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"Latar Belakang. Leptospirosis merupakan penyakit infeksi di daerah tropis yang terabaikan (neglected infectious diseases) akibat vektor hewan dan kondisi lingkungan. Prevalensi di negara beriklim tropis, khususnya Indonesia cukup tinggi namun tidak terdiagnosis dengan baik. Diagnostik pasti sulit didapat karena uji baku emas leptospirosis, MAT (microscopic agglutination test), di Indonesia masih terbatas aksesnya dan uji alternatif belum banyak diteliti efektifitasnya.Metode. Penelitian ini menguji kinerja dua jenis alat diagnostik terbaru yaitu tes cepat berbasis IgM (Leptodipstick), dan PCR (polymerase chain reaction) terhadap gen lipl32 dan secY Leptospira dibandingkan uji baku emas MAT pada populasi Indonesia. Subjek penelitian adalah pasien dewasa rawat inap di RSCM pada tahun 2016-2018 yang memenuhi kriteria klinis leptospirosis WHO dan diujikan dengan MAT, tes cepat Leptospira, dan PCR Leptospira. Penelitian ini bersifat potong lintang, dengan variable yang diteliti meliputi data demografi, manifestasi klinis, paparan pekerjaan, paparan vektor ataupun lingkungan, serta hasil pemeriksaan MAT, tes cepat, dan PCR terhadap Leptospira. Hasil: Terdapat total 30 subjek yang ikut dalam penelitian dengan 11 (36,7%) meninggal dunia. Berdasarkan uji baku emas MAT, 14 (46,67%) dinyatakan reaktif terhadap antibodi Leptospira. Tes cepat Leptospira mendapat nilai sensitivitas 85,71% (95%IK: 57,19-98,22%), serta spesifisitas 62,50% (95%IK: 35,43%-84,80%), dan area di bawah kurva ROC 0,741 (95%IK: 0,549-0,883). Pemeriksaan PCR terhadap gen lipl32, sensitivitas 85,71% (95%IK: 57,19-98,22%), spesifisitas 18,75% (95%IK: 4,05-45,65%), dan area di bawah kurva ROC 0,522 (95%IK: 0,333 – 0,707). Pemeriksaan PCR gen secY, sensitivitas 71,43% (95%IK: 41,90-91,61%), spesifisitas 12,50% (95%IK: 1,55-38,35%), dan area di bawah kurva ROC 0,580 (95%IK: 0,371-0,789). Secara umum, semua uji memiliki sensitivitas yang memuaskan, namun spesifisitas yang buruk dibandingkan dengan MAT. Spesifisitas yang rendah dapat dikaitkan dengan onset penyakit yang sebagian besar berada pada fase akut sehingga belum dapat terdeteksi dengan alat uji yang berbasis antibodi.Kesimpulan. Spesifisitas tes cepat Leptospira dengan IgM maupun PCR terhadap gen lipl32 dan secY Leptospira sebagai alat penapisan cukup baik. Perlu dilakukan penelitian kinerja diagnostik lanjutan berdasarkan onset penyakit.

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Background. Leptospirosis is a neglected infectious disease transmitted by animal vectors and environment. The prevalency in  tropical Asia-Pacific country, including Indonesia was high. The gold standard test for leptospirosis was microscopic agglutination test (MAT), very limited distribution in Indonesia.

Methods. This study compares the diagnostic profiles of two new diagnostic tools, an IgM-based rapid test (Leptodipstick) and polymerase chain reaction (PCR) of the genes lipl32 and secY of the Leptospira genome, to MAT as the gold standard, in the Indonesian population. Adult inpatients of RSCM in the years 2016-2018 with conformity to the WHO clinical criteria for leptospirosis and undergo MAT test, Leptospira rapid test, and Leptospira PCR were enrolled in the study. The study was cross-sectional with the examined variable were demographic data, clinical manifestation, occupational exposure, exposure towards animal vectors or environmental conditions, and results of the MAT, Leptospira rapid test, and Leptospira PCR.

