|Year : 2018 | Volume
| Issue : 2 | Page : 122-124
Identification of Candida species by direct inoculation of cornmeal tween™ 80 agar from blood culture bottles: Rapid and cost-effective approach
Noureen Saeed, Madiha Sattar, Sadia Shakoor, Joveria Farooqi, Raunaq Mehboob, Afia Zafar, Kauser Jabeen
Department of Pathology and Laboratory Medicine, The Aga Khan University Hospital, Karachi, Pakistan
|Date of Web Publication||14-Jun-2018|
Dr. Kauser Jabeen
Department of Pathology and Laboratory Medicine, The Aga Khan University Hospital, Karachi
Source of Support: None, Conflict of Interest: None
Background: This study aimed to compare the accuracy of Candida species identification by direct inoculation of positive blood culture specimens into CornMeal Tween™ (CMT) 80 agar with the conventional methods. Methods: This study was performed in two phases: Phase 1: Inoculation of direct CMT from artificially spiked blood culture bottles with identified Candida species. Phase 2: Direct inoculation of smear-positive bottles on CMT agar for identification. Routine identification method was carried out using urease production, sensitivity to cycloheximide, CMT inoculation, and colony morphology on BiGGY agar. All isolates were also evaluated for sugar assimilation on the biochemical test panel Analytical profile index (API) 20C AUX (BioMerieux). Statistical Analysis Used: The correlation between both methods was determined as percentage agreement rate using kappa score. Results: In the first phase 50 artificially spiked blood culture bottles were included in the study. The sample set consisted of 24% C albicans, 38% C tropicalis, and 22% C glabrata isolates. There was 100% agreement between the directly inoculated and CMT inoculated from colonies at 48 hours. Phase 2: The overall agreement between directly inoculated CMT and conventional method of identification at 72 h was 92%. Conclusion: Direct inoculation of CMT agar can easily differentiate common Candida species microscopically on the basis of chlamydospores, blastopores, and arrangement of pseudo hyphae. However, identification of rare species from specimens containing two different species using direct method is challenging. Therefore, conventional methods including macroscopic/microscopic characteristics and biochemical profile with the aid of API with API 20 AUX would be a better choice for such cases.
Keywords: API 20 C Aux, Candida identification, Candida species, Corn meal tween 80
|How to cite this article:|
Saeed N, Sattar M, Shakoor S, Farooqi J, Mehboob R, Zafar A, Jabeen K. Identification of Candida species by direct inoculation of cornmeal tween™ 80 agar from blood culture bottles: Rapid and cost-effective approach. Biomed Biotechnol Res J 2018;2:122-4
|How to cite this URL:|
Saeed N, Sattar M, Shakoor S, Farooqi J, Mehboob R, Zafar A, Jabeen K. Identification of Candida species by direct inoculation of cornmeal tween™ 80 agar from blood culture bottles: Rapid and cost-effective approach. Biomed Biotechnol Res J [serial online] 2018 [cited 2021 Sep 16];2:122-4. Available from: https://www.bmbtrj.org/text.asp?2018/2/2/122/234457
| Introduction|| |
Accurate and rapid identification of Candida species is critical for appropriate management of candidemia. Blood cultures are the most common microbiological investigation requested from a patient with suspected candidemia. The conventional method for identification of yeast from a positive blood culture bottle requires growth of Candida species on a solid agar followed by microscopic evaluation on Corn Meal Tween ™ (CMT) 80 agar as well as biochemical studies from the colonies spanning at least 48–72 h for final identification. Several rapid tests have been developed recently including commercial peptide nucleotide analog probe fluorescent in situ hybridization method and nucleic acid-based detection systems that can identify Candida spp. directly from positive blood culture bottles., In resource-limited settings, identification mostly relies on biochemical tests whose sensitivity and specificity are known to be insufficient for identification of closely related or newly described species. Species identification/differentiation with the newly introduced matrix-assisted laser desorption ionization-time of flight mass spectrometry method is markedly superior to that with conventional phenotypic tests; however, the daily use of such a purely biophysical instrument currently is limited to European countries and/or is confined to large clinical microbiology laboratories.,
In several resource-limited settings with a significant burden of candidiasis, there is insufficient biotechnology infrastructure and expertise to enable the integration of molecular methods in the clinical microbiology laboratory. It is, therefore, imperative that turnaround time for conventional methods also be decreased to facilitate faster identification of microorganisms to the species level in such laboratories.
