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Suratno Lulut Ratnoglik
Tinjauan sistematik: Uji multiplex real-time PCR untuk deteksi bakteri penyebab Rickettsiosis = Multiplex real-time PCR assay for detection rickettsiosis agents: a systematic review
"Latar belakang: Tingginya insiden penyakit rickettsial di Asia Tenggara termasuk Indonesia, memerlukan alat diagnostik yang cepat dan akurat untuk berbagai agen rickettsiosis yang termasuk dalam typhus group, spotted fever group dan scrub typhus group. Tujuan: Penelitian ini bertujuan untuk memberikan gambaran komprehensif mengenai akurasi uji diagnostik multiplex real-time PCR untuk mendeteksi typhus group rickettsia (TGR), spotted fever group rickettsia (SFGR) dan Orientia tsutsugamushi (Scrub Typhus Group/STG), pada pasien / sampel dengan penyakit demam akut. Sumber data: Pencarian daring yang sistematis di database PubMed, EBSCOhost, Scopus, dan Google Cendekia, menggabungkan istilah pencarian ‘multipleks real-time PCR’ ,‘ typhus group’, ‘spotted fever group’, dan ‘scrub typhus’. Kriteria kelayakan penelitian: Studi klinis yang memenuhi syarat adalah studi yang meneliti reliabilitas uji multipleks real time PCR pada pasien / sampel dengan penyakit demam akut. Penilaian studi: Kualitas metodologis dari studi yang dimasukkan dinilai dengan menggunakan The Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) Hasil: Diperoleh 2 artikel penelitian dengan kualitas sedang dan 1 artikel penelitian dengan kualitas baik. Hanya satu penelitian yang melaporkan nilai akurasi diagnostik metode multiplex real – time PCR yang digunakan dengan nilai sensitivitas/spesifitas klinis 20% / 99% untuk TGR, 25%/98% untuk SFGR dan 27%/100% untuk STG (OT), dengan nilai prediksi positif/ nilai predeksi negatif sebesar 63%/ 92% untuk SFGR, 75% / 87% untuk TGR dan 100% / 88% untuk OT(STG). Sementara kedua penelitian yang lain memberikan proporsi positif masing – masing 9 dari 12 (75%) sampel klinis terkonfirmasi positif dan 11 dari 319 (3%) sampel klinis pasien demam akut. Kesimpulan / signifikansi: Nilai sensitifitas klinis uji multiplex real-time PCR untuk mendeteksi TGR, SFGR dan STG (Orientia tsutsugamushi) dari penelitian – penelitian yang diikutkan tinjauan sistematik ini masih rendah, namun memiliki spesifisitas klinis yang tinggi. Untuk ke depan, penting untuk mengembangkan uji multiplex real-time PCR dengan sensitifitas klinis yang tinggi untuk mendeteksi bakteri Rickettsia / Orientia pada fase akut sehingga pasien mendapat perawatan yang cepat dan tepat.
Background: The high incidence of rickettsiosis in Southeast Asia, including Indonesia, necessitates rapid and accurate diagnostic tools for a broad range of rickettsiosis agents, including typhus and spotted fever group and scrub typhus group. Objectives: This study aimed to provide a comprehensive overview of multiplex realtime PCR for detecting typhus group rickettsia (TGR), spotted fever group rickettsia (SFGR) and Orientia tsutsugamushi (Scrub Typhus Group/STG) simultaneously, in patients/samples with acute febrile illness. Data sources: A systematic computerized search conduct in PubMed, EBSCOhost, Scopus, and Google Scholar databases, combining the search term ‘multiplex real-time PCR,’ ‘spotted fever group,’ ‘typhus group,’ and ‘scrub typhus.’ Study eligibility criteria: Clinical studies that investigate the reliability multiplex real time PCR assay in patients/samples with acute febrile illness were eligible. Study appraisal and synthesis methods: The methodological quality of the included studies was assessed with the use of the The Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) checklist Results: It was obtained two moderate quality articles and one good quality article. Only one study reported diagnostic accuracy values of multiplex real-time PCR methods used with clinical sensitivity / specificity values of 20% / 99% for TGR, 25% / 98% for SFGR and 27% / 100% for STG (OT), with positive predictive value / negative predictive value of 63% / 92% for SFGR, 75% / 87% for TGR and 100% / 88% for OT (STG). While the other two studies provided a positive proportion each 9 out of 12 (75%) clinical samples were confirmed positive and 11 out of 319 (3%) clinical samples were acute fever patients. Conclusion/significance: The clinical sensitivity values of real-time multiplex PCR assay for detecting TGR, SFGR, and STG (Orientia tsutsugamushi) from studies included in this systematic review are still low but have high clinical specificity. On the forward, it is crucial to develop a multiplex real-time PCR test with high clinical sensitivity to detect Rickettsia / Orientia bacteria in the acute phase so the patients receive appropriate care."
