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"Leptospirosis adalah penyakit infeksi akut yang dapat menyerang rnanusia maupun hewan yang disebabkan bakteri Leprospfra spp dan digolongkan sebagai zoonosis. Gejala klinis leptospirosis yang tidak spesiiik dan sulitnya uji laboratorium untuk konfirmasi diagnosis mengakibatkan penyakit ini seringkali tidak terdiagnosis. Oleh karena itu dalam ponelirian ini dilakukan optimasi uji diagnostik molekuler menggimakan real-time PCR sebagai deteksi cepat, sensitif dan spesiflk untuk Leptospira patogen pada manusia DNA bakten di dalam spesimen darah diekstraksi menggunak:an QIAamp DNA Blood Mini Kit, Qiagen dan spesimen urin diekstraksi menggunakan QIAamp DNA Stool Mini Kit,Qiagen dengan prosodur sesuai dengan petunjuk manualnya. Primer dan probe yang digunakan berdasarkan publikasi penelitian oleh Smythe dkk, 2002. Dari hasil uji optimasi kondisi optimal real-time PCR didapat suhu annealing 60°c, konsentrasi primer 0,9 uM dan konsentrmi probe 0,2 uM. Spesifisitas primer diuji menggunakan DNA balcteri patogen lain Hasii uji sensitiiitas real-time PCR untuk mendeteksi konsentrasi DNA terendah bakteri Leprospim spp adalah 0,75 fypl, hasil uji spesitisitas real-time PCR menunjukkan bahwa primer yang digunakan untuk deteksi balderi Leprospira spp tidak beraksi silang dengan genom bakteri-bakteri uji, konsentrasi minimal DNA bakteri yang masih terdeteksi dalam darah mencapai 150 fg/pl, sedangkan dalam urin mencapai 1470 fg/pl yang masih dapat dideteksi dengan pemeriksaan real-time PCR. Metode real-time PCR ini dapat digunakan sebagai alternatif pemeriksaan mikrobiologi yang cepat dan tepat untuk mendiagnosis leptospirosis.

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Leptospirosis is an emerging infectious disease in human and animals caused by Leptospira spp. and considered endemic in Indonesia due to its tropical climate. The International Leptospirosis Society (2001) declared Indonesia has high incidence of leptospirosis and ranked the third in the world for mortality (16.7%) The clinical features are not specific and may result in a missed or delayed diagnosis. The microbiology diagnostic method e.g. culture and microscopic agglutination test (MAT) are sensitive and specific but time-consuming and high cost. The other method to detect the antibody result false positive reactions and need confirmation by the MAT. Therefore in this study we optimized the real-time PCR assay, which has been used to detect a large number of microbes. It has high sensitivity and specificity, thus making it ideal as a rapid and accurate method to detect pathogen Leptospira spp. in human specimens. The amplification of the DNA control was performed optimally with the following conditions: annealing temperature is 60°C, primer volume is 0.5p1 (final concentration: 0.9 phd); probe volume is 0.2 ul (final concentration 0.2 pM). This method may detect the DNA in the Mastermix Mix with the concentration of 0.75 fg/ul, however in blood specimen the limit of detection of the DNA 150 fg/pl and in urine is 1470 fg/pl. The primer used in this assay is not complementary with the DNA of other pathogenic Leptospira spp. The real-time PCR assay is a rapid and accurate method to detect pathogenic Leptospira in human specimens. Further studies are needed to know the sensitivity and specificity of the real-time PCR assay compared to other diagnostic methods in clinical settings."

