scholarly journals Perbandingan deteksi Plasmodium falciparum dengan metode pemeriksaan mikroskopik dan teknik real-time polymerase chain reaction

2016 ◽  
Vol 4 (1) ◽  
Author(s):  
Elril T. Langi ◽  
Janno B. B. Bernadus ◽  
Greta J. P. Wahongan
Keyword(s):  
Polymerase Chain Reaction ◽  
Plasmodium Falciparum ◽  
Real Time ◽  
Real Time Pcr ◽  
Gold Standard ◽  
Dna Amplification ◽  
World Health ◽  
Chain Reaction ◽  
Blood Samples ◽  
Polymerase Chain

Abstract: Plasmodium falciparum is one of the species of parasites causing tropical malaria disease. Plasmodium falciparum was reported as often being the major source of pain and even death in most cases. The data released by WHO shows that, globally, 198 millions of malaria cases occurred in 2013 with 548 thousands as cause of death. Microscopic examination is a gold standard for detecting Plasmodium falciparum. Although this method has certain limitations in diagnosing complication infection, phases of parasitemia, and also the capability of laboratory's medical staff factor. Nowadays, there has been innovation in biomolecular department, that is examination using PCR which can accurately detect the plasmodium, due to the DNA amplification. This method however, has not often used by doctors in diagnose malaria disease. The aim of this research is to determine the comparison of malaria detection using microscopic verification of plasmodium falciparum with real-time PCR verification. The method used in this research is diagnostic with 35 blood samples of patient suffering malaria disease. The blood samples from patient's vena were then divided into thick and thin microscopic sample, and some were putted into EDTA tube for DNA extraction in the laboratory using real-time PCR verification. The result of this research shown that sensitivity and specificity rate of PCR is 100% accurate. Conclusion: detection result of plasmodium falciparum using real-time PCR verification produced equal result as microscopic verification.Keywords: Plasmodium falciparum, Microscopic method, Real-time Polymerase Chain Reaction (PCR)Abstrak: Plasmodium falciparum adalah salah satu spesies parasit penyebab penyakit malaria, yaitu malaria tropika. Plasmodium falciparum dilaporkan sebagai spesies yang paling banyak menyebabkan angka kesakitan dan kematian pada manusia akibat penyakit malaria. World Health Organization (WHO) melaporkan secara global, diperkirakan 198 juta kasus malaria terjadi secara keseluruhan pada tahun 2013 dan menyebabkan 584 ribu kematian. Pemeriksaan mikroskopik adalah pemeriksaan gold standard untuk mendeteksi Plasmodium falciparum. Namun pemeriksaan ini memiliki keterbatasan dalam hal mendiagnosis infeksi campuran, infeksi dengan keadaan parasitemia, dan tidak terlatihnya tenaga kesehatan laboratorium. Saat ini dalam bidang biomolekuler telah dikembangkan pemeriksaan real-time polymerase chain reaction (PCR) yang akurat untuk mendeteksi plasmodium, karena didasarkan pada amplifikasi DNA plasmodium, namun pemeriksaan ini belum rutin digunakan untuk mendiagnosis malaria. Penelitian ini bertujuan untuk mengetahui perbandingan deteksi Plasmodium falciparum dengan pemeriksaan mikroskopik dan pemeriksaan real-time PCR. Metode penelitian ini ialah uji diagnostik. Sampel pada penelitian ini yaitu 35 sampel darah pasien suspek malaria. Sampel darah vena yang diambil langsung dibuat sedian darah tipis dan sediaan darah tebal untuk diperiksa di mikroskop, sedangkan darah yang tersisa dimasukkan dalam tabung EDTA, dan dibawa ke Laboratorium untuk dibuat ekstraksi DNA dan dilanjutkan dengan pemeriksaan real-time PCR. Hasil penelitian menunjukkan tingkatsensitivitas dan spesifisitas real-time PCR sebesar 100%. Simpulan: Hasil deteksi Plasmodium falciparum dengan pemeriksaan real-time PCR memiliki efektivitas yang setara dengan metode pemeriksaan mikroskopik sebagai gold standart.Kata kunci: Plasmodium falciparum, Pemeriksaan Mikroskopik, Real-time Polymerase Chain Reaction (PCR)

scholarly journals Deteksi Plasmodium falciparum dengan menggunakan metode real-time polymerase chain reaction di daerah Likupang dan Bitung