Jakarta: Fakultas Kedokteran Universitas Indonesia, 2020
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UI - Tugas Akhir  Universitas Indonesia Library
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Verawati Sulaiman
Optimasi uji multiplex real time PCR untuk deteksi bakteri atipik pada sputum pasien Community Acquired Pneumonia (CAP) = Optimization of multiplex real time PCR to detect atipycal bacteria in sputum of patients with Community Acquired Pneumonia (CAP)
"Latar Belakang: Community Acquired Pneumonia (CAP) merupakan salah satu penyebab utama morbiditas dan mortalitas di dunia. Bakteri atipikal (Chlamydia pneumoniae, Mycoplasma pneumaniae, Legionella pneumophila) sebagai penyebab penting CAP. Sejauh ini belum ada pemeriksaan mikrobiologi yang rutin dilakukan sehingga perlu pengembangan uji, salah satunya metode molekuler multiplex real time PCR. .
Tujuan: Melakukan optimasi uji multiplex real time PCR untuk mendeteksi secara simultan dan cepat C.pneumoniae, L.pneumophila dan M.pneumoniae pada sputum pasien CAP.
Metode: Penelitian ini merupakan uji eksperimental laboratorium yang terdiri atas 3 tahap. Tahap 1 meliputi optimasi suhu penempelan, primer, probe, volume elusi akhir dan cetakan DNA. Tahap 2 untuk menentukan batas ambang deteksi DNA dan reaksi silang. Tahap 3 adalah penerapan uji multiplex real time PCR pada spesimen sputum pasien CAP.
Hasil: Uji multiplex real time PCR telah berhasil dioptimasi dengan ambang batas minimal deteksi DNA untuk Chlamydia pneumoniae, Legionella pneumophila dan Mycoplasma pneumaniae adalah 1855, 3185 dan 130 kopi DNA. Uji ini tidak bereaksi silang dengan mikroorganisme yang berpotensi menimbulkan reaksi positif palsu. Sebanyak 134 sputum telah diuji dan ditemukan positif M.pneumoniae sebanyak 1 spesimen (0,74 %).
Kesimpulan: Uji multiplex real time PCR dapat mendeteksi C.pneumoniae, M.pneumoniae, dan L.pneumophila secara simultan pada sputum pasien CAP.
Background: Community Acquired Pneumonia (CAP) is one of the leading causes of morbidity and mortality in the world. Atypical bacteria (Chlamydia pneumoniae, Legionella pneumophila, Mycoplasma pneumaniae) are the important causes of CAP. In daily clinical practice, detection of atypical bacteria are sometimes neglected due to the limited standard test available. The real time multiplex PCR methode can be used as an alternative test for the detection of atypical bacteria.
Objective: Optimization of the multiplex real time PCR test to simultaneously detect C.pneumoniae, L.pneumophila and M.pneumoniae in CAP patients.
Methods: This study is experimental laboratory test that conducted in three phases. The first is optimization of annealing temperature, primers dan probe concentration, final elution of DNA extraction and volume of PCR templete. The second is determination of minimal detection of DNA and cross reaction of optimized real time PCR multiplex. The third is application of real time PCR multiplex in sputum clinical specimen patient with CAP.
Results: The multiplex real time PCR test was successfully optimized for annealing temperature, concentration of primer both forward and reverse, probes concentration and inhibitor. Limit detection of the DNA Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumaniae were 1855 copies, 3185 copies and 130 copies DNA. This test also showed no cross reaction to microorganisms that have potential to cause false positives. A total of 134 sputum clinical specimens have been tested with this method and only one sample (0,74%) was positive M.pneumoniae.