"Leptospira spp. dan digolongkan sebagai zoonosis. Gejala klinis leptospirosis yang tidak spesifik dan sulitnya uji laboratorium untuk konfirmasi diagnosis mengakibatkan penyakit ini seringkali tidak terdiagnosis. Oleh karenanya perlu dilakukan optimasi uji diagnostik molekuler menggunakan real-time PCR sebagai deteksi cepat Leptospira patogen yang sensitif dan spesifik. Penelitian ini di disain untuk mengoptimatisasi uji diagnostik molekuler menggunakan real time-PCR sebagai metode yang sensitif dan spesifik untuk mendeteksi Leptospira. Metode: DNA bakteri di dalam spesimen diektraksi menggunakan DNA extraction kit dengan prosedur sesuai dengan petunjuk manualnya. Primer dan probe yang digunakan berdasarkan penelitian terpublikasi terdahulu. Penelitian ini menggunakan mesin PCR-IQTM5, iCycler Multicolor real-time PCR detection system. Spesifisitas primer diuji menggunakan DNA bakteri patogen lain yang mungkin dapat ditemukan pada kedua spesimen dan dapat memberikan gejala klinis yang mirip. Hasil: Hasil uji real-time PCR menunjukkan bahwa kadar DNA Leptospira spp. terendah yang dapat terdeteksi adalah 0,375 fg/μl. Hasil uji juga menunjukkan bahwa primer yang digunakan untuk deteksi bakteri Leptospira spp. tidak beraksi silang dengan genom bakteri uji lainnya. Konsentrasi minimal DNA bakteri standar yang masih dapat dideteksi dengan pemeriksaan ini adalah 150 fg/μl, sedangkan dalam urin adalah 1470 fg/μl. Kesimpulan: Uji real-time PCR adalah metode yang cepat dan akurat untuk deteksi Leptospira spp. patogen pada spesimen manusia. Penelitian lebih lanjut diperlukan untuk mengetahui sensitivitas dan spesifisitas dari uji real-time PCR dibandingkan dengan metode diagnostik lain.

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AbstractBackground: Leptospirosis is an acute infectious disease in humans caused by Leptospira spp. and classified as a zoonosis. Clinical symptoms of leptospirosis are nonspecific and the current available laboratory method for detecting Leptospira spp. is difficult, which resulted to the misdiagnosis of this disease. Therefore, the rapid and accurate method is needed to diagnose the disease. This study was aimed to optimize molecular diagnostic test using real-time PCR assay as a rapid, sensitive and specific method for the detection of pathogenic Leptospira spp. in humans. Methods: Bacterial DNA was extracted by DNA extraction kit according to the manufacturer?s instructions. Primers and probes used in this study was based on previous and published research. The assay is performed using PCR-IQTM5, iCycler Multicolor real-time PCR detection system. Specificity of the primer used was evaluated towards some bacterial pathogens. Results: Limit detection of the DNA was 0.375 fg/ml and the primers used does not cross-react with the genomes of the pathogens tested. Limit detection of DNA in blood is 150 fg/μl, and in urine is 1470 fg/μl. Conclusion: Real-time PCR test is a rapid and accurate method for detecting pathogenic Leptospira spp. in human specimens. Further research is needed to determine the sensitivity and specificity of real-time PCR tests compared with other diagnostic methods in clinical settings"

"Latar belakang: Difteri merupakan penyakit infeksi bakteri yang diperantarai olehtoksin. Corynebacterium diphtheriae adalah penyebab tersering difteri. Diagnostiklaboratorium harus dilakukan dengan cepat untuk menunjang diagnosis klinis difteri.Pemeriksaan mikroskopik tidak direkomendasikan karena tidak spesifik, kultur dan ujitoksin yang merupakan uji baku emas cukup memakan waktu, membutuhkanketerampilan dan pengalaman serta hanya dilakukan di laboratorium rujukan. PCRmerupakan metode pemeriksaan yang cepat, sensitif dan spesifik. Duplex real-time PCRdapat 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 tenggorokpasien tersangka difteri.Metode: Duplex real-time PCR menggunakan dua pasang primer dan probe dengantarget gen rpoB C.diphtheriae dan toksin difteri subunit A Tox. Parameter yangdioptimasi adalah suhu penempelan, konsentrasi masing-masing primer dan probe,inhibitor, reaksi silang dengan patogen lain dan ambang batas deteksi uji. Kemudian ujidiaplikasikan pada spesimen usap tenggorok pasien tersangka difteri yang dirawat diRSPI Sulianti Saroso pada periode 2018-2019. Sebagai perbandingan dilakukan uji Elekuntuk konfirmasi toksigenitas dan analisa data klinis pasien.Hasil: Kondisi optimal uji didapat pada suhu penempelan 55oC, konsentrasi primer Cd0,4 μM, primer Tox 0,6 μM, probe Cd 0,5 μM dan probe Tox 0,625 μM, volume elusiekstraksi 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 PCRadalah 21,3%, sedangkan proporsi positif C.diphtheriae toksigenik menggunakan ujibaku emas adalah 11,2%.Kesimpulan: Duplex real time PCR untuk deteksi C.diphtheriae potensial toksigeniktelah dioptimasi dan diaplikasikan pada pasien tersangka difteri. Diharapkan uji inidapat meningkatkan diagnosis laboratorium kasus difteri.

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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."