Results. The study enrolled 30 participants, with 11 (36,7%) deceased in the study period. Based on MAT, 14 participants (46,67%) were considered reactive for Leptospira antibodies. The Leptospira rapid test has a sensitivity of 85.71% (95%CI: 57.19-98.22%), and specificity of 62.50% (95%CI: 35.43%-84.80%), and the area under the ROC curve of 0.741 (95%CI: 0.549-0.883). PCR for the gene lipl32 showed a sensitivity of 85.71% (95%CI: 57.19-98.22%), specificity of 18.75% (95%CI: 4.05-45.65%), and area under the ROC curve of 0.522 (95%CI: 0.333-0.707). PCR for the gene secY showed a sensitivity of 71.43% (95%CI: 41.90-91.61%), specificity of 12.50% (95%CI: 1.55-38.35%), and area under the ROC curve of 0.580 (95%CI: 0.371-0.789). Generally, all tests showed a satisfactory sensitivity, yet very low specificity compared to MAT. Area under the curve showed a low (PCR) to moderate (rapid test) diagnostic value for each test. Low specificity may be tied to the onset of the disease of the study sample that most of them are on acute phase which cannot detected by the alternative test with antibody basis.

Conclusion. The diagnostic parameters of the Leptospira IgM rapid test, and Leptospira PCR with the genes lipl32 and secY are still satisfactory to be used as early diagnostic tools in cases of leptospirosis."

"ABSTRAKSifilis merupakan penyakit multistadium yang ditularkan terutama melalui hubungan seksual. Saat ini penggunaan uji polymerase chain reaction (PCR) untuk Treponema pallidum telah banyak digunakan dan diharapkan mampu mengurangi masalah dalam uji diagnostik sifilis. Hasil uji PCR Treponema pallidum dipengaruhi oleh jenis spesimen, metode PCR dan gen target. Penelitian ini ditujukan untuk menilai penggunaan darah dan serum untuk uji multiplex nested PCR dengan gen target 23S rRNA Treponema pallidum. Studi potong lintang dilakukan dari bulan April 2015 - April 2016. Pengambilan sampel secara konsekutif dari pasien dengan gambaran klinis sifilis sekunder yang datang ke poliklinik Infeksi Menular Seksual (IMS) di Jakarta. Uji PCR dilakukan terhadap 122 spesimen klinis (61 darah dan 61 serum). Uji serologi rapid plasma reagin (RPR) dan Treponema pallidum Haemagglutination Assay (TPHA) dilakukan pada semua serum. Hasil positif uji PCR darah sebesar 22,95% dan serum sebesar 6,56%, sedangkan hasil positif uji serologi sebesar 68,85%. Pada hasil uji serologi positif, proporsi hasil positif uji multiplex nested PCR Treponema pallidum darah sebesar 30,95% dibandingkan serum 9,52%. Uji PCR terhadap darah mampu mendeteksi 3,25 kali lebih tinggi daripada serum. Penggunaan darah memberikan nilai kepositivan yang lebih tinggi dibandingkan serum pada uji multiplex nested PCR Treponema pallidum menggunakan gen target 23S rRNA

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ABSTRACTSyphilis is a multistage disease transmitted primarily through sexual intercourse. Nowadays, polymerase chain reaction (PCR) test for Treponema pallidum has been widely used and expected to overcome problems in diagnostic test for syphilis. The PCR Treponema pallidum are influenced by type of specimens, PCR methods and gene targets. This study is aim to assess the use of blood and serum using multiplex nested PCR Treponema pallidum targeting 23S rRNA. Cross-sectional study was conducted from April 2015 - April 2016. Sampling was carried out consecutively from patients with clinical features of secondary syphilis who came to sexual transmitted infection (STI) clinics in Jakarta. PCR test performed on 122 clinical specimen ( 61 blood and 61 serum). All serum were tested with RPR and TPHA assay. The positive results of PCR test on blood was 22,95% and serum was 6,56%, while the positive results of serology was 68,85%. On positive serological test results, the proportion of positive results of multiplex nested PCR Treponema pallidum on blood was 30,95% compared to serum 9,52%. PCR test on blood is able to detect 3,25 times higher than serum. The use of blood give a higher positivity compared to serum in multiplex nested PCR Treponema pallidum using 23S rRNA gene target."