Conclusion: The multiplex real time PCR assay can detect C. pneumoniae, M. pneumoniae, and L. pneumophila simultanously in sputum of patients with Community Acquired Pneumonia (CAP)"
Jakarta: Fakultas Kedokteran Universitas Indonesia, 2020
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UI - Tugas Akhir  Universitas Indonesia Library
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Syahira Andini
Deteksi molekuler tuberkulosis paru dari sampel urine menggunakan multiplex PCR dengan gen deteksi ESAT6, IS6110, dan MPT64 dan real-time PCR dengan gen deteksi ESAT6 = Molecular detection of pulmonary tuberculosis from urine samples using multiplex PCR with ESAT-6, IS6110, and MPT64 detection genes and and real-time PCR with ESAT6 detection gene
"Kendala utama dalam diagnosis penyakit tuberkulosis (TB) paru di Indonesia adalah sulitnya pengeluaran sputum sebagai spesimen diagnostik dari pengidap TB paru, terutama pada kelompok anak dan lansia. Pengembangan spesimen alternatif, seperti urine, sangat dibutuhkan untuk meningkatkan notifikasi kasus TB paru pada subjek yang belum terdiagnosis secara optimal. Penelitian ini mengevaluasi potensi urine pada 60 sampel terkonfirmasi TB paru positif, sebagai studi kasus-kontrol dengan subjek yang memiliki kondisi target. Tujuan penelitian ini meliputi evaluasi multiplex polymerase chain reaction (PCR), untuk mendeteksi gen ESAT6, IS6110, dan MPT64, serta real-time PCR untuk deteksi gen ESAT6 dalam menunjang diagnosis TB paru. Selain itu, penelitian ini diharapkan dapat menggambarkan nilai sensitivitas masing-masing metode dan gen diagnostik yang digunakan. Metode penelitian meliputi preparasi sampel urine, isolasi DNA, kuantifikasi DNA, amplifikasi DNA (multiplex PCR dan real-time PCR), elektroforesis DNA, dan analisis data. Hasil evaluasi menunjukkan kemurnian total DNA yang diisolasi dari urine berdasarkan A260/A280   (Mean 3,08  SD 1,08). Evaluasi multiplex PCR dengan gen deteksi ESAT6, IS6110, dan MPT64 memberikan nilai sensitivitas sebesar 68,3% (41/60), dan real-time PCR dengan gen deteksi ESAT6 sebesar 71,67% (43/60). Nilai sensitivitas real-time PCR diperoleh dengan ketentuan limit of detection (LOD) sebesar 13,04 kopi/μl. Nilai sensitivitas kedua metode tersebut menunjukkan bahwa urine dapat menjadi spesimen alternatif untuk pendeteksian Mycobacterium tuberculosis (Mtb) secara molekuler.
The main obstacle in the diagnosis of pulmonary tuberculosis (TB) in Indonesia is the difficulty of extracting sputum, as a diagnostic specimen for people with pulmonary TB, especially in the group of children and the elderly. The development of alternative specimens, such as urine, is urgently needed to increase the notification of pulmonary TB cases in subjects who have not been diagnosed optimally. This study evaluated the urine potency of 60 samples of confirmed positive pulmonary TB, as a case-control study with subjects with the target condition. The objectives of this study include the evaluation of multiplex polymerase chain reaction (PCR), to detect the ESAT6, IS6110, and MPT64 genes, as well as real-time PCR to detect the ESAT6 gene in supporting the diagnosis of pulmonary TB. In addition, this study is expected to describe the sensitivity value of each diagnostic method and gene used. Research methods include urine sample preparation, DNA isolation, DNA quantification, DNA amplification (multiplex PCR and real-time PCR), DNA electrophoresis, and data analysis. The evaluation results showed the total purity of DNA isolated from urine based on A260/A280   (Mean 3,08  SD 1.08). Evaluation of multiplex PCR with ESAT6, IS6110, and MPT64 detection genes gave a sensitivity value of 68,3% (41/60), and real-time PCR with ESAT6 detection genes of 71,67% (43/60). Real-time PCR sensitivity value was obtained with the limit of detection (LOD) of 13,04 copies/μl. The sensitivity values of both methods indicate that urine can be an alternative specimen for molecular detection of Mycobacterium tuberculosis."
Depok: Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Indonesia, 2022
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UI - Skripsi Membership  Universitas Indonesia Library
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Eka Octaviani Budiningtyas
Uji diagnostik metode Real-Time Multiplex Polymerase Chain Reaction Strip terhadap Real-Time Polymerase Chain Reaction Tunggal untuk deteksi Multi-Patogen pada uveitis : analisis Humor Akuos = Diagnostic study of Real-Time Multiplex Polymerase Chain Reaction Strip assay in comparison with Single Real-Time Polymerase Chain Reaction for Multi-Pathogen detection in uveitis : Aqueous Humor analysis
"Latar Belakang: Uveitis infeksi di Indonesia berkisar antara 30-60% dari total kasus uveitis. Identifikasi patogen etiologi infeksi sangat penting agar dapat diberikan terapi antimikroba yang sesuai dengan segera sehingga komplikasi kebutaan dapat diminimalisir. Dewasa ini, perkembangan teknologi biologi molekuler menggunakan metode Polymerase Chain Reaction (PCR) untuk deteksi uveitis infeksi sedang berkembang pesat.