"Leptospirosis merupakan penyakit akibat infeksi bakteri Leptospira spp. patogen yang umumnya terjadi di negara tropis dan subtropis serta memiliki karakteristik berupa gejala klinis yang kurang spesifik. Vektor dari penyakit tersebut adalah tikus maupun hewan peliharaan, seperti anjing dan sapi. Microscopic agglutination test (MAT) merupakan uji baku emas leptospirosis yang telah ditetapkan WHO. Akan tetapi, uji tersebut kurang sensitif di fase awal terjadinya infeksi dan memiliki prosedur yang rumit. Konfirmasi melalui metode real-time polymerase chain reaction (RT-PCR) dengan gen secY menggunakan darah utuh dan serum dapat dilakukan untuk pengembangan diagnosis leptospirosis. Tujuan dari penelitian ini adalah mendeteksi gen secY pada sampel darah utuh dan serum pada pasien terduga leptospirosis yang meliputi pasien suspek dan probable di Klaten, Jawa Tengah dengan metode RT-PCR. Selain itu, penelitian ini juga bertujuan untuk membandingkan positivity rate hasil RT-PCR sampel darah utuh dan serum dari 111 pasien terduga leptospirosis di Klaten, Jawa Tengah. Metode penelitian ini meliputi isolasi DNA, kuantifikasi DNA, amplifikasi DNA dengan RT-PCR menggunakan probe dan pewarna ROX, serta analisis hasil RT-PCR. Hasil RT-PCR menunjukkan terdeteksinya gen secY pada sampel darah utuh dan serum, dengan positivity rate darah utuh sebesar 5,41% (6/111) dan serum sebesar 18,92% (21/111), sedangkan positivity rate pada pasien suspek adalah sebesar 19,51% (16/82) dan pada pasien probable sebesar 27,59% (8/29). Dengan demikian, darah utuh dan serum dapat digunakan untuk mendeteksi leptospirosis dengan RT-PCR menggunakan gen secY dengan serum sebagai sampel yang lebih sensitif dibandingkan darah utuh. Akan tetapi, perlu dilakukan upaya untuk meningkatkan kualitas metode deteksi, berupa proses ekstraksi, optimasi primer, maupun penggunaan gen pendamping. Selain itu, hasil RT-PCR tersebut juga dapat dikonfirmasi melalui analisis sekuens sebagai analisis lanjutan.

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Leptospirosis is a disease caused by infection with pathogenic Leptospira spp. bacteria that commonly occurs in tropical and subtropical countries and has characteristics in the form of less specific clinical symptoms. The vectors of the disease are rats and domestic animals, such as dogs and cattle. Microscopic agglutination test (MAT) is the WHO gold standard test for leptospirosis. However, the test is less sensitive in the early phase of infection and has a complicated procedure. Confirmation through real-time polymerase chain reaction (RT-PCR) method with secY gene using whole blood and serum can be done for the development of leptospirosis diagnosis. The aim of this study is to detect secY gene in whole blood and serum samples of presumed leptospirosis patients including suspected and probable patients in Klaten, Central Java using RT-PCR method. In addition, this study also aimed to compare the positivity rate of RT-PCR results of whole blood and serum samples from 111 presumed leptospirosis patients in Klaten, Central Java. The method of this research includes DNA isolation, DNA quantification, DNA amplification by RT-PCR using ROX probes and dyes, and analysis of RT-PCR results. RT-PCR results showed the detection of secY gene in whole blood and serum samples, with a positivity rate in whole blood is 5.41% (6/111) and in serum is 18.92 (21/111), meanwhile the positivity rate in suspected patients is 19.51% (16/82) and in probable patients is 27.59% (8/29). Thus, whole blood and serum can be used to detect leptospirosis by RT-PCR using the secY gene with serum as the more sensitive sample than whole blood. However, efforts need to be made to improve the quality of the detection method, in the form of the extraction process, primer optimization, and the use of companion genes. In addition, the RT-PCR results can also be confirmed through sequence analysis as a follow-up analysis."

"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.

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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."

