 |
 |
 |
 |
 |
 |
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 JAINLABDIN1,2, ANG LIM CHUA3, TZAR MOHD NIZAM4 & JACINTA SANTHANAM1*
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
Diserahkan: 17 Oktober
2016/Diterima: 23 Oktober 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
RUJUKAN
Abbas,
J., Bodey, G.P., Hanna, H.A., Mardani, M., Girgawy, E., Abi-Said, D., Whimbey,
E., Hachem, R. & Raad, I. 2000. Candida krusei fungemia an escalating
serious infection in immunocompromised patients. Archives of Internal
Medicine 160(17): 2659.
Alastruey-Izquierdo,
A., Cuenca-Estrella, M., Monzón, A., Mellado, E. & Rodríguez-Tudela, J.L.
2008. Antifungal susceptibility profile of clinical Fusarium spp.
isolates identified by molecular methods. Journal of Antimicrobial
Chemotherapy 61(4): 805-809.
Amini, F.,
Kachuei, R., Noorbakhsh, F. & Imani Fooladi, A.A. 2015. A multiplex PCR
method for detection of Aspergillus spp. and Mycobacterium
tuberculosis in BAL specimens. Journal of Medical Mycology 25(2):
59-64.
Avni, T.,
Leibovici, L. & Paul, M. 2011. PCR diagnosis of invasive candidiasis:
Systematic review and meta-analysis. Journal of Clinical Microbiology 49(2):
665-670.
Azab, M.M.,
Taleb, A.F.A., Mohamed, N.A.E. & Omran, F.H. 2015. Rapid diagnosis of
invasive fungal infections. International Journal Current Microbiology and
Applied Sciences 4(11): 470-486.
Bašková,
L. & Buchta, V. 2012. Laboratory diagnostics of invasive fungal infections:
An overview with emphasis on molecular approach. Folia Microbiologica 57(5):
421-430.
Berrouane,
Y.F. & Hollis, R.J. 1996. Strain variation among and antifungal
susceptibilities of isolates of Candida krusei. Journal of Clinical
Microbiology 34(7): 1856-1858.
Blum, G.,
Hörtnagl, C., Jukic, E., Erbeznik, T., Pümpel, T., Dietrich, H., Nagl, M.,
Speth, C., Rambach, G. & Lass-Flörl, C. 2013. New insight into Amphotericin
B resistance in Aspergillus terreus. Antimicrobial Agents and
Chemotherapy 57(4): 1583-1588.
Cerikçioğlu,
N., Aksu, B., Dal, T.D., Deniz, U., Bilgen, H.S., Ozek, E. & Söyletir, G.
2010. Seminested PCR for detection and identification of Candida species
directly from blood culture bottles. The New Microbiologica 33(1):
57-62.
Chang, H.C.,
Leaw, S.N. & Huang, A.H. 2001. Rapid identification of yeasts in positive
blood cultures by a multiplex PCR method. Journal of Clinical Microbiology 39(10):
3466-3471.
Cuenca-Estrella,
M., Bernal-Martinez, L., Buitrago, M.J., Castelli, M.V., Gomez-Lopez, A.,
Zaragoza, O. & Rodriguez- Tudela, J.L. 2008. Update on the epidemiology and
diagnosis of invasive fungal infection. International Journal of
Antimicrobial Agents 32(2): 143-147.
Denning,
D.W. 1998. Invasive aspergillosis. Clinical Infectious Diseases 26:
781-803.
Dignani,
M.C. & Anaissie, E. 2004. Human fusariosis. Clinical microbiology and
infection: The Official Publication of the European Society of Clinical
Microbiology and Infectious Diseases 10(1): 67-75.
Emam, S.M.
& Abd El-salam, O.H. 2015. Real-time PCR: A rapid and sensitive method for
diagnosis of dermatophyte induced onychomycosis, a comparative study. Alexandria
Journal of Medicine 52(1): 1-8.
Galimberti,
R., Torre, A.C., Baztán, M.C. & Rodriguez- Chiappetta, F. 2012. Emerging
systemic fungal infections. Clinics in Dermatology 30(6): 633-650.
Gregory, T.R.,
Nicol, J.A., Tamm, H., Kullman, B., Kullman, K., Leitch, I.J., Murray,
B.G., Kapraun, D.F., Greilhuber, J. & Bennett, M.D. 2007. Eukaryotic
genome size databases. Nucleic Acids Research 35(Database
issue): D332-D338. Henry, T.,
Iwen, P.C. & Hinrichs, S.H. 2000. Identification of Aspergillus
species using internal transcribed spacer regions 1 and 2. Journal
of Clinical Microbiology 38(4): 1510-1515. Horváth, Á.,
Pető, Z., Urbán, E., Vágvölgyi, C. & Somogyvári, F. 2013. A novel,
multiplex, real-time PCR-based approach for the detection of the commonly
occurring pathogenic fungi and bacteria. BMC Microbiology 13: 300.
Landlinger,
C., Preuner, S., Willinger, B., Haberpursch, B., Racil, Z., Mayer, J. &
Lion, T. 2009. Species-specific identification of a wide range of clinically
relevant fungal pathogens by use of Luminex xMAP technology. Journal of
Clinical Microbiology 47(4): 1063-1073.
Lenka, B.,
Christine, L., Sandra, P. & Thomas, L. 2007. The pan-AC assay: A
single-reaction real-time PCR test for quantitative detection of a broad range
of Aspergillus and Candida species. Journal of Medical
Microbiology 56(9): 1167-1173.