Tujuan: Melakukan uji validasi diagnostik pada metode PCR Multiplex dibandingkan dengan metode PCR Tunggal.
Metodologi: Uji diagnostik untuk menentukan sensitivitas dan spesifisitas dari alat Real-Time PCR Multiplex terhadap PCR Tunggal dari spesimen cairan intraokular humor akuos yang diambil dari parasentesis bilik mata depan. Dilakukan pemeriksaan PCR terhadap patogen Mycobacterium tuberculosis (M.tuberculosis), Toxoplasma gondii (T.gondii), Herpes Simplex Virus (HSV), Varicella Zoster Virus (VZV), Cytomegalovirus, dan Treponema pallidum.
Hasil: Dilakukan analisis uji diagnostik pada 46 subjek penelitian. Didapatkan hasil sensitivitas sebesar 57.14% dan spesifisitas sebesar 100%. Positivity rate terbanyak didapatkan untuk patogen VZV (n=4), dan tidak didapatkan hasil positif terhadap deteksi patogen M.tuberculosis. Patogen T.pallidum berhasil dideteksi sebanyak 4.34% (n=2) oleh PCR Multiplex.
Kesimpulan: Metode PCR Multiplex pada penelitian ini memiliki sensitivitas yang rendah dengan spesifisitas yang tinggi. Hasil positif pada PCR Multiplex dapat bermanfaat untuk mendiagnosis pasien dengan uveitis infeksi.
Background: In Indonesia, infectious uveitis represents 30-60% of the country’s total uveitis cases. The identification of etiological pathogens is imperative to immediately select and administer the appropriate antimicrobial therapy in infectious uveitis, thereby complications of blindness can be minimized. Currently, the development of molecular biology technology using the Polymerase Chain Reaction (PCR) method for detection of infectious uveitis pathogens is growing rapidly.
Objective: To compare the diagnostic validation test results of the Multiplex PCR method and Single PCR method.
Method: Diagnostic test to determine the sensitivity and specificity of the Multiplex Real-Time PCR device against the Single PCR of aqueous humor intraocular fluid specimens taken from anterior chamber paracentesis. PCR examinations were carried out to identify the pathogens of Mycobacterium tuberculosis (M.tuberculosis), Toxoplasma gondii (T.gondii), Herpes Simplex Virus (HSV), Varicella Zoster Virus (VZV), Cytomegalovirus, dan Treponema pallidum.
Result: A diagnostic test analysis was performed on 46 study subjects. The results obtained 57.14% sensitivity and 100% specificity. Highest positivity rate was obtained for VZV pathogens, while positive results were not obtained for M.tuberculosis. There were 4.34% of subjects (n = 2) of T. pallidum were detected by PCR Multiplex. 
Conclusion: The PCR Multiplex method in this study has low sensitivity with high specificity. A positive result on Multiplex PCR can be useful for diagnosing patients with infectious uveitis."
Depok: Fakultas Kedokteran Universitas Indonesia , 2020
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UI - Tesis Membership  Universitas Indonesia Library
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Nunik Utami
Deteksi mikroba penyebab uveitis menggunakan uji real time pcr pada cairan akuos = Microbial detection cause uveitis using real time pcr of aqueous humor
"Uveitis jarang terjadi dengan insidens sekitar 52/100.000 penduduk/tahun namun dapat mengakibatkan kebutaan. Diagnosis uveitis di Indonesia selama ini berdasarkan gambaran klinis dan belum dibuktikan dengan pemeriksaan deteksi mikroba sehingga belum diketahui prevalensi patogen uveitis. Penelitian ini bertujuan untuk meningkatkan peran uji real time PCR sebagai pendukung diagnosis etiologi sehingga dapat diketahui proporsi Mycobacterium tuberculosis, Toxoplasma gondii, Rubella, Herpes simplex, Varicella zoster, Epstein barr, Cytomegalovirus sebagai penyebab uveitis dan analisis kesesuaian diagnosis klinis dengan hasil pemeriksaan real time PCR. Pengambilan sampel cairan akuos dilakukan di Departemen Ilmu Kesehatan Mata FKUI-RSCM, sedangkan untuk uji real time PCR dilakukan di Laboratorium Mikrobiologi Klinik FKUI-RSCM selama rentang waktu Oktober 2016 sampai Mei 2017. Terdapat total 81 pasien dengan diagnosis klinis uveitis infeksi 32, 22 uveitis non-infeksi, dan 27 idiopatik. Berdasarkan uji real time PCR diperoleh hasil bahwa patogen terbanyak yaitu CMV diikuti oleh Toxoplasma Gondii dan Mycobacterium tuberculosis. Mikroba terdeteksi pada 14 spesimen diantara 32 uveitis infeksi, 1 spesimen diantara 22 uveitis non-infeksi, dan 2 diantara 27 uveitis idopatik. Dari 17 hasil positif real time PCR 13 sampel menunjukkan hasil PCR yang sesuai dengan klinis sedangkan 4 sampel tidak sesuai. Deteksi mikroba menggunakan pemeriksaan PCR pada cairan akuos dapat membantu dalam penegakan diagnosis dan tatalaksana uveitis dengan tepat.