2016 ◽  
Vol 4 (1) ◽  
Author(s):  
David C. Tooy ◽  
Janno B. Bernadus ◽  
Angle Sorisi
Keyword(s):  
Polymerase Chain Reaction ◽  
Plasmodium Falciparum ◽  
Real Time ◽  
Real Time Pcr ◽  
18S Rrna ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Negative Results ◽  
Pcr Method ◽  
Polymerase Chain

Abstract: Malaria is one of the most important parasitic disease which is caused by Plasmodium spp. There are approximately 1,2 billion people in the world with high risk of getting malaria. Plasmodium falciparum (P. falciparum) is the cause of tropical malaria or falciparum malaria, and is responsible for most of the mortality rate. Currently, real-time polymerase chain reaction (RT-PCR) is being studied as an alterative of conventional malarian examination. Mangold et al reported that RT-PCR have 94.1% sensitivity and 100% specificity compared to microscopic examination in detecting P. falciparum. The aim of this research is to detect the presence of P. falciparum using RT-PCR in Likupang and Bitung region. This research were using descriptive design to find out the capability of real-time PCR method to detect P. falciparum in Likupang dan Bitung region. The researcher have examined 71 samples which are fulfill the research sample’s criteria. Postive results of P. falciparum found in 18 samples (25,3%) and negative results in 53 samples (74,6%) of total 71 samples with using RT-PCR. No positive results were found in samples from Likupang. There are positive result of P. falciparum in samples from Bitung. It is concluded that RT-PCR method can detect the presence of P. falciparum from the samples obtained from Likupang and Bitung based on the presence of its DNA. This detection efford is done by using 18S rRNA as target gene and ajust specific temperature on the RT-PCR instrument.Keywords: Plasmodium falciparum, Real-time Polymerase Chain Reaction (PCR), DetectionAbstrak: Malaria merupakan salah satu penyakit penting yang disebabkan oleh parasit Plasmodium spp. Kira-kira 1,2 miliar penduduk dunia memiliki risiko tinggi untuk mendapat malaria. Di Indonesia sendiri, terdapat 343.527 kasus terkonfirmasi dan 45 kematian karena malaria. Plasmodium falciparum (P. Falciparum) merupakan penyebab dari malaria tropika atau malaria falsiparum, dan bertanggung jawab atas sebagian besar angka mortalitas. Saat ini Real-Time Polymerase Chain Reaction (RT-PCR) telah banyak diteliti sebagai alternatif dari pemeriksaan malaria. Mangold dkk melaporkan bahwa real-time PCR memiliki nilai sensitivitas 94,1% dan nilai spesifisitas 100% terhadap pemeriksaan mikroskopis dalam mendeteksi P. falciparum. Penelitian bertujuan untuk mendeteksi P. falciparum dengan menggunakan RT-PCR di daerah Likupang dan Bitung. Penelitian ini menggunakan rancangan penelitian deskriptif untuk mengetahui kemampuan metode real-time PCR dalam mendeteksi P. falciparum di daerah Likupang dan Bitung. Tujuan penelitian ini ialah untuk mendeteksi keberadaan P. falciparum dengan menggunakan metode real-time PCR di daerah Likupang dan Bitung. Peneliti memeriksa 71 sampel darah yang memenuhi kriteria sampel penelitian. Hasil positif P. falciparum ditemukan pada 18 sampel (25,3 %) dan hasil negatif pada 53 sampel (74,6 %) dari total 71 sampel dengan menggunakan RT-PCR. Tidak ditemukannya hasil positif P. falciparum pada sampel dari Likupang. Ditemukan hasil positif P. falciparum pada sampel dari Bitung. Simpulan: Metode RT-PCR dapat mendeteksi P. falciparum berdasarkan keberadaan DNA-nya pada sampel yang diperoleh dari daerah Likupang dan Bitung. Deteksi ini berhasil dilakukan dengan menggunakan 18S rRNA sebagai gen target dan pengaturan suhu tertentu pada instrument RT-PCR.Kata kunci: P. falciparum, Real-time Polymerase Chain Reaction (PCR), Detection

scholarly journals Performance comparison of two malaria rapid diagnostic test with real time polymerase chain reaction and gold standard of microscopy detection method

