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Use of Mycological, nested PCR, and Real-time PCR Methods on BAL Fluids for Detection of Aspergillus fumigatus and A. flavus in Solid Organ Transplant Recipients

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Abstract

Invasive aspergillosis continues to be a significant cause of morbidity and mortality in solid organ transplant (SOT) recipients. A reliable and early diagnostic method is needed to improve survival. In this study, four methods direct microscopy, culture, nested PCR on internal transcribed spacer region, and TaqMan real-time PCR targeted β-tubulin gene were examined for the detection of Aspergillus fumigatus and A. flavus in sixty-four bronchoalveolar lavage (BAL) fluids that were obtained from SOT recipients. Direct examination with 20 % KOH (potassium hydroxide) and culture on mycological media were also performed. Of the 64 samples, seven (10.9 %) were positive in direct examination (five with septate hyphae and two with aseptate hyphae), and 15 (23 %) had positive culture including five A. flavus, four A. niger, two Penicillium spp., two Rhizopus spp., one Fusarium spp. and one mixed A. flavus/A. niger. Twenty five (39 %) samples had positive nested PCR with A. flavus and 6 (9.4 %) with A. fumigatus-specific primers. Only eight (12.5 %) had positive real-time PCR for A. flavus and nine (14 %) for A. fumigatus. The incidence of aspergillosis in these patients included proven invasive pulmonary aspergillosis (IPA) in two (3 %), probable IPA in 14 (22 %), possible IPA in 38 (59 %), and not IPA in 10 (16 %). A. flavus was the most common cause of pulmonary aspergillosis (PA) in the study. The results suggest that because nested PCR is too sensitive it may increase the number of false-positive results and is not recommended for BAL samples for diagnosis of PA. Although further studies with significant number of proved positive/negative standard BAL samples are necessary for better evaluation, the novel multiplex real-time PCR developed in the study could be promising as a valid diagnostic method for IPA.

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Acknowledgments

We thank the staff of Teikyo University Institute of Medical Mycology (TIMM) in Japan and all personnel at respiratory ward of Shariati Hospital and Medical Mycology laboratory in School of Public Health in Tehran University of Medical Sciences (TUMS), for their helps.

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Authors and Affiliations

  1. Department of Medical Parasitology and Mycology, School of Medicine, Ghaem Hospital, Mashhad University of Medical Sciences, Mashhad, Iran

    Hossein Zarrinfar

  2. Department of Medical Parasitology and Mycology, National Institute of Health Research, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

    Hossein Mirhendi & Hossein Khodadadi

  3. Teikyo University Institute of Medical Mycology, Tokyo, Japan

    Koichi Makimura & Kazuo Satoh

  4. Department of Pulmonology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

    Omolbanin Paknejad

  5. Allergy Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

    Hossein Zarrinfar

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  1. Hossein Zarrinfar
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  2. Hossein Mirhendi
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  3. Koichi Makimura
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  4. Kazuo Satoh
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Correspondence to Hossein Mirhendi.

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Zarrinfar, H., Mirhendi, H., Makimura, K. et al. Use of Mycological, nested PCR, and Real-time PCR Methods on BAL Fluids for Detection of Aspergillus fumigatus and A. flavus in Solid Organ Transplant Recipients. Mycopathologia 176, 377–385 (2013). https://doi.org/10.1007/s11046-013-9657-9

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  • DOI: https://doi.org/10.1007/s11046-013-9657-9

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