Uveitis is a rare disease with an incidence of 52 100,000 population year but can cause blindness. The diagnosis of uveitis in Indonesia has been upheld primarily based on clinical features and has not been proven by microbial detection, so it is never known precisely the prevalence of uveitis pathogens. This study aims to increase the role of microbiological examination of real time PCR as supporting the etiology diagnosis so that it can be known the proportion of Mycobacterium tuberculosis, Toxoplasma gondii, Rubella, Herpes simplex, Varicella zoster, Epstein barr, Cytomegalovirus as cause of uveitis and assess a clinical diagnosis accordance with real time PCR results. Aqueous tap was conducted at the Department of Opthalmology FMUI RSCM, while for real time PCR was conducted at Clinical Microbiology Laboratory FMUI RSCM during October 2016 until May 2017. There were a total of 81 patients with clinical diagnosis consisting of 32 infectious uveitis, 22 non infectious uveitis, and 27 idiopathic uveitis. Based on real time PCR results obtained that the most common pathogens are CMV followed by Toxoplasma Gondii and Mycobacterium tuberculosis. Microbes were detected in 14 specimens among 32 infectious uveitis, 1 sample among 22 non infectious uveitis, and 2 of 27 idiopathic uveitis. Out of 17 positive results of real time PCR 13 samples showed a clinically accordance with the real time PCR result whereas 4 samples did not. Microbial detection using PCR of aqueous humor is helpful in diagnosing and management of uveitis."
2018
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UI - Tesis Membership  Universitas Indonesia Library
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Rizka Ariani
Deteksi molekuler virus penyebab demam menyerupai gejala demam dengue menggunakan real time RT-PCR = Moleculer detection of viruses causing dengue-like fever using real time RT-PCR
"Demam akut yang disertai gejala mirip demam Dengue nyeri kepala, nyeri sendi, ruam, perdarahan perifer seperti mimisan, ptekie adalah gejala yang paling sering dikeluhkan oleh pasien. Gejala-gejala tersebut seringkali diakibatkan infeksi arbovirus yang sangat endemik di Indonesia sebagai negara tropis. Deteksi agen penyebab infeksi tersebut sangat diperlukan untuk penatalaksanaan yang tepat. Studi ini melakukan uji optimasi untuk deteksi molekuler virus penyebab demam mirip demam Dengue, meliputi DENV, ZIKV, WNV, JEV, YFV, CHIKV, dan Hantavirus menggunakan RT PCR. Primer pada studi ini dirancang menggunakan perangkat lunak online Primer-BLAST dari NCBI dan primer-primer tersebut memenuhi kriteria untuk reaksi RT PCR. Kontrol positif pada real time RT-PCR menggunakan DNA sintetik yang dirancang sesuai dengan amplicon target virus. DNA sintetik sepanjang 1.047 pasang basa dirancang untuk digunakan pada virus ZIKV, JEV, YFV, WNV, CHIKV, dan Hantavirus. Suhu penempelan optimum pada primer-primer adalah 600C kecuali primer flavivirus universal yaitu 560C. Limit deteksi primer JEV mencapai 4.355 salinan DNA setiap reaksi real time RT PCR. Tidak terdapat reaksi silang maupun positif palsu pada sampel RNA DENV serotipe 2 maupun pada sampel orang sehat yang digunakan pada studi ini. Sebagai kesimpulan, studi ini menghasilkan primer dan protokol real time RT-PCR yang berpotensi untuk dikembangkan lebih lanjut untuk digunakan dalam uji diagnostik pada sampel pasien demam akut menyerupai gejala demam Dengue.