Logotheti,
M., Kotsovili-Tseleni, A., Arsenis, G. & Legakis, N.I. 2009. Multiplex PCR
for the discrimination of A. fumigatus, A. flavus, A. niger and A. terreus. Journal of Microbiological Methods 76(2): 209-211.
Lortholary,
O., Desnos-Ollivier, M., Sitbon, K., Fontanet, A., Bretagne, S., Dromer, F.,
Bouges-Michel, C. & the French Mycosis Study Group. 2011. Recent exposure
to caspofungin or fluconazole influences the epidemiology of candidemia: A
prospective multicenter study involving 2,441 patients. Antimicrobial Agents
and Chemotherapy 55(2): 532-538.
Mallus, F.,
Martis, S., Serra, C., Loi, G., Camboni, T. & Manzin, A. 2013. Usefulness
of capillary electrophoresis-based multiplex PCR assay for species-specific
identification of Candida spp. Journal of Microbiological Methods 92(2):
150-152.
Neal,
C.O.S., Richardson, A.O., Hurst, S.F., Tortorano, A.M., Viviani, M.A., Stevens,
D.A. & Balajee, S.A. 2011. Global population structure of Aspergillus
terreus inferred by ISSR typing reveals geographical subclustering. BMC
Microbiology 11: 203-209.
Oren, I.
& Paul, M. 2014. Up to date epidemiology, diagnosis and management of
invasive fungal infections. Clinical Microbiology and Infection: The
Official Publication of the European Society of Clinical Microbiology and
Infectious Diseases 20(6): 1-4.
Pfaller,
M.A. & Diekema, D.J. 2007. Epidemiology of invasive candidiasis: A
persistent public health problem. Clinical Microbiology Reviews 20(1):
133-163.
Pfaller,
M.A. & Castanheira, M. 2016. Nosocomial candidiasis: Antifungal stewardship
and the importance of rapid diagnosis. Medical Mycology 54(1): 1-22.
Preuner, S.
& Lion, T. 2009. Towards molecular diagnostics of invasive fungal
infections. Expert Review of Molecular Diagnostics 9(5): 397-401.
Richardson,
M. & Lass-Flörl, C. 2008. Changing epidemiology of systemic fungal
infections. Clinical Microbiology and Infection 14(4): 5-24.
Rishi, H.D.
& Clark, J. 2011. Fungal infections in the critically ill. Trends in
Anaesthesia and Critical Care 1(4): 210-218.
Steinbach,
W.J. & Perfect, J.R. 2003. Newer antifungal therapy for emerging fungal
pathogens. International Journal of Infectious Diseases: Official Publication
of the International Society for Infectious Diseases 7(1): 5-20.
Sulaiman,
I., Jacobs, E., Simpson, S. & Kerdahi, K. 2014. Molecular identification of
isolated fungi from unopened containers of greek yogurt by dna sequencing of
internal transcribed spacer region. Pathogens 3(3): 499-509.
Than, L.T.,
Chong, P.P., Ng, K.P. & Seow, H.F. 2012. Detection of 10 medically
important Candida species by seminested polymerase chain reaction. Diagnostic
Microbiology and Infectious Disease 72(2): 196-198.
Tortorano, A.M.,
Prigitano, A., Esposto, M.C., Arsic Arsenijevic, V., Kolarovic, J., Ivanovic,
D.,Paripovic, L., Klingspor, L., Nordøy, I., Hamal, P., Arikan Akdagli, S.,
Ossi, C., Grancini, A., Cavanna, C., Lo Cascio, G., Scarparo, C., Candoni,
A., Caira, M. & Drogari Apiranthitou, M. 2014. European Confederation of
Medical Mycology (ECMM) epidemiological survey on invasive infections due to Fusarium species in Europe. European Journal of Clinical Microbiology and
Infectious Diseases 33(9): 1623-1630.
Vahidnia, A., Bekers,
W., Bliekendaal, H. & Spaargaren, J. 2015. High throughput multiplex-PCR
for direct detection and diagnosis of dermatophyte species, Candida albicans and Candida parapsilosis in clinical specimen. Journal of
Microbiological Methods 113: 38-40.
Vazquez, J.A., Miceli,
M.H. & Alangaden, G. 2013. Invasive fungal infections in transplant
recipients recipients. Therapeutic Advances in Infectious Disease 1(3):
85-105.
Walsh, T.J., Wissel,
M.C., Grantham, K.J., Petraitiene, R., Petraitis, V., Kasai, M., Francesconi,
A., Cotton, M.P., Hughes, J.E., Greene, L., Bacher, J.D., Manna, P., Salomoni,
M., Kleiboeker, S.B. & Reddy, S.K. 2011. Molecular detection and
species-specific identification of medically important Aspergillus species
by real-time PCR in experimental invasive pulmonary aspergillosis. Journal
of Clinical Microbiology 49(12): 4150-4157.
Wisplinghoff, H.,
Ebbers, J., Geurtz, L., Stefanik, D., Major, Y., Edmond, M.B., Wenzel, R. P.
& Seifert, H. 2014. Nosocomial bloodstream infections due to Candida spp.
in the USA: Species distribution, clinical features and antifungal
susceptibilities. International Journal of Antimicrobial Agents 43(1):
78-81.
Yeo, S.F. & Wong, B.
2002. Current status of nonculture methods for diagnosis of invasive fungal
infections. Clinical Microbiology Reviews 15(3): 465-484.
*Pengarang untuk surat-menyurat; email: jacinta@ukm.edu.my
|
|||
|
