recover Zygomycetes organisms from tissue specimens, despite the presence of ... In addition, data on consecutive clinical specimens sub- mitted for fungal ...
Microbiology and Infectious Disease / ZYGOMYCETES CULTURE RECOVERY
Increased Culture Recovery of Zygomycetes Under Physiologic Temperature Conditions Dimitrios P. Kontoyiannis, MD,1,2 Georgios Chamilos, MD,1 Saad A. Hassan, MD, PhD,3 Russell E. Lewis, PharmD,1,2 Nathaniel D. Albert,1 and Jeffrey J. Tarrand, MD3 Key Words: Zygomycetes; Diagnosis; Physiology; In vitro growth DOI: 10.1309/7KU5XWURYM0151YN
Abstract Poor recovery of Zygomycetes hyphae from tissue specimens may result from failure of current culture methods to mimic physiologic conditions found in hyphae-laden infected tissue. We describe the use of an in vitro model simulating Zygomycetes growth under necrotic or hypoxic tissue conditions. We preconditioned hyphae of clinical Zygomycetes isolates in flasks under anaerobic conditions using Ana-Packs (Becton Dickinson Microbiology Systems, Sparks, MD) at 37°C for 48 hours, thus simulating in vivo growth in an infracted hypoxic lesion, and compared the recovery of paired inocula at 37°C and 25°C. Incubation of stock culture isolates at 37°C resulted in significantly better culture recovery (about 10-fold) when compared with incubation at 25°C (P < .0001). In addition, we similarly evaluated 25,291 consecutive clinical specimens. Among 41 specimens, the yield of Zygomycetes cultures incubated at 37°C (23/41 [56%]) was significantly higher than that incubated at 25°C (9/41 [22%]; P = .0001). Overall, we found that culture recovery was significantly (254%) enhanced at 37°C.
Zygomycetes fungi can produce invasive infections, particularly in immunocompromised hosts.1,2 The incidence of infections by these opportunistic pathogens is on the rise, partially reflecting changes in antifungal prophylaxis and treatment.1,3,4 Routine laboratory methods fail to effectively recover Zygomycetes organisms from tissue specimens, despite the presence of large amounts of hyphae in the specimens.5 For example, Kontoyiannis et al4 showed that in more than one third of patients with cancer who had histopathologically documented zygomycosis, the organism failed to grow in culture. Another study reported a similar discrepancy for Aspergillus species.6 For clinical mycology laboratories, the current recommended incubation temperature of less than 30°C is thought to be the optimum temperature, and although higher temperatures are used as part of identification keys, initial cultures are frequently incubated at room temperature.7 Tarrand et al8 demonstrated enhanced yield of anaerobically “stressed” hyphae at physiologic temperatures for the Aspergillus species typically associated with invasive infections in immunosuppressed hosts. We hypothesized that a similar physiologic adaptation of Zygomycetes hyphae to 37°C may occur in vivo and that mimicking the physiologic temperature may enhance the recovery of Zygomycetes fungi from clinical specimens.
Materials and Methods Yield of Cultures of Anaerobically Stressed Hyphae All Zygomycetes isolates used in these studies were obtained from laboratory stocks of clinical isolates at our 208 208
Am J Clin Pathol 2007;127:208-212 DOI: 10.1309/7KU5XWURYM0151YN
© American Society for Clinical Pathology
Microbiology and Infectious Disease / ORIGINAL ARTICLE
institution derived from clinical isolates. The genus of 17 strains was identified using standard morphologic criteria and confirmed using a DNA sequencing method ❚Table 1❚. Two strains obtained from patients with cancer who had known documented invasive zygomycosis—Cunninghamella bertholletiae Lab16 and Rhizopus oryzae Lab15—were identified at the species level. Isolates were subcultured on Sabouraud dextrose agar with the Emmons modification (SDAE) or on YAG (0.5% yeast extract, 1.0% dextrose, 0.2% vitamin mix, 0.1% trace elements, 1.5% agar, 1% magnesium sulfate) plates for a minimum of 3 passages at 37°C for 2 days; conidia were then collected using sterile glass wool. To induce hyphal growth without sporulation, standardized inocula containing 103 to 104 conidia in RPMI 1640–MOPS (3-[N-morpholino]propanesulfonic acid), pH 7.2, in liquid media were incubated under microaerobic conditions (6% oxygen, 5%-10% carbon dioxide) using CampyPak gas packs (Becton Dickinson Microbiology Systems, Sparks, MD) at 37°C for 24 hours. The growth of Zygomycetes fungi exclusively as hyphae and the absence of ungerminated conidia in all test cultures were confirmed under a microscope (Olympus BX51, 100× magnification; Olympus, Melville, NY). The Zygomycetes hyphae were then incubated in flasks under anaerobic conditions using Ana-Packs (Becton Dickinson Microbiology Systems) at 37°C for an additional 48 hours, thus simulating in vivo growth in an infracted hypoxic lesion.8 Hyphae were then harvested in 5 mL of cold, sterile normal saline and ground immediately