Acute fever with Dengue like fever symptoms headache, rash, joint pain, perifer bleeding like ptechie, rinhorrhea is general symptoms that often being complained by patient. Usually the etiology agent of the symptoms are arbovirals which are endemic in Indonesia as a tropical country. In this case, molecular detection is very important to confirm the etiology of disease for prompt and adequate management. This study optimized viral molecular detection as an etiology agent for Dengue like fever symptoms using real time RT PCR. The viruses that were investigated were DENV, ZIKV, WNV, JEV, YFV, CHIKV, and Hantavirus. Primer were designed used Primer BLAST software from NCBI. Those primers fulfilled the good primer requirements and could be used in real time RT PCR reaction. Synthetic DNA with 1.047 base pairs was designed based on amplicon target to be used as control positive for ZIKV, JEV, YFV, WNV, CHIKV, and Hantavirus. The optimal annealing temperature for all primers were at 600C except for flavivirus universal primer was at 560C. The limit of detection of JEV primer was 4355 copies DNA per reaction. Cross reactivity between all primers with DENV serotype 2 RNA and healthy person sample were not found. This study still need RNA viruses as negative or positive control and clinical sample to determine the sensitivity and specificity. As a conclusion, this study provided primers and real time RT PCR protocol that potentially be further developed as diagnostic tools for patient with Dengue like fever symptoms. "
Jakarta: Fakultas Kedokteran Universitas Indonesia, 2018
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UI - Tesis Membership  Universitas Indonesia Library
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Luh Inta Prilandari
Optimasi uji duplex real-time PCR untuk deteksi Corynebacterium Diphtheriae Potensial Toksigenik dan penerapan pada spesimen usap tenggorok pasien tersangka difteri = Optimization of duplex real-time PCR assay for detection of potentially Toxigenic Corynebacterium Diphtheriae and application using throat swab of suspected diphtheria patient.
"Latar belakang: Difteri merupakan penyakit infeksi bakteri yang diperantarai oleh
toksin. Corynebacterium diphtheriae adalah penyebab tersering difteri. Diagnostik
laboratorium harus dilakukan dengan cepat untuk menunjang diagnosis klinis difteri.
Pemeriksaan mikroskopik tidak direkomendasikan karena tidak spesifik, kultur dan uji
toksin yang merupakan uji baku emas cukup memakan waktu, membutuhkan
keterampilan dan pengalaman serta hanya dilakukan di laboratorium rujukan. PCR
merupakan metode pemeriksaan yang cepat, sensitif dan spesifik. Duplex real-time PCR
dapat mendeteksi bakteri penyebab tersering dan gen pengkode toksin secara simultan.
Tujuan penelitian: Melakukan optimasi uji duplex real time PCR untuk deteksi C.
diphtheriae potensial toksigenik dan menerapkannya pada spesimen usap tenggorok
pasien tersangka difteri.
Metode: Duplex real-time PCR menggunakan dua pasang primer dan probe dengan
target gen rpoB C.diphtheriae dan toksin difteri subunit A Tox. Parameter yang
dioptimasi adalah suhu penempelan, konsentrasi masing-masing primer dan probe,
inhibitor, reaksi silang dengan patogen lain dan ambang batas deteksi uji. Kemudian uji
diaplikasikan pada spesimen usap tenggorok pasien tersangka difteri yang dirawat di
RSPI Sulianti Saroso pada periode 2018-2019. Sebagai perbandingan dilakukan uji Elek
untuk konfirmasi toksigenitas dan analisa data klinis pasien.
Hasil: Kondisi optimal uji didapat pada suhu penempelan 55oC, konsentrasi primer Cd
0,4 μM, primer Tox 0,6 μM, probe Cd 0,5 μM dan probe Tox 0,625 μM, volume elusi
ekstraksi DNA 50 μL, volume cetakan DNA 5 μL dan ambang batas deteksi 2 CFU/ml.
Uji tidak bereaksi silang dengan mikroorganisme lain yang dicobakan. Dari 89 sampel,
proporsi positif C.diphtheriae potensial toksigenik dengan uji duplex real-time PCR
adalah 21,3%, sedangkan proporsi positif C.diphtheriae toksigenik menggunakan uji
baku emas adalah 11,2%.
Kesimpulan: Duplex real time PCR untuk deteksi C.diphtheriae potensial toksigenik
telah dioptimasi dan diaplikasikan pada pasien tersangka difteri. Diharapkan uji ini
dapat meningkatkan diagnosis laboratorium kasus difteri.
Background: Diphtheria is toxin-mediated bacterial infection. The most common
etiology is Corynebacterium diphtheriae. Laboratory diagnostic should be done
immediately to support clinical diagnosis. Microscopic examination is not
recommended, culture followed by toxin test is consider gold standard but timeconsuming,
require experience and only done in referral laboratory. PCR is fast,
sensitive and specific. Duplex real-time PCR can detect bacteria and toxin-encoding
gene simultaneously.
