J Korean Med Sci.  2007 Aug;22(4):672-676. 10.3346/jkms.2007.22.4.672.

Comparison of the Real-time Nucleic Acid Sequence-based Amplification (RTi-NASBA) with Conventional NASBA, and Galactomannan Assay for the Diagnosis of Invasive Aspergillosis

• Yoo JH
• Choi SM
• Lee DG
• Park SH
• Choi JH
• Kwon EY
• Shin WS

• KMID: 1127085
• DOI: http://doi.org/10.3346/jkms.2007.22.4.672

Abstract

We compared a real time-nucleic acid sequence-based amplification (RTi-NASBA) with conventional NASBA, galactomannan enzyme immunosorbent assay (GMEIA), and Mycology Study Group of the European Organization for Research and Treatment of Cancer (EORTC/MSG) criteria for the diagnosis of invasive aspergillosis (IA). From May 2004 to May 2005, blood samples (314 in total) were collected twice a week from 78 patients with hematologic diseases during neutropenic fever after chemotherapy or hematopoietic stem cell transplantation. Results were compared with each other on the basis of EORTC/ MSG criteria. The cutoff of conventional NASBA was set to be 3.5; GM 0.5; RTi-NASBA, 20% above the negative control. There were 22 patients with IA (7 probables and 15 possibles) and 56 patients with nonfungal infection. The Kappa statistic for RTi-NASBA versus conventional NASBA was 0.80 (0.66-0.82; p<0.001) indicating that there was fairly good accordance between two tests. RTi-NASBA showed sensitivity 0.96, specificity 0.43, positive- and negative-predictive value 0.40 and 0.96, respectively. GM showed good specificity (0.98), while the sensitivity (0.45) was poor. When we use the combination of GM with either of two NASBAs, the sensitivity was improved up to 100%. In conclusion, RTi-NASBA could be a good alternative to the conventional one for the screening of IA.

Keyword

Aspergillosis; NASBA; Galactomannan

MeSH Terms

Aspergillosis/blood/*diagnosis/microbiology
Aspergillus/*genetics/metabolism
Enzyme-Linked Immunosorbent Assay
Female
Humans
Male
Mannans/*blood
Nucleic Acid Amplification Techniques/*methods
RNA, Fungal/genetics/isolation & purification
Reproducibility of Results
Sensitivity and Specificity

Figure

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  Fig. 1 Scheme of real time-nucleic acid sequence-based amplification (RTi-NASBA); upper: Sequence of RTi-NASBA; lower left: structure of molecular beacon and the process of detection by emission of fluorescence from R (recipient) on receiving light and freed from the inhibitory action of Q (quencher); lower right: a sample of the result from RTi-NASBA.

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  Fig. 2 Receiver-operating characteristic (ROC) curve and a table of statistics for selected thresholds for the cutoff NASBA value on the group of patients with probable and possible invasive aspergillosis as a standard (AUC: area under the ROC curve, 95% C.I.: 95% confidence interval).

Reference

1. Denning DW. Invasive aspergillosis. Clin Infect Dis. 1998. 26:781–805.

2. Yeo SI, Wong B. Current status of non-culture methods for diagnosis of invasive fungal infections. Clin Microbiol Rev. 2002. 15:465–484.

3. Stevens DA. Diagnosis of fungal infections: current status. J Antimicrob Chemother. 2002. 49:Suppl 1. 11–19.

4. Verweij PE, Poulain D, Obayashi T, Patterson TF, Denning DW, Ponton J. Current trends in the detection of antigenaemia, metabolites, and cell wall markers for the diagnosis and therapeutic monitoring of fungal infections. Med Mycol. 1998. 36:Suppl 1. 146–155.

5. Stynen D, Goris A, Sarfati J, Latge JP. A new sensitive sandwich enzyme-linked immunosorbent assay to detect galactofuran in patients with invasive aspergillosis. J Clin Microbiol. 1995. 33:497–500.

6. Boutboul F, Albert C, Leblanc T, Sulahian A, Gluckman E, Derouin F, Ribaud P. Invasive aspergillosis in allogeneic stem cell transplant recipients: increasing antigenemia is associated with progressive disease. Clin Infect Dis. 2002. 34:939–943.

7. Ascioglu S, Rex JH, de Pauw B, Bennett JE, Bille J, Crokaert F, Denning DW, Donnelly JP, Edwards JE, Erjavec Z, Fiere D, Lortholary O, Maertens J, Meis JF, Patterson TF, Ritter J, Selleslag D, Shah PM, Stevens DA, Walsh TJ. Defining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international consensus. Clin Infect Dis. 2002. 34:7–14.

8. Compton J. Nucleic acid sequence-based amplification. Nature. 1991. 350:91–92.

9. Yoo JH, Choi JH, Choi SM, Lee DG, Shin WS, Min WS, Kim CC. Application of nucleic acid sequence-based amplification for diagnosis of and monitoring the clinical course of invasive aspergillosis in patients with hematologic diseases. Clin Infect Dis. 2005. 40:392–398.

10. Leone G, van Schijndel H, van Gemen B, Kramer FR, Schoen CD. Molecular beacon probes combined with amplification by NASBA enable homogeneous, real-time detection of RNA. Nucleic Acids Res. 1998. 26:2150–2155.

11. McClernon DR, Vavro C, St Clair M. Evaluation of a real-time nucleic acid sequence-based amplification assay using molecular beacons for detection of human immunodeficiency virus type 1. J Clin Microbiol. 2006. 44:2280–2282.

12. Ginocchio GC, Kemper M, Stellrecht KA, Witt DJ. Multicenter evaluation of the performance characteristics of the NucliSens HIV-1 QT assay used for quantitation of human immunodeficiency virus type 1 RNA. J Clin Microbiol. 2003. 41:164–173.

13. Yates S, Penning M, Goudsmit J, Frantzen I, van de Weijer B, van Strijp D, van Gemen B. Quantitative detection of hepatitis B virus DNA by real-time nucleic acid sequence-based amplification with molecular beacon detection. J Clin Microbiol. 2001. 39:3656–3665.

14. Moore C, Hibbitts S, Owen N, Corden SA, Harrison G, Fox J, Gelder C, Westmoreland D. Development and evaluation of real-time nucleic acid sequence-based amplification assay for rapid detection of influenza A. J Med Virol. 2004. 74:619–628.

15. In : Ginocchio CC, Sillekens P, Loeffler J, van Aarie P, editors. Real-time detection of Aspergillus spp. Using nucleic acid sequence based amplification (NASBA) and molecular beacons. 2002. 12th ECCMID; Milan, Italy. –poster 1454.

16. Loeffler J, Hebart H, Cox P, Flues N, Schumacher U, Einsele H. Nucleic acid sequence-based amplification of Aspergillus RNA in blood samples. J Clin Microbiol. 2001. 39:1626–1629.

17. Hibbits S, Rahman A, John R, Westmoreland D, Fox JD. Development and evaluation of NucliSens basic kit NASBA for diagnosis of parainfluenza virus infection with 'end-point' and 'real-time' detection. J Virol Methods. 2003. 108:145–155.

18. Pinel C, Fricker-Hidalgo H, Lebeau B, Garban F, Hamidfar R, Ambroise-Thomas P, Grillot R. Detection of circulating Aspergillus fumigatus galactomannan: value and limits of the Platelia test for diagnosing invasive aspergillosis. J Clin Microbiol. 2003. 41:2184–2186.