"Translokasi bakteri merupakan kejadian yang diinisiasi oleh adanya reaksi inflamasi pada permukaan usus dan dapat menyebabkan terjadinya sepsis. Bifidobacterium anima/is subspesies lactis merupakan salah satu bakteri probiotik yang dapat memberikan efek anti-inflamasi, sehingga dapat menghambat terjadinya translokasi bakteri. Gen dnaK merupakan sekuens penanda yang dapat digunakan untuk deteksi B. anima/is subsp. lactis. Optimasi dilakukan untuk mendapatkan pasangan primer optimal dalam kuantifikasi B. anima/is subsp. lactis dengan metode kuantitatif Real-time PCR. Isolat DNA diisolasi dari sampel feses bayi menggunakan metode fenol-kloroform. Pasangan primer dirancang berdasarkan sekuen gen dnaK B.ani malis subsp.lacti s [ABOTO1000010.1] menggunakan program pri mer3. Optimasi primer dilakukan menggunakan 5 konsentrasi berbeda, yaitu 50/50, 100/100, 300/300, 500/500, dan 1.000/1.000 nM. Konsentrasi optimal pasangan primer F_HN019_dnaK dan R_HN019_dnaK untuk kurva standar adalah 1.000/ 1.000 nM dengan nilai efisiensi 95.397% dan R2 0,998. Konsentrasi pasangan primer 50/50--1.000/1.000 nM dapat digunakan untuk kuantifikasi DNA target dengan kisaran nilai Ct sebesar 16,13--31,89. Konsentrasi primer dan DNA sampel tidak berpengaruh dan berkorelasi terhadap nilai Ct. Konsentrasi sampel DNA target terkecil yang dapat terkuantifikasi dengan baik oleh pasangan primer F_HN019_dnaK dan R_HN019_dnaK 300/ 300 nM sampai dilusi 10-4 Pasangan primer F_HN019_dnaK dan R_HN019_dnaK dapat dikembangkan untuk kuantifikasi B. anima/is subsp. lactis dalam sampel feses bayi pada kejadian sepsis.

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Bacterial translocation is an event that is initiated by the presence of an inflammatory reaction at the surface of the intestine and can lead to sepsis. Bifzdobacterium anima/is subspecies lactis is a probiotic bacterium that can provide anti-inflammatory effect, so as to prevent the occurrence of bacterial translocation. dnaK is a marker gene sequences that can be used for detection of B. anima/is subsp. lactis. Optimization is performed to obtain optimal primer pair in quantifying B. anima/is subsp. lactis by the method of real-time quantitative PCR. Isolate DNA were isolated from infant feces samples using phenol­ chloroform method. Primer pairs designed based on gene sequences B.anima/is subsp.lactis dnaK f ABOTO1000010.11 using primer3 program. The primary optimization is done using 5 different concentrations, namely 50/50, 100/100, 300/300, 500/500, and 1.000/1.000 nM. F HN019 dnaK optimal primer pair concentrations and R HN019 dnaK for standard curve were 1,000 I 1,000 nM with 95,397% efficiency values and R2 0.998. 50/50--1.000/1.000 nM concentration of primer pairs can be used for quantification of the target DNA with a range of Ct values of 16.13 to 31, 89. Primer concentration and DNA samples have no effect and relation with the Ct value. The smallest concentration of the target DNA sample that can be quantified well by F_HN019_dnaK and R HN019 dnaK primer pair 300/300 nM is up to dilution 10-4 R HN019 dnaK F_HN019_dnaK primer pairs can be developed for the quantification of B. anima/is subsp. lactis in fecal samples of infants on the incidence of sepsis."

"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.

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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)"

