IJCM  Vol.4 No.10 , October 2013
The Effect Analysis of Different Experimental Methods for the Diagnosis of Invasive Pulmonary Aspergillosis in a Rat Model
ABSTRACT
Background: Consensus on the most reliable assays to detect invasive aspergillosis from minimally or noninvasive samples has not been reached. In this study, we compared the efficacy of an enzyme-linked immunosorbent assay (ELISA) for galactomannan (GM) detection and quantitative real-time PCR assay (qRT-PCR) for the diagnosis of invasive pulmonary aspergillosis in a rat model. Methods: Neutropenic, male Sprague-Dawley rats (specific pathogen free; 8 weeks old; weight, 200 ± 20 g) were immunosuppressed with cyclophosphamide and infected with Aspergillus fumigatus intratracheally. Tissue and whole blood samples were harvested on days 1, 3, 5, and 7 post-infection and examined with GM ELISA and qRT-PCR. Results: On day 7, A. fumigatus DNA was amplified from 14 of 48 whole blood samples from immunosuppressed infected rats: day 1 (0/12), day 3 (0/12), day 5 (6/12), day 7 (8/12) post infection. The sensitivity and specificity of the qRT-PCR assay were 29.2% and 100%, respectively. Receiver operating characteristic curve (ROC) analysis indicated a Ct cut-off value of 15.35, and the area under the curve (AUC) was 0.627. The GM assay detected antigen in sera obtained on day 1 (5/12), day 3 (9/12), day 5 (12/12), and day 7 (12/12) post-infection, and thus had a sensitivity of 79.2% and a specificity of 100%. The ROC of the GM assay indicated that the optimal cut-off value was 1.40 (specificity, 100%; AUC, 0.919). Conclusions: The GM assay was more sensitive than qRT-PCR assay in diagnosing invasive pulmonary aspergillosis in rats.

Cite this paper
J. Lin, W. Xu, M. Li, Y. Xin, Y. Niu, C. Zhang and Z. Guo, "The Effect Analysis of Different Experimental Methods for the Diagnosis of Invasive Pulmonary Aspergillosis in a Rat Model," International Journal of Clinical Medicine, Vol. 4 No. 10, 2013, pp. 472-478. doi: 10.4236/ijcm.2013.410083.
References
[1]   C. Hahn-Ast, A. Glasmacher, S. Muckter, A. Schmitz, A. Kraemer, G. Marklein, P. Brossart and M. von LilienfeldToal, “Overall Survival and Fungal Infection-Related Mortality in Patients with Invasive Fungal Infection and Neutropenia after Myelosuppressive Chemotherapy in a Tertiary Care Centre from 1995 to 2006,” Journal of Antimicrobial Chemotherapy, Vol. 65, No. 4, 2010, pp. 761-768. http://dx.doi.org/10.1093/jac/dkp507

[2]   A. H. Groll and L. McNeil Grist, “Current Challenges in the Diagnosis and Management of Invasive Fungal Infections: Report from the 15th International Symposium on Infections in the Immunocompromised Host: Thessaloniki, Greece, 22-25 June 2008,” International Journal of Antimicrobial Agents, Vol. 33, No. 2, 2009, pp. 101-104.
http://dx.doi.org/10.1016/j.ijantimicag.2008.08.014

[3]   M. von Eiff, N. Roos, R. Schulten, M. Hesse, M. Zuhlsdorf and J. van de Loo, “Pulmonary aspergillosis: Early Diagnosis Improves Survival,” Respiration, Vol. 62, No. 6, 1995, pp. 341-347.
http://dx.doi.org/10.1159/000196477

[4]   G. Chamilos, M. Luna, R. E. Lewis, G. P. Bodey, R. Chemaly, J. J. Tarrand, A. Safdar, I. I. Raad and D. P. Kontoyiannis, “Invasive Fungal Infections in Patients with Hematologic Malignancies in a Tertiary Care Cancer Center: An Autopsy Study over a 15-Year Period (1989-2003),” Haematologica, Vol. 91, No. 7, 2006, pp. 986-989.

[5]   I. Hadrich, C. Mary, F. Makni, M. Elloumi, H. Dumon, A. Ayadi and S. Ranque, “Comparison of PCR-ELISA and Real-Time PCR for Invasive Aspergillosis Diagnosis in Patients with Hematological Malignancies,” Medical Mycology, Vol. 49, No. 5, 2011, pp. 489-494.