"Latarbelakang:Sebagian besar wanita hamil mengalami kolonisasi Streptococcus haemolyticus grup B (SGB) di saluran urogenital yang mempengaruhi kesehatan ibu hamil dan bayi. Deteksi SGBintrapartum perlu pemeriksaan yang cepat dan sensitif. Pemeriksaan mikrobiologi untuk mendeteksi SGB menggunakan metode kultur dan real time polymerase chain reaction(RT-PCR) telah digunakan untuk mendukung diagnosis, namun penggunaannya untuk skrining pada ibu hamil belum pernah diuji keakuratannya di Indonesia. Penelitian ini bertujuan untuk mencari metode terbaik untuk mendeteksi kolonisasi SGB pada ibu hamil sekaligus menilai akurasi uji RT-PCR.Metode: Penelitian dengan metode potong lintang padawanita hamil kurang dari 37 minggu dengan ketuban pecah diRumah Sakit Umum Pusat Nasional Cipto Mangunkusumo(RSCM), Rumah Sakit Umum Daerah Pasar Rebo, dan Rumah Sakit Budi Kemuliaan. serta bayi baru lahir dengan tersangka sepsis neonatorum awitan dini yang lahir dari ibu tersebut di RSCM. Swab rektovaginal pada ibu dan darah pada bayi untuk pemeriksaan tes RT-PCRdan kultur pada 3 media: agar darah (AD), agar darah Columbia (ADC), dan CHROMagar (CA).Hasil: Ada 50 ibu dan 25 bayi direkrut dalam penelitian ini.Prevalensi SGB pada ibu hamil 24%, 2 bayi meninggal. Dibandingkan dengan kultur dengan media ADC, tes RT-PCR mempunyai sensitivitas 83,33%, spesifisitas 86,84 %, NPP 66,67 %, NPN 94,29 %, dan akurasi 86,00 %.Media CAmenunjukkan hasil yang lebih tinggi dalam hal sensitivitas 100%, spesifisitas 100%, NPP 100%, NPN 100%, dan akurasi 100 %, dengan hasil lebih singkat, lebih praktis, dan lebih murah.Simpulan: Pemeriksaan RT-PCR menjadi pilihan dalam skrining SGB intrapartum, dengan alternatif media CA.

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Background: Most pregnant women were colonized of group B Streptococcus haemolyticus (GBS) in urogenital tract which affects the health of pregnant women and babies. Detection of intrapartum GBS requires rapid and sensitive examination. Microbiological examination to detect GBS using culture and real time polymerase chain reaction (RT-PCR) has been used to support the diagnosis, but its use for screening in pregnant women has never been tested for accuracy in Indonesia. This study aims to find the best method for detecting GBS colonization in pregnant women as well as assessing the accuracy of the RT-PCR test.

Methods: This was a cross-sectional study in pregnant women less than 37 weeks with ruptured membranes at the Cipto Mangunkusumo National Central General Hospital (RSCM), Pasar Rebo Regional General Hospital, and Budi Kemuliaan Hospital, And newborns with suspected early-onset neonatal sepsis born to these mothers at RSCM. Rectovaginal swab in mother and blood in infant for RT-PCR assay and culture on 3 media: blood agar (BA), Columbia blood agar (CBA), and CHROMagar (CA).