Direct inoculation of identification media has been employed for bacteria grown in a blood culture bottle to reduce time. We took a similar approach and inoculated CMT 80 agar directly from blood culture bottles positive for yeasts. Direct inoculation of CMT 80 Agar from positive blood culture has not been previously evaluated and was expected to save at least 24 h that is required for the growth of Candida spp. on solid media.
| Methods|| |
This study was performed at the Aga Khan University (AKU) Laboratory, Karachi, Pakistan. The study was conducted in two phases. In the first phase, blood culture bottles were artificially spiked with well-characterized Candida spp. strains. In the second phase, positive blood culture bottles from actual patients were studied. During both phases, BACTEC 9240 system was used for blood culture.
Preparation of Corn Meal Tween ™ 80 agar and sterility checks
Agar was prepared in-house from CMT by Oxoid as per manufacturer's recommendation. Prepared agar was checked for purity and performance using Candida albicans ATCC 10231 and Candida krusei ATCC 6258.
Phase 1: Inoculation of Corn Meal Tween™ directly from blood culture bottles artificially spiked with well-characterized Candida spp.
Aerobic BACTEC bottles were spiked with well-characterized Candida species revived from AKU collection of Candida strains. These isolates were identified by API yeast and confirmed by molecular identification performed at the CDC, Atlanta, USA. A total of fifty strains (C. albicans, Candida tropicalis, and Candida glabrata) were revived on Sabouraud's dextrose agar (SDA). After 24 h of incubation, a suspension with turbidity of 0.5 McFarland in 0.85% saline was made. One milliliter of this inoculum was inoculated in blood culture bottles. These bottles were then incubated in BACTEC ™ system. These bottles were pulled out after they flagged positive. 10 ul of suspension from positive blood bottle in hash form was inoculated on CMT agar and was covered with a sterile cover slip. The same amount was also inoculated and streaked in four quadrants on SDA and sheep blood agar to establish purity. The media were incubated for 24–72 h at 25°C. The plates were reviewed daily microscopically for morphological examination of production of chlamydospores, blastospores, and pseudohyphae (i.e., at intervals of 24, 48, and 72 h, respectively). The results were interpreted independently by two laboratory personnel (one resident microbiologist and one trained mycology technologist) blinded to each other's observations.
Phase 2: Direct inoculation of smear-positive bottles in routine conditions
10 ul of blood from blood culture bottles flagged positive in BACTEC ™ 9240 system showing yeasts on gram stain was inoculated using the similar methodology as described above on CMT 80 agar and incubated at 25°C ± 2°C for 72 h. The plates were examined daily and growth on CMT was observed microscopically for morphological examination of production of chlamydospores, blastospores, and pseudohyphae (i.e., at intervals of 24, 48, and 72 h, respectively). The results were interpreted independently by two laboratory personnel (one resident microbiologist and one trained mycology technologist) blinded to each other's observations.
Routine identification of yeast grown on a solid media was performed by germ tube production, colony morphology on BiGGY Agar (Becton Dickinson), Urea agar (Oxoid), sensitivity to cycloheximide (Becton Dickinson), inoculation on CMT 80 agar. All isolates were also evaluated for sugar assimilation on the biochemical test panel API 20C AUX (BioMerieux) (12).
| Results|| |
In the first phase, fifty artificially spiked blood culture bottles were included in the study. The sample set consisted of 24% C. albicans, 38% C. tropicalis, and 22% C. glabrata isolates. There was 100% agreement between direct inoculation and CMT inoculation from colonies at 48 h [Table 1].
|Table 1: Comparison of directly inoculated corn meal agar DICA in artificially spiked blood culture bottles|
Click here to view
A total of 129 positive blood cultures from 127 patients were included in the study. There were 58 male and 69 female patients of different age groups ranging from 1 month to 83 years.
The final identification of the organisms using conventional method revealed C. albicans (n = 30), C. parapsilosis (n = 36), C. tropicalis (n = 33), C. glabrata (n = 18), and C. krusei (n = 3). The overall agreement between directly inoculated CMT and conventional method of identification at 72 h was 92%. This agreement was 32% at 24 h and 75% at 48 h [Table 2]. The agreement rates according to Candida species are shown in [Table 3].
|Table 2: Comparison of directly inoculated corn meal agar DICA with time duration|
Click here to view
|Table 3: Comparison of common Candida species identified at 48 and 72 h by Corn Meal Tween™|
Click here to view
There were 11 specimens which yielded uncommon Candida species (Candida lusitaniae, 5; Candida guilliermondii, 3; Candida pelliculosa, 1; and Candida famata, 2), of which only five isolates were correctly identified using direct inoculation of CMT. Three Candida species could not be identified using conventional identification methods. Eight specimens yielded two different species and could be identified correctly in 4 (50%) samples using a direct inoculation on CMT.
| Conclusion|| |
Fungal bloodstream infections, primarily those caused by Candida species, are an emerging cause of sepsis in both developed and less developed settings. Most clinicians start antifungal therapy without proper speciation of yeast predisposing to emergence of resistant strains and in turn increasing morbidity and mortality of the patients. Sugar assimilation studies on the biochemical test panel is the gold standard in the phenotypic identification of yeast; only few laboratories go beyond the scope of germ tube testing and limit the identification of yeast to Candida and non-Candida species only. Use of biochemical test panels is not cost-effective and practical for most laboratories in resource-limited settings as health-care costs transmit directly to patients.