Objective: Optimizing duplex real-time PCR assay for detection of potentially
toxigenic C.diphtheriae and applicate the assay on throat swab of suspected diphtheria
patient.
Method: Two pair of primers and specific probe targeting rpoB gene of C.diphtheriae
and A-subunit of diphtheria toxin gene were used in this study. Parameters including
annealling temperature, concentration of primers and probes, inhibitors, cross reaction
and detection limit were being optimized to receive optimal condition. The optimized
assay was applicated on throat swab of suspected diphtheria patient in Sulianti Saroso
Infectious Disease Hospital at 2018-2019. Elek toxigenity test was used for comparison
and clinical data of the patient were analyzed.
Result: The optimum condition for duplex real-time PCR was received upon the
annealing temperature 60oC, concentration of Cd primer 0,4 μM, Tox primer 0,6 μM,
Cd probe 0,5 μM, Tox probe 0,625 μM, DNA elution volume 50 μL, DNA template
volume 5 μL and detection limit 2 CFU/ml. There was no cross reaction found with
other tested microorganisms. Of 89 samples, proportion of potentially toxigenic
C.diphtheriae was 21,3% and proportion of toxigenic C.diphtheriae confirmed by gold
standard was 11,2%.
Conclusion: Duplex real time PCR has been optimized for detection of potentially
toxigenic C.diphtheriae. This method can be used to detect C.diphtheriae and Tox
simultaneosly and increase supporting diagnosis."
Jakarta: Fakultas Kedokteran Universitas Indonesia, 2020
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UI - Tugas Akhir  Universitas Indonesia Library
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Via Ekawati
Penggunaan Swab Bukal sebagai Spesimen Untuk Deteksi SARS-COV2 Menggunakan Metode Real Time RT-PCR = The Use of Buccal Swab as Specimen for Detection of SARS-COV-2 by Using Real-Time RT-PCR Method
"Latar Belakang : COVID- 19 disebabkan SARS-COV-2. WHO menerbitkan protokol pemeriksaan laboratorium untuk deteksi virus menggunakan metode real time RT-PCR dari spesimen swab nasofaring dan orofaring. Metode ini cukup invasif. Diperlukan tehnik pemeriksaan yang relatif aman dan nyaman untuk pasien. Penelitian ini bertujuan untuk melihat efetivitas swab bukal sebagai alternatif pemeriksaan SARS-COV-2.
Metode : Studi uji diagnostik ini dilaksanakan sejak tahun 2020 - 2021, mengambil spesimen swab nasofaring, swab orofaring dan swab bukal dari pasien positif COVID- 19. Dilakukan optimasi, ekstraksi RNA virus dan real time RT-PCR .
Hasil Penelitian : Hasil studi mengumpulkan 68 spesimen dari pasien COVID-19. Hasil uji nasofaring, orofaring dan bukal positif adalah 24 spesimen. Hasil uji nasofaring dan orofaring positif dengan uji bukal negatif adalah 23 spesimen. Berdasarkan nilai Ct < 20 dan Ct <25, hasil kesesuaian positif dan negatif adalah 100%. Nilai Ct < 30 hasil kesesuaian positif 85,3 % dan negatif adalah 100%. Nilai Ct < 40 , hasil kesesuaian positif 51,1 % dan negatif adalah 100%. 
Kesimpulan : Swab bukal dapat digunakan sebagai pemeriksaan alternatif pada pemeriksaan SARS- CoV-2.
Background: COVID-19 caused by the SARS-COV-2 virus. WHO published protocol for the detection of the virus using the real time RT-PCR from nasopharyngeal and oropharynx swab specimens. This method is invasive. Required an examination technique that is relatively safe and comfortable. This study aims to see the effectiveness of the buccal swab as an alternative to the SARS-CoV-2 examination.
Methods: This diagnostic test study from 2020 to 2021, specimens of nasopharyngeal, oropharyngeal and buccal swabs from COVID-19. Specimens underwent an optimization, viral RNA extraction and real time RT-PCR.
Result : This study collected 68 specimens from COVID- 19 patients. The results of positive nasopharyngeal, oropharynx and buccal tests were 24 specimens. The results of a positive nasopharynx and oropharynx test with a negative buccal test were 23 specimens. Based on the values ​​of Ct < 20 and Ct < 25 , the results of positive agreement and negative are 100%. The value of Ct < 30 and Ct < 40  results in a positive agreement are 85.3% and 51,1 %. The negative  results are 100%.