"ABSTRAK Latar belakang : Cytomegalovirus (CMV) merupakan salah satu infeksi oportunistikpada pasien dengan sindrom immunodefisiensi (AIDS). Gejala klinis dan CT scantidak dapat menegakkan diagnosa definitif ensefalitis CMV. Oleh karena itudiperlukan uji alternatif untuk menegakkan diagnosis infeksi CMV pada pasien HIVdengan infeksi otak. Salah satu uji yang sensitif dan spesifik adalah Real TimePolymerase Chain Reaction (rPCR).Tujuan : Mendapatkan uji deteksi molekular CMV pada pasien HIV dengantersangka infeksi otak.Metode : Penelitian dilakukan dalam 3 tahap. Tahap 1 adalah optimasi konsentrasiprimer, probe, suhu annealing, volume elusi ekstraksi DNA, dan volume cetakan.Tahap 2 adalah uji spesifisitas (reaksi silang) dan uji sensitivitas (ambang batasdeteksi DNA) rPCR dan tahap 3 adalah penerapan uji rPCR yang sudah dioptimasiterhadap sampel plasma, urin, dan LCS.Hasil : Kondisi optimal uji rPCR telah diperoleh dengan konsentrasi primer danprobe 0,1 μM, dengan kondisi suhu reaksi rPCR: aktivasi enzim pada 950C selama 3menit; 45 siklus pada 950C selama 15 detik (denaturasi) dan 560C selama 1 menit(annealing dan ekstensi). Volume elusi ekstraksi DNA yang optimal untuk ketigajenis sampel (LCS, plasma dan urin) adalah 40 μL, dan volume cetakan rPCR untukLCS, plasma, dan urin, masing-masing adalah 5, 4, dan 3 μL. Uji rPCR mampumendeteksi DNA pada 50.000 jumlah kopi/mL dan tidak bereaksi silang denganStaphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus,Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycobacteriumtuberculosis, Candida spp, Toxoplasma gondii, EBV,HSV,dan VZV. Penerapan ujirPCR pada sampel klinis memberikan hasil negatif pada semua sampel LCS, 72,22%positif pada sampel plasma, dan 72,22% positif pada sampel urin.Kesimpulan: Telah dilakukan optimasi uji rPCR dengan minimal deteksi DNACMV 50.000 jumlah kopi/mL dan tidak bereaksi silang dengan mikroorganisme yangberpotensi menyebabkan positif palsu (false positive).ABSTRACT Background: Cytomegalovirus (CMV) is one of opportunistic infections in patientswith Aquired Immunodeficiency Syndrome (AIDS). Clinical manifestations are nottypical, and CT scans can not define encephalitis CMV specifically. Therefore, it isimportant to apply an alternative assay for sensitive and specific detection of CMVinfection in HIV patients with suspected central nervous system (CNS) infections.One of the assays is real time polymerase chain reaction (rPCR).Objective: To obtain a molecular assay for detection of CMV in HIV patients withsuspect CNS infections.Methods: This study was conducted in three phases. The first is optimization ofconcentrations of primers, probe, annealing temperature, final elution of DNAextraction, and volume of PCR template. The second is determinations of sensitivity(minimal detection of DNA) and specificity (cross-reaction) of the optimized rPCR,and the third is application of the rPCR for clinical samples of plasma, urine, andliquor cerebrospinal (LCS).Results: The rPCR reaction showed optimal concentrations of primers and probe at0.1 μM, with thermal cycler: 950C for 3 min (enzyme activation), followed by 45cycles of 950C for 15 sec (denaturation) and 560C for 1 min (annealing andextension). Final elution of DNA extraction was 40 μL and volume of PCR templatesfor urine, plasma, and LCS was 3, 4, and 5 μL, respectively. The rPCR had minimaldetection of DNA at 50,000 copies/mL and was not cross-reacted withStaphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus,Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycobacteriumtuberculosis, Candida spp, Toxoplasma gondii, Epstein-Bar Virus (EBV), HerpesSimplex Virus (HSV) and Varicella Zoster Virus (VZV). Application of rPCR forclinical samples showed that the rPCR yielded 72.22% positive for plasma or urine,and negative for all LCS samples.Conclusion: The rPCR has been optimized in this study with minimal DNA detectionat 50,000 copies/mL and was not cross-reacted with other microorganisms that arepotential to cause false positive results.;Background: Cytomegalovirus (CMV) is one of opportunistic infections in patientswith Aquired Immunodeficiency Syndrome (AIDS). Clinical manifestations are nottypical, and CT scans can not define encephalitis CMV specifically. Therefore, it isimportant to apply an alternative assay for sensitive and specific detection of CMVinfection in HIV patients with suspected central nervous system (CNS) infections.One of the assays is real time polymerase chain reaction (rPCR).Objective: To obtain a molecular assay for detection of CMV in HIV patients withsuspect CNS infections.Methods: This study was conducted in three phases. The first is optimization ofconcentrations of primers, probe, annealing temperature, final elution of DNAextraction, and volume of PCR template. The second is determinations of sensitivity(minimal detection of DNA) and specificity (cross-reaction) of the optimized rPCR,and the third is application of the rPCR for clinical samples of plasma, urine, andliquor cerebrospinal (LCS).Results: The rPCR reaction showed optimal concentrations of primers and probe at0.1 μM, with thermal cycler: 950C for 3 min (enzyme activation), followed by 45cycles of 950C for 15 sec (denaturation) and 560C for 1 min (annealing