The conventional method for identification of yeast requires growth of Candida species on a solid agar followed by microscopic evaluation on CMT agar as well as biochemical studies from the colonies taking at least 72-96 hours for final identification from the time sample is received. Our study had the objective of demonstrating an alternative to the expensive methods for early identification of yeasts. CMT was found to be a suitable and affordable medium for observing microscopic morphology by directly inoculating it from positive blood culture bottles. Approximate cost per culture for complete identification of Candida species using CMT is US$ 1.6 (Rs. 160), while identification of Candida species via conventional methods (SDA, SBA, Biggy, Urea, germ tube test, CMT, and API) usually costs around US$ 20.40 (Rs. 2000).
We found that direct inoculation of CMT agar can easily differentiate common Candida species microscopically on the basis of chlamydospores, blastopores, and arrangement of pseudohyphae. However, identification of rare species from specimens containing two different species using direct method is challenging. Therefore, conventional methods including macroscopic/microscopic characteristics and biochemical profile with the aid of API with API 20 AUX would be a better choice for such cases.
The authors would like to acknowledge Mycotic diseases branch, Centers for Disease Control and Prevention, Atlanta, for their support.
Financial support and sponsorship
This study was financially supported by HEC and US State Department grant.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Larone D. Yeast and yeast-like organisms. Medically Important Fungi: A Guide to Identification. ASM press; 2002. p. 53-85.
Ariff S, Saleem AF, Soofi SB, Sajjad R. Clinical spectrum and outcomes of neonatal candidiasis in a tertiary care hospital in Karachi, Pakistan. J Infect Dev Ctries 2011;5:216-23.
Shepard JR, Addison RM, Alexander BD, Della-Latta P, Gherna M, Haase G, et al.
Multicenter evaluation of the Candida albicans
peptide nucleic acid fluorescent in situ
hybridization method for simultaneous dual-color identification of C. albicans
and C. glabrata
directly from blood culture bottles. J Clin Microbiol 2008;46:50-5.
Sow D, Fall B, Ndiaye M, Ba BS, Sylla K, Tine R, et al.
Usefulness of MALDI-TOF mass spectrometry for routine identification of Candida
species in a resource-poor setting. Mycopathologia 2015;180:173-9.
Santos C, Lima N, Sampaio P, Pais C. Matrix-assisted laser desorption/ionization time-of-flight intact cell mass spectrometry to detect emerging pathogenic Candida
species. Diagn Microbiol Infect Dis 2011;71:304-8.
Posteraro B, De Carolis E, Vella A, Sanguinetti M. MALDI-TOF mass spectrometry in the clinical mycology laboratory: Identification of fungi and beyond. Expert Rev Proteomics 2013;10:151-64.
Clark AE, Kaleta EJ, Arora A, Wolk DM. Matrix-assisted laser desorption ionization-time of flight mass spectrometry: A fundamental shift in the routine practice of clinical microbiology. Clin Microbiol Rev 2013;26:547-603.
Archibald LK, Tuohy MJ, Wilson DA, Nwanyanwu O, Kazembe PN, Tansuphasawadikul S, et al.
Antifungal susceptibilities of Cryptococcus neoformans
. Emerg Infect Dis 2004;10:143-5.
Wisplinghoff H, Bischoff T, Tallent SM, Seifert H, Wenzel RP, Edmond MB, et al.
Nosocomial bloodstream infections in US hospitals: Analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis 2004;39:309-17.
Nadeem SG, Hakim ST, Kazmi SU. Use of CHROMagar Candida
for the presumptive identification of Candida
species directly from clinical specimens in resource-limited settings. Libyan J Med 2010;5:2144.
Sandven P. Laboratory identification and sensitivity testing of yeast isolates. Acta Odontol Scand 1990;48:27-36.
Lyon GM, Karatela S, Sunay S, Adiri Y; Candida
Surveillance Study Investigators. Antifungal susceptibility testing of Candida
isolates from the Candida
surveillance study. J Clin Microbiol 2010;48:1270-5.
Neppelenbroek KH, Seó RS, Urban VM, Silva S, Dovigo LN, Jorge JH, et al.
Identification of Candida
species in the clinical laboratory: A review of conventional, commercial, and molecular techniques. Oral Dis 2014;20:329-44.
[Table 1], [Table 2], [Table 3]