2020 ◽  
Author(s):  
Puspa Wardhani ◽  
Trieva Verawaty Butarbutar ◽  
Christophorus Oetama Adiatmaja ◽  
Amarensi Milka Betaubun ◽  
Nur Hamidah ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Diagnostic Test ◽  
Real Time Pcr ◽  
Predictive Value ◽  
Gold Standard ◽  
Detection Method ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

Background: The diagnostic test for malaria is mostly based on Rapid Diagnostic Test (RDT) and detection by microscopy. Polymerase Chain Reaction (PCR) is also a sensitive detection method that can be considered as a diagnostic tool. The outcome of malaria microscopy detection depends on the examiner's ability and experience. Some RDT has been distributed in Indonesia, which needs to be evaluated for their results. Objective: This study aimed to compare the performance of RightSign RDT and ScreenPlus RDT for detection of Plasmodium in human blood. We used specific real-time polymerase chain reaction abTESTMMalaria qPCRII) and gold standard of microscopy detection method to measure diagnostic efficiency. Methods: Blood specimens were evaluated using RightSign RDT, ScreenPlus RDT, Microscopy detection, and RT-PCR as the protocol described. The differences on specificity (Sp), sensitivity (Sn), positive predictive value (PPV), and negative predictive value (NPV) were analyzed using McNemar and Kruskal Wallis analysis. Results: A total of 105 subjects were recruited. Based on microscopy test, RightSign RDT had sensitivity, Specificity, PPV, NPV, 100%, 98%, 98.2%, 100%, respectively. ScreenPlus showed 100% sensitivity, 98% specificity, 98.2% PPV, 100% NPV. The sensitivity of both RDTs became lower (75%) and the specificity higher (100 %) when using real-time PCR. Both RDTs showed a 100% agreement. RT-PCR detected higher mix infection when compared to microscopy and RDTs. Conclusion: RightSign and ScreenPlus RDT have excellent performance when using microscopy detection as a gold standard. Real-time PCR method can be considered as a confirmation tool for malaria diagnosis.

scholarly journals Development of a Novel Cytochrome b Real-Time PCR Assay for Identification of Plasmodium malariae

2020 ◽  
Vol 36 (3) ◽  
Author(s):  
Dinh Thi Thu Hang ◽  
Vu Thi Nga ◽  
Hoang Van Tong ◽  
Hoang Xuan Su ◽  
Le Quoc Tuan ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Plasmodium Falciparum ◽  
Real Time ◽  
Cytochrome B ◽  
Real Time Pcr ◽  
Clinical Microbiology ◽  
Plasmodium Malariae ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain

This article aims to establish a novel cytochrome b real-time PCR assay using Taqman probe for identification of P. malariae and its discrimination from other Plasmodium human infecting species. The optimization of real-time PCR assay with 1X QuantiTect Probe PCR Master Mix, primers and probe used at concentrations of 0.4 μM and 0.1 μM, respectively; and 2.5 mM MgCl2, 5 μl DNA template and deionized H2O of 20 μl, was performed using a real-time PCR instrument. The developed real-time PCR assay was evaluated for the limit of detection, stability on standard panels (109-100 copies/ µl), as well as the sensitivity, specificity on control groups. The probit analysis demonstrates that the 95% detection limit was <0.5 parasite/μl, both the sensitivity and specificity of the assay were 100% when evaluated on the control groups.  Additionally, the assay initially evaluated on 41 clinical samples including 21 malaria samples and 20 samples of volunteer blood donors, identified 1 positive sample with P. malariae from the disease group, which shows a concordant result with nested PCR. This novel Cyt b real-time PCR assay for identifying P. malariae may also facilitate earlier discrimination of P. malariae from other Plasmodium parasites with high sensitivity. Keywords Cytochrome b, malaria parasite, plasmodium malariae, mitochondria, real-time PCR. References [1] B. Singh, C. Daneshvar, Human infections and detection of Plasmodium knowlesi, Clinical microbiology reviews 26(2) (2013) 165-84. https://doi.org/10.1128/cmr.00079-12.[2] World Health Organization, Regional and global trends in burden of malaria cases and deaths, World malaria report 2019, Geneva, pp. 4-12.[3] World Health Organization, Progress towards elimination during the RBM decade 2000-2010, Eliminating malaria: learning from the past, looking ahead, Geneva (2011), pp. 39-70.[4] J.M. Vinetz, J. Li, T.F. McCutchan, et al., Plasmodium malariae infection in an asymptomatic 74-year-old Greek woman with splenomegaly, N Engl J Med 338(6) (1998) 367-71. https://doi.org/10.1056/NEJM199802053380605.[5] E. Lo, K. Nguyen, J. Nguyen, et al., Plasmodium malariae Prevalence and csp Gene Diversity, Kenya, 2014 and 2015, Emerg Infect Dis 23(4) (2017) 601-610. https://doi.org/10.3201/eid2304.161245.[6] W.E. Collins, G.M. Jeffery, Plasmodium malariae: parasite and disease, Clinical microbiology reviews 20(4) (2007) 579-92. https://doi.org/10.1128/CMR.00027-07.[7] M. Adams, S.N. Joshi, G. Mbambo, et al., An ultrasensitive reverse transcription polymerase chain reaction assay to detect asymptomatic low-density Plasmodium falciparum and Plasmodium vivax infections in small volume blood samples, Malar J 14 (2015) 520. https://doi.org/10.1186/s12936-015-1038-z.[8] W. Xu, U. Morris, B. Aydin-Schmidt, et al., SYBR Green real-time PCR-RFLP assay targeting the plasmodium cytochrome B gene-a highly sensitive molecular tool for malaria parasite detection and species determination, PloS one 10(3) (2015) e0120210. https://doi.org/10.1371/journal.pone.0120210.[9] E.M. Burd, Validation of laboratory-developed molecular assays for infectious diseases, Clinical microbiology reviews 23(3) (2010) 550-76. https://doi.org/10.1128/CMR.00074-09.[10] G. Snounou, S. Viriyakosol, X.P. Zhu, et al., High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction, Molecular and biochemical parasitology 61(2) (1993) 315-20. https://doi.org/10.1016/0166-6851(93)90077-b.[11] C.G. Haanshuus, K. Morch, B. Blomberg, et al., Assessment of malaria real-time PCR methods and application with focus on low-level parasitaemia, PloS one 14(7) (2019) e0218982. https://doi.org/10.1371/journal.pone.0218982.[12] F. Perandin, N. Manca, A. Calderaro, et al., Development of a real-time PCR assay for detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for routine clinical diagnosis, Journal of clinical microbiology 42(3) (2004) 1214-9. https://doi.org/10.1128/jcm.42.3.1214-1219.2004.[13] C.E. Oriero, J.P. van Geertruyden, J. Jacobs, et al., Validation of an apicoplast genome target for the detection of Plasmodium species using polymerase chain reaction and loop mediated isothermal amplification, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 21(7) (2015) 686 e1-7. https://doi.org/10.1016/j.cmi.2015.02.025.[14] D.F. Echeverry, N.A. Deason, J. Davidson, et al., Human malaria diagnosis using a single-step direct-PCR based on the Plasmodium cytochrome oxidase III gene, Malaria journal 15 (2016) 128. https://doi.org/10.1186/s12936-016-1185-x.[15] P. Li, Z. Zhao, H. Xing, et al., Plasmodium malariae and Plasmodium ovale infections in the China-Myanmar border area, Malaria journal 15(1) (2016) 557. https://doi.org/10.1186/s12936-016-1605-y.[16] E.T.J. Chong, J.W.F. Neoh, T.Y. Lau, et al., Genetic and haplotype analyses targeting cytochrome b gene of Plasmodium knowlesi isolates of Malaysian Borneo and Peninsular Malaysia, Acta tropica 181 (2018) 35-39. https://doi.org/10.1016/j.actatropica.2018.01.018.[17] C. Farrugia, O. Cabaret, F. Botterel, et al., Cytochrome b gene quantitative PCR for diagnosing Plasmodium falciparum infection in travelers, Journal of clinical microbiology 49(6) (2011) 2191-5. https://doi.org/10.1128/JCM.02156-10.[18] C. Wongsrichanalai, M.J. Barcus, S. Muth, et al., A review of malaria diagnostic tools: microscopy and rapid diagnostic test (RDT), The American journal of tropical medicine and hygiene 77(6 Suppl) (2007) 119-27.[19] H.V. Nguyen, P.V.D. Eede, C. van Overmeir, et al., Marked age-dependent prevalence of symptomatic and patent infections and complexity of distribution of human Plasmodium species in central Vietnam, The American journal of tropical medicine and hygiene 87(6) (2012) 989-995. https://doi.org/10.4269/ajtmh.2012.12-0047.  

