimens was compared Table 2. Among the Aspergillus species, A flavus was isolated more frequently from tissue samples (7/16) than from BAL and BW specimens (4/34) (χ 2 = 4.75; P <.03). Although the difference was not statistically significant, A fumigatus was isolated more frequently from BAL and BW specimens (15/34 [44%]) than from tissue samples (4/16 [25%]). Discussion There is substantial controversy concerning the optimal diagnostic criteria for invasive mold infections. Consensus criteria recently developed by the European Organization for Research and Treatment of Cancer incorporate clinical, radiologic, microscopic, and culture results and provide a consistent approach to management and diagnosis of these devastating Table 2 Septate Molds Recovered by Source of Specimen infections. 6 Nevertheless, microscopic criteria, while perhaps less sensitive than clinical criteria, still remain critical for the objective diagnosis of invasive mold infections. The invasive septate molds most commonly seen are hyaline Hyphomycetes, generally characterized by parallel cell walls, septation, lack of pigmentation, and progressive, dichotomous branching Image 1. Zygomycetes show large, ribbon-like, multinucleated cells with nonparallel walls without septation. 7 The present study, limited to a comparison of histologic or cytologic findings of septate molds with fungal culture from clinical specimens, demonstrates the low yield of culture in histopathologically confirmed cases of invasive septate mold infection. The culture yield of an Aspergillus or another septate mold from autopsy tissue identified as having an invasive septate mold infection has been generally low, from 30% to 50%. 1,8 The yield of 52% from our 23 autopsy cases is consistent with these previous reports. We also found that the culture yield of septate molds from surgical and biopsy tissue specimens was even lower, 30% (9/30) for those with histologically evident invasive septate mold infections. There are no similar data for comparison. Our data for BAL and BW specimens also seem unique. The overall isolation rate of septate molds from BAL and BW samples (8.10%) was much higher than that from surgical and biopsy tissue specimens (1.54%). Cytologically positive BAL and BW specimens had a higher culture yield as well (58% [28/48]). Fusarium organisms grew well by culture. The 2 autopsy cases with Fusarium species had 3 and 2 antemortem blood cultures positive for Fusarium species. In contrast, among the 8 autopsy cases that grew Aspergillus organisms, no antemortem samples were positive. Fusarium organisms have adventitious sporulation resulting in direct production of blastoconidia in tissue. This is believed to explain their frequent isolation from blood cultures. 9 In contrast, in a review of the 10-year experience at our institution, isolation of Aspergillus organisms from blood culture was infrequent (37 in 300,000 cultures) and was correlated with clinical aspergillosis in only 34% of cases. 10 The fact Specimen Source Bronchoalveolar Organism Autopsy and Surgery or Biopsy Lavage or Bronchial Wash Total Aspergillus fumigatus Aspergillus terreus Aspergillus flavus Aspergillus niger Fusarium species Scedosporium apiospermum Total Am J Clin Pathol 2003;119: DOI: /EXBVYAUPENBM285Y

4 Microbiology and Infectious Disease / ORIGINAL ARTICLE A B C Image 1 Invasive septate mold infections can show variable morphologic features. A, Aspergillus fumigatus in a lung biopsy specimen showing progressive, dichotomous, acute branching septate hyphae (H&E, 100). B, Aspergillus species from a bronchoalveolar lavage specimen showing few branches and numerous empty and distorted cells (Gram, 400). C, Scedosporium apiospermum from a bronchoalveolar lavage specimen showing acute branching (Gram, 200). D, Fusarium species from skin showing swollen distorted forms, rudimentary branching, and intercalary chlamydospores (arrows) (H&E, 400). that one cannot reliably diagnose invasive aspergillosis by blood or other culture reinforces the need to obtain tissue- or fluid-based microscopic analysis. The reason for the poor recovery of Aspergillus species from infected tissue is not well understood. 1-3,8,11,12 There are a few plausible explanations. To be seen histologically, approximately 10 3 to 10 5 microorganisms per gram of tissue must be present; therefore, at least in this subset, culture failure would suggest nonviability of these organisms due to various factors, such as the effect of antifungal treatment or the environment of anoxic necrotic tissue. 13 Alternatively, there is a possible difference in the physiologic state of the D mold in tissue vs in culture media. The mold adapted to growth in tissue cannot rapidly adapt its metabolism to current in vitro culture conditions (eg, media, temperature, oxygen content). This notion is supported by unpublished data indicating that culture conditions with a microaerophilic atmosphere and a temperature of 35 C improve the growth and recovery of Aspergillus species. 14 Our study also indicates a possible changing pattern of the occurrence of various Aspergillus species. Previous studies have found A fumigatus to be the predominant species associated with invasive aspergillosis in autopsy series. 15,16 Our data from combined autopsy and surgical and biopsy Am J Clin Pathol 2003;119: DOI: /EXBVYAUPENBM285Y 857