Conclusion : Buccal swab can be used as an alternative test for SARS-CoV-2 examination."
Jakarta: Fakultas Kedokteran Universitas Indonesia, 2022
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UI - Tugas Akhir  Universitas Indonesia Library
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Amelia Nurfitriana
Aplikasi real time pcr dalam deteksi infeksi submikroskopis plasmodium falciparum dan plasmodium vivax di nangapanda ende = A real time pcr assay in detection sub microscopic infection of plasmodium falciparum and plasmodium vivax in nangapanda sub district ende
"Parasitemia rendah umumnya tidak dapat terdeteksi pada saat pemeriksaan secara mikroskopis. Penelitian dilakukan untuk mendeteksi infeksi submikroskopis Plasmodium falciparum dan Plasmodium vivax di Nangapanda, Ende menggunakan metode Real-time Polymerase Chain Reaction (PCR). Sebanyak 72 sampel darah anak (umur 5--18 tahun) digunakan dalam amplifikasi DNA menggunakan gen target 18S rRNA. Hasil yang diperoleh menunjukkan Realtime PCR mampu mendeteksi adanya infeksi submikroskopis P. falciparum dan P. vivax di Nangapanda. Persentase infeksi submikroskopis yang diperoleh dari sampel positif P. falciparum sebesar 11,1%, P. vivax sebesar 9,7% dan infeksi campuran sebesar 2,8%. Sensitivitas Real-time PCR mampu mendeteksi kategori nilai Ct low load of DNA (Ct>35) dengan persentase infeksi sebesar 9,7% untuk P. falciparum dan 8,3% untuk P. vivax. Hasil uji chi-square menunjukkan tidak adanya hubungan yang signifikan (p>0,05) antara infeksi submikroskopis dengan variabel jenis kelamin dan kelompok umur. Deteksi terhadap infeksi submikroskopis berguna terutama dalam program eliminasi malaria.
In malaria endemic areas, individuals are frequently asymptomatic are present at densities below the limit for conventional microscopy. An 18S rRNA based multiplex Real-time Polymerase Chain Reaction (PCR) had been done to determine sub-microscopic infection of Plasmodium falciparum and Plasmodium vivax. A total of 72 blood samples from children 5--18 years of age in Nangapanda sub-district, Ende were analysed. The sensitivity of Real-time PCR revealed that sub-microscopic infections are common in this area. The prevalence for P. falciparum, P. vivax and mixed infection of both species are 11,1%, 9,7% and 2,8%, respectively. A low load of Plasmodium species-specific DNA (Ct>35) was found for P. falciparum and P. vivax in 9,7% and 8,3% of the positive cases, respectively. There was no significant correlation (p>0,05) between malaria submicroscopic with gender and age groups. Real-time PCR provides more insight into the epidemiology of P. falciparum and P. vivax infections, which could be applied for monitoring and evaluation of novel programs for the elimination of malaria."
Depok: Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Indonesia, 2013
S46167
UI - Skripsi Membership  Universitas Indonesia Library
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Detection of human group and C rotaviruses in pediatric patients with acute gastroenteritis by real time RT-PCR assay : a preliminary study
"Rotavirus causes 25?55% of all hospital admissions for diarrhea and approximately 611.000 deaths every year in developing countries. Clinically, it is not possible to recognize the diarrhea caused by rotavirus and other infections. To know a causative agent of rotavirus gastroenteritis, availability of an accurate diagnosis assay is necessary. Therefore, we developed real time RT-PCR assay (rRT-PCR) assay for confirmation of infections of Group A or C rotaviruses simultaneously. A total of 54 stool samples obtained from pediatric patients (< 5 years old) was used in this study. All samples were tested for Group A rotavirus by Serological rapid test. Result of serological rapid test was compared with rRT-PCR assay to obtain the test accuracies of both assays.
Result of this study showed that rates of positive testing for Group A rotavirus by serological rapid test and the rRT-PCR assay were 22.22% and 18.50%, espectively. Forty-two serology-negative specimens for Group A rotavirus were also PCR negative (100% specificity). Two serology-positive specimens for Group A rotavirus was rRT-PCR negative (confirmed by electrophoresis gel); therefore, rRT-PCR assay represents the decrease of 3.70% in the number of specimens that are positive for Group A rotavirus. For Group C rotavirus, all tested samples were no rRT-PCR positive and the results need to be confirmed in the future."
Fakultas Kedokteran Universitas Indonesia, 2010
AJ-Pdf
Artikel Jurnal  Universitas Indonesia Library
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