andextension). Final elution of DNA extraction was 40 μL and volume of PCR templatesfor urine, plasma, and LCS was 3, 4, and 5 μL, respectively. The rPCR had minimaldetection of DNA at 50,000 copies/mL and was not cross-reacted withStaphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus,Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycobacteriumtuberculosis, Candida spp, Toxoplasma gondii, Epstein-Bar Virus (EBV), HerpesSimplex Virus (HSV) and Varicella Zoster Virus (VZV). Application of rPCR forclinical samples showed that the rPCR yielded 72.22% positive for plasma or urine,and negative for all LCS samples.Conclusion: The rPCR has been optimized in this study with minimal DNA detectionat 50,000 copies/mL and was not cross-reacted with other microorganisms that arepotential to cause false positive results.;Background: Cytomegalovirus (CMV) is one of opportunistic infections in patientswith Aquired Immunodeficiency Syndrome (AIDS). Clinical manifestations are nottypical, and CT scans can not define encephalitis CMV specifically. Therefore, it isimportant to apply an alternative assay for sensitive and specific detection of CMVinfection in HIV patients with suspected central nervous system (CNS) infections.One of the assays is real time polymerase chain reaction (rPCR).Objective: To obtain a molecular assay for detection of CMV in HIV patients withsuspect CNS infections.Methods: This study was conducted in three phases. The first is optimization ofconcentrations of primers, probe, annealing temperature, final elution of DNAextraction, and volume of PCR template. The second is determinations of sensitivity(minimal detection of DNA) and specificity (cross-reaction) of the optimized rPCR,and the third is application of the rPCR for clinical samples of plasma, urine, andliquor cerebrospinal (LCS).Results: The rPCR reaction showed optimal concentrations of primers and probe at0.1 μM, with thermal cycler: 950C for 3 min (enzyme activation), followed by 45cycles of 950C for 15 sec (denaturation) and 560C for 1 min (annealing andextension). Final elution of DNA extraction was 40 μL and volume of PCR templatesfor urine, plasma, and LCS was 3, 4, and 5 μL, respectively. The rPCR had minimaldetection of DNA at 50,000 copies/mL and was not cross-reacted withStaphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus,Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycobacteriumtuberculosis, Candida spp, Toxoplasma gondii, Epstein-Bar Virus (EBV), HerpesSimplex Virus (HSV) and Varicella Zoster Virus (VZV). Application of rPCR forclinical samples showed that the rPCR yielded 72.22% positive for plasma or urine,and negative for all LCS samples.Conclusion: The rPCR has been optimized in this study with minimal DNA detectionat 50,000 copies/mL and was not cross-reacted with other microorganisms that arepotential to cause false positive results.;Background: Cytomegalovirus (CMV) is one of opportunistic infections in patientswith Aquired Immunodeficiency Syndrome (AIDS). Clinical manifestations are nottypical, and CT scans can not define encephalitis CMV specifically. Therefore, it isimportant to apply an alternative assay for sensitive and specific detection of CMVinfection in HIV patients with suspected central nervous system (CNS) infections.One of the assays is real time polymerase chain reaction (rPCR).Objective: To obtain a molecular assay for detection of CMV in HIV patients withsuspect CNS infections.Methods: This study was conducted in three phases. The first is optimization ofconcentrations of primers, probe, annealing temperature, final elution of DNAextraction, and volume of PCR template. The second is determinations of sensitivity(minimal detection of DNA) and specificity (cross-reaction) of the optimized rPCR,and the third is application of the rPCR for clinical samples of plasma, urine, andliquor cerebrospinal (LCS).Results: The rPCR reaction showed optimal concentrations of primers and probe at0.1 μM, with thermal cycler: 950C for 3 min (enzyme activation), followed by 45cycles of 950C for 15 sec (denaturation) and 560C for 1 min (annealing andextension). Final elution of DNA extraction was 40 μL and volume of PCR templatesfor urine, plasma, and LCS was 3, 4, and 5 μL, respectively. The rPCR had minimaldetection of DNA at 50,000 copies/mL and was not cross-reacted withStaphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus,Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycobacteriumtuberculosis, Candida spp, Toxoplasma gondii, Epstein-Bar Virus (EBV), HerpesSimplex Virus (HSV) and Varicella Zoster Virus (VZV). Application of rPCR forclinical samples showed that the rPCR yielded 72.22% positive for plasma or urine,and negative for all LCS samples.Conclusion: The rPCR has been optimized in this study with minimal DNA detectionat 50,000 copies/mL and was not cross-reacted with other microorganisms that arepotential to cause false positive results."

"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.

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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."

"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.

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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."