[6]   M. Lengerova, I. Kocmanova, Z. Racil, K. Hrncirova, S. Pospisilova, J. Mayer, L. K. Najvar, N. P. Wiederhold, W. R. Kirkpatrick ad T. F. Patterson, “Detection and Measurement of Fungal Burden in a Guinea Pig Model of Invasive Pulmonary Aspergillosis by Novel Quantitative Nested Real-Time PCR Compared with Galactomannan and (1,3)-Beta-D-glucan Detection,” Journal of Clinical Microbiology, Vol. 50, No. 3, 2012, pp. 602-608.
http://dx.doi.org/10.1128/JCM.05356-11

[7]   V. R. Aquino, F. Nagel, H. F. Andreolla, F. de-Paris, M. O. Xavier, L. Z. Goldani, D. W. Denning and A. C. Pasqualotto, “The Performance of Real-Time PCR, Galactomannan, and Fungal Culture in the Diagnosis of Invasive Aspergillosis in Ventilated Patients with Chronic Obstructive Pulmonary Disease (COPD),” Mycopathologia, Vol. 174, No. 2, 2012, pp. 163-169.
http://dx.doi.org/10.1007/s11046-012-9531-1

[8]   M. J. Ruping, J. J. Vehreschild, A. Groll, C. Lass-Florl, H. Ostermann, M. Ruhnke and O. A. Cornely, “Current Issues in the Clinical Management of Invasive Aspergillosis—The AGIHO, DMykG, OGMM and PEG Web-Based Survey and Expert Consensus Conference 2009,” Mycoses, Vol. 54, No. 5, 2011, pp. e557-e568.
http://dx.doi.org/10.1111/j.1439-0507.2010.01989.x

[9]   Y. Zhao, S. Park, P. Warn, R. Shrief, E. Harrison and D. S. Perlin, “Detection of Aspergillus fumigatus in a Rat Model of Invasive Pulmonary Aspergillosis by Real-Time Nucleic Acid Sequence-Based Amplification,” Journal of Clinical Microbiology, Vol. 48, No. 4, 2010, pp. 1378-1383. http://dx.doi.org/10.1128/JCM.02214-09

[10]   M. Hoenigl, H. J. Salzer, R. B. Raggam, T. Valentin, A. Rohn, A. Woelfler, et al., “Impact of Galactomannan Testing on the Prevalence of Invasive Aspergillosis in Patients with Hematological Malignancies,” Medical Mycology, Vol. 50, No. 3, 2012, pp. 266-269.
http://dx.doi.org/10.3109/13693786.2011.603102

[11]   I. Hadrich, F. Makni, F. Cheikhrouhou, S. Neji, I. Amouri, H. Sellami, H. Trabelsi, H. Bellaaj, M. Elloumi and A. Ayadi, “Clinical Utility and Prognostic Value of Galactomannan in Neutropenic Patients with Invasive Aspergillosis,” Pathologie Biologie (Paris), Vol. 60, No. 6, 2012, pp. 357-361.
http://dx.doi.org/10.1016/j.patbio.2011.10.011

[12]   T. J. Walsh, M. C. Wissel, K. J. Grantham, R. Petraitiene, V. Petraitis, M. Kasai, A. Francesconi, M. P. Cotton, J. E. Hughes, L. Greene, et al., “Molecular Detection and Species-Specific Identification of Medically Important Aspergillus Species by Real-Time PCR in Experimental Invasive Pulmonary Aspergillosis,” Journal of Clinical Microbiology, Vol. 49, No. 12, 2011, pp. 4150-4157.
http://dx.doi.org/10.1128/JCM.00570-11

[13]   M. J. Becker, S. de Marie, D. Willemse, H. A. Verbrugh and I. A. Bakker-Woudenberg, “Quantitative Galactomannan Detection Is Superior to PCR in Diagnosing and Monitoring Invasive Pulmonary Aspergillosis in an Experimental Rat Model,” Journal of Clinical Microbiology, Vol. 38, No. 4, 2000, pp. 1434-1438.

[14]   M. M. Leeflang, Y. J. Debets-Ossenkopp, C. E. Visser, R. J. Scholten, L. Hooft, H. A. Bijlmer, J. B. Reitsma, P. M. Bossuyt and C. M. Vandenbroucke-Grauls, “Galactomannan Detection for Invasive Aspergillosis in Immunocompromized Patients,” Cochrane Database of Systematic Reviews, Vol. 8, No. 4, 2008, Article ID: 007394.

[15]   S. F. Yeo and B. Wong, “Current Status of Nonculture Methods for Diagnosis of Invasive Fungal Infection,” Clinical Microbiology Review, Vol. 15, No. 3, 2002, pp. 465-484.
http://dx.doi.org/10.1128/CMR.15.3.465-484.2002

[16]   L. Millon, F. Grenouillet, J. Crouzet, F. Larosa, S. Loewert, A. P. Bellanger, E. Deconinck and F. Legrand, “FalsePositive Aspergillus Real-Time PCR Assay Due to a Nutritional Supplement in a Bone Marrow Transplant Recipient with GVH Disease,” Medical Mycology, Vol. 48, No. 4, 2010, pp. 661-664.
http://dx.doi.org/10.3109/13693780903451836

[17]   T. J. Walsh, S. Shoham, R. Petraitiene, T. Sein, R. Schaufele, A. Kelaher, H. Murray, C. Mya-San, J. Bacher and V. Petraitis, “Detection of Galactomannan Antigenemia in Patients Receiving Piperacillin-Tazobactam and Correlations between in Vitro, in Vivo, and Clinical Properties of the Drug-Antigen Interaction,” Journal of Clinical Microbiology, Vol. 42, No. 10, 2004, pp. 4744-4748.
http://dx.doi.org/10.1128/JCM.42.10.4744-4748.2004

