Sains Malaysiana 47(3)(2018): 489–498

http://dx.doi.org/10.17576/jsm-2018-4703-08

Dual Panel Multiplex PCR Assay for Rapid Detection of Medically Important Fungi and Resistant Species of Candida and Aspergillus

(Asai PCR Multipleks Dual Panel untuk Pengesanan Segera Kulat yang Penting daripada

Segi Perubatan dan Spesies Rintang Candida dan Aspergillus)

MOHD HANIF JAINLABDIN^1,2, ANG LIM CHUA³, TZAR MOHD NIZAM⁴ & JACINTA SANTHANAM^1*

¹Biomedical Science Programme, School of Diagnostic and Applied Health Sciences

Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz

50300 Kuala Lumpur, Wilayah Persekutuan, Malaysia

²Department of Basic Medical Sciences for Nursing, Kulliyyah of Nursing, International Islamic University Malaysia, Indera Mahkota Campus, Jalan Sultan Ahmad Shah, 25200 Kuantan, Pahang Darul Makmur, Malaysia

³Medical Laboratory Sciences Cluster, Faculty of Medicine, Universiti Teknologi Mara (UiTM)

Sungai Buloh Campus, Jalan Hospital, 47000 Sungai Buloh, Selangor Darul Ehsan, Malaysia

⁴Department of Medical Microbiology & Immunology, Universiti Kebangsaan Malaysia Medical Centre, 56000 Kuala Lumpur, Wilayah Persekutuan, Malaysia

Received: 17 October 2016/Accepted: 23 October 2017

ABSTRACT

Invasive fungal infections (IFIs) have risen dramatically in recent years among high risk immunocompromised patients. Rapid detection of fungal pathogens is crucial to timely and accurate antifungal therapy. Two multiplex polymerase chain reaction (PCR) assays were developed to detect major fungal species that cause invasive infections and identify resistant species. Genus specific primers for Candida, Aspergillus, Fusarium and species specific primers for Candida glabrata, Candida krusei and Aspergillus terreus which are known to be clinically resistant species, were designed from the internal transcribed spacer (ITS) regions of ribosomal ribonucleic acid (rRNA) gene complex. Both assays were performed simultaneously to promote rapid detection of fungal isolates based on distinct amplicon sizes. Inclusion of the universal fungal primers ITS 1 and ITS 4 in the genus specific assay produced a second amplicon for each isolate which served to confirm the detection of a fungal target. The limit of detection for the genus specific assay was 1 nanogram (ng) deoxyribonucleic acid (DNA) for Aspergillus fumigatus and Candida albicans, 0.1 ng DNA for Fusarium solani, while the species-specific assay detected 0.1 ng DNA of A. terreus and 10 picogram (pg) DNA of C. krusei and C. glabrata. The multiplex PCR assays, apart from universal detection of any fungal target, are able to detect clinically important fungi and differentiate resistant species rapidly and accurately, which can contribute to timely implementation of effective antifungal regime.

Keywords: Aspergillus; Candida; detection; Fusarium; multiplex PCR

ABSTRAK

Jangkitan kulat invasif telah meningkat sejak kebelakangan ini dalam kalangan pesakit terimunokompromi. Pengesanan segera patogen kulat adalah amat perlu supaya terapi antikulat yang bersesuaian dapat diberikan. Dua asai tindak balas rantai polimerase multipleks telah dibangunkan untuk mengesaan spesies utama patogen kulat yang menyebabkan infeksi invasif dan mengenal pasti spesies resistan. Primer khusus untuk genus Candida, Aspergillus, Fusarium dan khusus untuk spesies Candida glabrata, Candida krusei dan Aspergillus terreus yang merupakan spesies rintang telah direka cipta berdasarkan jujukan mentranskripsi jarak dalaman (ITS) kompleks gen rRNA. Kedua-dua asai dijalankan serentak untuk mempercepatkan pengesanan pencilan kulat berdasarkan saiz amplikon yang terhasil. Dalam asai khusus untuk pengesanan genus, primer universal kulat disertakan bersama supaya amplikon kedua terhasil bagi setiap pencilan yang mengesahkan kehadiran kulat. Tahap pengesanan untuk asai khusus genus adalah 1 nanogram (ng) asid deoksiribonukleik (DNA) Aspergillus fumigatus dan Candida albicans serta 0.1 ng DNA Fusarium solani, manakala asai khusus spesies dapat mengesan 0.1 ng DNA A. terreus dan 10 pikogram (pg) DNA C. krusei serta C. glabrata. Selain daripada pengesanan semua kulat secara am, asai tindak balas rantai polimerase multipleks yang dibangunkan dapat mengesan kulat berkepentingan klinikal dan membezakan spesies rintang secara pantas dan tepat, justeru boleh berperanan dalam penentuan awal ubatan antikulat yang berkesan bagi pesakit.

Kata kunci: Aspergillus; Candida; Fusarium; PCR multipleks; pengesanan

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*Corresponding author; email: jacinta@ukm.edu.my