5 Tarrand et al / DIAGNOSIS OF SEPTATE MOLD INFECTIONS tissue specimens showed that only 25% (4/16) of the Aspergillus species were A fumigatus. It has been noted that susceptibility to amphotericin B may vary substantially among Aspergillus species. 16 The frequent prophylactic, preemptive, and targeted use of azoles and amphotericin B in our center may have resulted in the selection of Aspergillus species more resistant than A fumigatus. The present study shows more frequent isolation of A flavus from tissue specimens than from BAL and BW samples. In contrast, A fumigatus was the most common isolate in BAL and BW specimens, possibly reflecting more frequent colonization or transient exposure. With the possible exception of nasal biopsy tissue, the fungal culture yield from antemortem sources was low. In thrombocytopenic patients, robust samples are difficult to obtain. However, even in autopsy cases, in which abundant fungus was seen, culture recovery was poor. Although both microscopic analysis and culture methods may be less sensitive than clinical criteria, a stringent histologic definition of invasive septate mold infection may provide a stable benchmark for evaluation of new diagnostic techniques, including antigen detection and Aspergillus polymerase chain reaction assays. From the 1 Division of Pathology and Laboratory Medicine and 2 Department of Infectious Diseases, Infection Control and Employee Health, the University of Texas M.D. Anderson Cancer Center, Houston. Supported by the Division of Pathology and Laboratory Medicine, the University of Texas M.D. Anderson Cancer Center. Address reprint requests to Dr Tarrand: Division of Pathology and Laboratory Medicine, Box 84, the University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX Acknowledgment: We thank Don Norwood for scientific editorial assistance. References 1. Bodey G, Bueltman B, Duguid W, et al. Fungal infections in cancer patients: an international autopsy survey. Eur J Clin Microbiol Infect Dis. 1992;11: Mirsk HS, Cuttner J. Fungal infections in acute leukemia. Cancer. 1972;30: Yu VL, Muder RR, Poorsattar A. Significance of isolation of Aspergillus from the respiratory tract in the diagnosis of invasive pulmonary aspergillosis. Am J Med. 1986;81: Larone DH. Medically Important Fungi: A Laboratory Guide to Identification. 3rd ed. Washington, DC: ASM Press; Colton T. Statistics in Medicine. Boston, MA: Little, Brown; 1984: Ascioglu S, Rex JH, de Pauw B, et al. Defining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international consensus. Clin Infect Dis. 2002;34: Chandler FW, Watts JC. Pathologic Diagnosis of Fungal Infections. Chicago, IL: ASCP Press; Young RC, Bennette JE, Vogel CL, et al. Aspergillosis: the spectrum of the disease in 98 patients. Medicine (Baltimore). 1970;49: Liu K, Howell DN, Perfect JR, et al. Morphologic criteria for the preliminary identification of Fusarium, Paecilomyces, and Acremonium species by histopathology. Am J Clin Pathol. 1998;109: Kontoyiannis DP, Sumoza D, Tarrand JJ, et al. Significance of aspergillemia in patients with cancer: a 10-year study. Clin Infect Dis. 2002;31: Patterson TF, Kirkpatrick WR, White M, et al. Invasive aspergillosis: disease spectrum, treatment practices, and outcomes. Medicine (Baltimore). 2000;79: Bartlett JG. Aspergillosis update. Medicine (Baltimore). 2000;79: Groll AH, Shah PM, Menzel C, et al. Trends in the postmortem epidemiology of invasive fungal infections at a university hospital. J Infect. 1996;33: Tarrand JJ, Kontoyiannis DP, Han XY, et al. Culture incubation conditions affect the growth of Aspergillus spps. in an in vitro model system of tissue phase fungal growth. Presented at the 42nd Annual Interscience Conference on Antimicrobial Agents and Chemotherapy; September 27-30, 2002; San Diego, CA. Abstract M Kaiser I, Huguenin T, Lew PD, et al. Invasive aspergillosis: clinical features of 35 proven cases at a single institution. Medicine (Baltimore). 1998;77: Iwen PC, Rupp ME, Langnas AN, et al. Invasive pulmonary aspergillosis due to Aspergillus terreus: 12-year experience and review of the literature. Clin Infect Dis. 1998;26: Am J Clin Pathol 2003;119: DOI: /EXBVYAUPENBM285Y