[18]   E. McCulloch, G. Ramage, R. Rajendran, D. F. Lappin, B. Jones, P. Warn, R. Shrief, W. R. Kirkpatrick, T. F. Patterson and C. Williams, “Antifungal Treatment Affects the Laboratory Diagnosis of Invasive Aspergillosis,” Journal of Clinical Pathology, Vol. 65, No. 1, 2012, pp. 83-86.
http://dx.doi.org/10.1136/jcp.2011.090464

[19]   J. M. Scotter and S. T. Chambers, “Comparison of Galactomannan Detection, PCR-Enzyme-Linked Immunosorbent Assay, and Real-Time PCR for Diagnosis of Invasive Aspergillosis in a Neutropenic Rat Model and Effect of Caspofungin Acetate,” Clinical and Diagnostic Laboratory Immunology, Vol. 12, No. 11, 2005, pp. 1322-1327.

[20]   F. Suarez, O. Lortholary, S. Buland, M. T. Rubio, D. Ghez, V. Mahe, G. Quesne, S. Poiree, A. Buzyn, B. Varet, et al., “Detection of Circulating Aspergillus fumigatus DNA by Real-Time PCR Assay of Large Serum Volumes Improves Early Diagnosis of Invasive Aspergillosis in HighRisk Adult Patients under Hematologic Surveillance,” Journal of Clinical Microbiology, Vol. 46, No. 11, 2008, pp. 3772-3777. http://dx.doi.org/10.1128/JCM.01086-08

[21]   M. L. Luong, C. J. Clancy, A. Vadnerkar, E. J. Kwak, F. P. Silveira, M. C. Wissel, K. J. Grantham, R. K. Shields, M. Crespo, J. Pilewski, et al., “Comparison of an Aspergillus Real-Time Polymerase Chain Reaction Assay with Galactomannan Testing of Bronchoalvelolar Lavage Fluid for the Diagnosis of Invasive Pulmonary Aspergillosis in Lung Transplant Recipients,” Clinical Infectious Diseases, Vol. 52, No. 10, 2011, pp. 1218-1226.
http://dx.doi.org/10.1093/cid/cir185

[22]   P. L. White, M. D. Perry, A. Moody, S. A. Follett, G. Morgan and R. A. Barnes, “Evaluation of Analytical and Preliminary Clinical Performance of Myconostica MycAssay Aspergillus When Testing Serum Specimens for Diagnosis of Invasive Aspergillosis,” Journal of Clinical Microbiology, Vol. 49, No. 6, 2011, pp. 2169-2174.
http://dx.doi.org/10.1128/JCM.00101-11

[23]   C. R. Zhang, Y. C. Tang, K. Kawakami, T. T. Zhang, K. X. Zhang and J. X. Zhu, “An Experimental Study of the Therapeutic Effect of Interleukin-2 and Interleukin-12 with and without Amphotericin B on Pulmonary Fungal Infection,” Zhonghua Jie He He Hu Xi Za Zhi, Vol. 27, No. 4, 2004, pp. 234-236.

[24]   A. C. Leenders, S. de Marie, M. T. ten Kate, I. A. BakkerWoudenberg and H. A. Verbrugh, “Liposomal Amphotericin B (AmBisome) Reduces Dissemination of Infection as Compared with Amphotericin B Deoxycholate (Fungizone) in a Rate Model of Pulmonary Aspergillosis,” Journal of Antimicrobial Chemotherapy, Vol. 38, No. 2, 1996, pp. 215-225. http://dx.doi.org/10.1093/jac/38.2.215

[25]   J. Loffler, H. Hebart, U. Schumacher, H. Reitze and H. Einsele, “Comparison of Different Methods for Extraction of DNA of Fungal Pathogens from Cultures and Blood,” Journal of Clinical Microbiology, Vol. 35, No. 12, 1997, pp. 3311-3312.

[26]   J. Liu, Y. Shi, D. He, P. Liu, Y. Zhang, L. Xu and L. Wang, “Development of Real-Time PCR Method to Detect Aspergillus fumigates,” Biotechnology, Vol. 19, No. 1, 2009, pp. 34-36.

[27]   R. Torelli, M. Sanguinetti, A. Moody, L. Pagano, M. Caira, E. De Carolis, L. Fuso, G. De Pascale, G. Bello, M. Antonelli, et al., “Diagnosis of Invasive Aspergillosis by a Commercial Real-Time PCR Assay for Aspergillus DNA in Bronchoalveolar Lavage Fluid Samples from High-Risk Patients Compared to a Galactomannan Enzyme Immunoassay,” Journal of Clinical Microbiology, Vol. 49, No. 12, 2011, pp. 4273-4278.
http://dx.doi.org/10.1128/JCM.05026-11

[28]   M. A. Mennink-Kersten, J. P. Donnelly and P. E. Verweij, “Detection of Circulating Galactomannan for the Diagnosis and Management of Invasive Aspergillosis,” Lancet Infectious Disease, Vol. 4, No. 6, 2004, pp. 349-357.
http://dx.doi.org/10.1016/S1473-3099(04)01045-X

 
 
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