using a tissue homogenizer (50-mL disposable; Kendall, Mansfield, MA) to generate hyphal fragments. By using these hyphal fragments, serial 10-fold dilutions in ice cold saline were prepared, and the dilutions were immediately plated on SDAE agar. For all of these experiments, the plates were inoculated and spread as matched pairs (pairs of plates were quickly spread before proceeding to the next dilution). In a separate series of experiments using Lab15 and Lab16, the inoculum was distributed using a spreader plate method ❚Table 2❚; in all of the other experiments, dilutions of each of the Zygomycetes isolates were inoculated simultaneously as pairs with 5 to 8 equal drops per plate, alternating drops between paired plates. Finally, the paired plates were incubated at 25°C (room temperature) or 37°C. Differences in growth were analyzed by counting the colony-forming units (CFUs) or the spots showing growth at 18, 24, and 48 hours. Plates without growth were finalized at 2 weeks. Yield of Cultures of Clinical Specimens Incubated at 25°C vs 37°C In addition, data on consecutive clinical specimens submitted for fungal culture from January 2002 to June 2006 were analyzed. During this period, samples were equally inoculated on 5 media: a Sabouraud dextrose agar tube, Mycosel, a brain-heart infusion tube with gentamicin and chloramphenicol, and 2 SDAE plates (Becton Dickinson Microbiology Systems). Only the SDAE plates were analyzed in the present study: one SDAE plate was incubated at
❚Table 1❚ Strain Identification and Recovery at 25°C vs 37°C Designation Lab1 Lab2 Lab3 Lab6 Lab7 Lab8 Lab9 Lab10 Lab12 Lab13 Lab14 Lab15 Lab16 Lab17 Lab18 Lab19 Lab20
Genus*
25°C†
37°C†
Dilution‡
Mucor Mucor Mucor Rhizopus Rhizopus Rhizopus Rhizopus Mucor Rhizopus Rhizopus Rhizopus Rhizopus Cunninghamella Rhizopus Rhizopus Rhizopus Rhizopus
3/5, 1/5 8/8, 8/8 2/8, 2/8 2/8, 4/8 0/8, 0/8 0/5, 2/5 2/8, 3/8 1/5, 1/5 0/5, 0/5 0/5, 0/5 0/8, 3/8 1/8, 1/8 1/8, 2/8 1/8, 2/8 6/8, 7/8 1/8, 2/8 0/5, 0/5
4/5, 3/5 8/8, 8/8 1/8, 2/8 8/8, 8/8 0/8, 7/8 2/5, 2/5 8/8, 8/8 4/5, 4/5 3/5, 2/5 5/5, 5/5 1/8, 4/8 5/8, 6/8 1/8, 3/8 8/8, 8/8 8/8, 8/8 8/8, 8/8 2/5, 2/5
10–3 101 103 103 105 103 102 103 103 101 104 105 105 105 103 101 103
P§ NS 1 1 .0002 .007 .63 .0001 .63 .032 .0001 .68 .003 1 .0001 .23 .0001 .09
NS, not significant. * Strains were genotypically confirmed using sequencing.3 † Results are interpreted as follows: 1/5, 1 spot with growth of 5 spots inoculated. Each temperature condition shows 2 data groups derived from 2 plates. Plates were read at 24 and 48 hours, but only the 48-hour value is shown. Growth-negative plates were reread at 5 to 7 days, and all negative plates remained negative. ‡ The final limiting dilution at which growth could be read. Thus, for each strain’s dilution series, lower dilution plates showed overgrowth, and higher dilution plates were negative growth. § Fisher exact test (independent 2-tailed contingency). When results for all isolates are combined, P < .001.
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DOI: 10.1309/7KU5XWURYM0151YN
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Kontoyiannis et al / ZYGOMYCETES CULTURE RECOVERY
❚Table 2❚ Recovery of Anaerobically Preconditioned Zygomycetes Hyphae at 25°C and 37°C Median No. of CFUs* Dilution 101 102 103 104
Isolate
Cunninghamella bertholletiae Rhizopus oryzae C bertholletiae R oryzae C bertholletiae R oryzae C bertholletiae R oryzae
25°C/37°C (12 h)
25°C/37°C (24 h)
25°C/37°C (48 h)
P
2/750 1/700 1/200 1/300 1/25 1/30 1/1 1/1
150/2,520 300/2,400 110/420 100/600 2/40 2/30 2/7 1/6
1,600/5,000† 1,500/5,000 1,400/2,500 1,500/2,500 4/1,500 4/1,500 2/950 2/950