scholarly journals DETEKSI CLOSTRIDIUM DIFFICILE TOKSIGENIK MENGGUNAKAN UJI CEPAT TOKSIN DAN REAL TIME POLYMERASE CHAIN REACTION (Toxigenic Clostridium Difficile Detection Using Toxin Rapid Test and Real Time Polymerase Chain Reaction)

2018 ◽  
Vol 22 (1) ◽  
pp. 22
Author(s):  
Ika Yasma Yanti ◽  
Dalima Ari Wahono Astrawinata
Keyword(s):  
Polymerase Chain Reaction ◽  
Clostridium Difficile ◽  
Antibiotic Therapy ◽  
Real Time ◽  
Real Time Pcr ◽  
Rapid Test ◽  
Chain Reaction ◽  
Chi Square ◽  
Polymerase Chain ◽  
Clostridium Difficile Associated Diarrhea

Toxigenic Clostridium difficile infection, causing a Pseudo Membrane Colitis (PMC) and Clostridium Difficile Associated Diarrhea(CDAD) has increased sharply. The largest risk factor is the use of antibiotics. The purpose of this study was to know how to determinethe prevalence and characteristics of subjects with Toxigenic Clostridium difficile and to assess the ability of the toxin rapid test comparedto real-time PCR. Ninety adult subjects with antibiotic therapy more than two (2) weeks were enrolled in this study. The results of toxinrapid test and real-time PCR were presented in a 2x2 table, statistical test used was Chi square. The prevalence of Toxigenic Clostridiumdifficile based on the toxin rapid test and by real-time PCR was 27.3% and 37.5%, respectively. There were significant differences betweenstool consistency and number of antibiotics used with the detection of Toxigenic Clostridium difficile. There was a relationship betweenthe duration of antibiotic therapy with the detection of Toxigenic Clostridium difficile using real-time PCR (p=0.010, RR=2.116). Thesensitivity, specificity, PPV, NPV, PLR and NLR rapid test against real-time PCR were 69.7%; 98.2%; 95.8%; 84.4%; 39.2 and 0.31,respectively. This study concluded that the prevalence of Clostridium difficile in RSCM was higher compared to that in Malaysia, Thailandand India; the subjects with antibiotic therapy for more than four (4) weeks had a double risk to have Toxigenic Clostridium difficilethan subjects with antibiotic therapy for less than that time (4 weeks). Thus, in this study, toxin rapid test could be used as a tool todetect Toxigenic Clostridium difficile.

Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

2010 ◽  
Vol 134 (3) ◽  
pp. 444-448 ◽  
Author(s):  
Zhengming Gu ◽  
Jianmin Pan ◽  
Matthew J. Bankowski ◽  
Randall T. Hayden
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Bk Virus ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

scholarly journals In Planta Distribution of ‘Candidatus Liberibacter asiaticus’ as Revealed by Polymerase Chain Reaction (PCR) and Real-Time PCR

2008 ◽  
Vol 98 (5) ◽  
pp. 592-599 ◽  
Author(s):  
Satyanarayana Tatineni ◽  
Uma Shankar Sagaram ◽  
Siddarame Gowda ◽  
Cecile J. Robertson ◽  
William O. Dawson ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Citrus Tree ◽  
Candidatus Liberibacter Asiaticus ◽  
In Planta ◽  
Chain Reaction ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Polymerase Chain

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide, and is caused by a phloem-limited fastidious prokaryotic α-proteobacterium that is yet to be cultured. In this study, a combination of traditional polymerase chain reaction (PCR) and real-time PCR targeting the putative DNA polymerase and 16S rDNA sequence of ‘Candidatus Liberibacter asiaticus,’ respectively, were used to examine the distribution and movement of the HLB pathogen in the infected citrus tree. We found that ‘Ca. Liberibacter asiaticus’ was distributed in bark tissue, leaf midrib, roots, and different floral and fruit parts, but not in endosperm and embryo, of infected citrus trees. Quantification analysis of the HLB bacterium indicated that it was distributed unevenly in planta and ranged from 14 to 137,031 cells/μg of total DNA in different tissues. A relatively high concentration of ‘Ca. Liberibacter asiaticus’ was observed in fruit peduncles. Our data from greenhouse-infected plants also indicated that ‘Ca. Liberibacter asiaticus’ was transmitted systemically from infection site to different parts of the plant. Understanding the distribution and movement of the HLB bacterium inside an individual citrus tree is critical for discerning its virulence mechanism and to develop management strategies for HLB.

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