Results: There were 50 mothers and 25 infants recruited in this study. The prevalence of GBS in pregnant women was 24%, 2 neonates died. Compared with culture with CBA media, the RT-PCR test had a sensitivity of 83,33%, specificity 86,84%, PPN 66,67%, NPN 94,29%, and accuracy 86,00%. CA media showed higher results in terms of 100% sensitivity, 100% specificity, 100% NPP, 100% NPN, and 100% accuracy, with shorter results, more practical, and cheaper.

Conclusions: RT-PCR examination is an option in intrapartum GBS screening, with CA media as an alternative. "

"Skrining darah pendonor di Indonesia terhadap malaria belum dilakukan dengan pemeriksaan laboratorium. Kemungkinan resiko penularan malaria melalui darah donor dapat terjadi dan membahayakan jiwa resipien. Malaria di kota Ambon berdasarkan Annual Parasite Incidence adalah 4,49? termasuk High Case Incidence (HCI). Penelitian ini bertujuan mengetahui prevalensi malaria dengan berbagai pemeriksaan laboratorium di kota Ambon. Dikumpulkan sebanyak 550 donor di Unit transfusi darah PMI Ambon dalam kurun waktu 3 bulan dan dilakukan berbagai pemeriksaan. Hasilnya memperlihatkan tidak satupun terdeteksi positif dengan pemeriksaan mikroskopik maupun rapid test antigen Pf HRP2-pan aldolase atau Pf HRP-2- PvLDH. Duapuluh dua donor terbukti mengandung immunoglobulin P. falciparum dengan rapid test antibodi. Lima donor lain positif dengan PCR menggunakan 18S rRNA. Penelitian ini membuktikan adanya potensi penularan malaria dari darah donor sebesar 4.9% di Pulau Ambon.

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Screening of blood donors in Indonesia against malaria with laboratory tests have not been done. Possible risk of malaria transmission through donated blood may occur and endanger the lives of recipients. Malaria in the city of Ambon by Annual Parasite Incidence was 4.49 - including High Case Incidence (HCI). This study aims to determine the prevalence of malaria with a several laboratory tests in the city of Ambon. Collection of total 550 donors at Red Cross blood transfusion unit Ambon, was carried out for a period of 3 months and followed by various examinations. The results showed none detected positive by microscopic examination or antigen rapid test PfHRP2-aldolase or PfHRP2-LDH. Twenty-two donors were found to contain P. falciparum with immunoglobulin antibody rapid test, in addition five other donors positive by PCR using 18S rRNA. This study showed that the potency of malaria transmission by blood donors was 4.9% in the island of Ambon."

"Diagnostik medis merupakan tahapan awal dalam mengidentifikasi kondisi dan penyakit seseorang. Salah satu metode diagnostik yang banyak dipakai sekarang ini adalah Polymerase Chain Reaction (PCR). Penggunaan material perangkat thermal cycler dalam reaksi PCR mempengaruhi waktu yang dibutuhkan dalam prosesnya. Dengan kemajuan teknologi mikrofluida, chip thermal cycler dengan sistem mikrofluida banyak dikembangkan untuk meningkatkan kecepatan reaksi PCR. Studi ini mensimulasikan beberapa material dan geometri yang digunakan sebagai thermal cycler reaksi PCR Studi pada penelitian ini akan menggunakan aplikasi COMSOL Multiphysics 5.3 untuk dua desain PCR, thermal cycle tipe blok (TCP desain 1) dan thermal cycle tipe mikrofluida (TCP desain 2). Hasil yang didapatkan adalah waktu jenuh untuk TCP desain 1 menggunakan material aluminium selama 29 detik untuk pemanasan dan 26 detik untuk pendinginan, tembaga selama 37 detik untuk pemanasan dan 35 detik untuk pendinginan, nikel selama 51 detik untuk pemanasan dan 53 detik untuk pendinginan, perak selama 26 detik untuk pemanasan dan pendinginan, serta PDMS selama 1480 detik untuk pemanasan dan pendinginan. Pada TCP desain 1, saat menggunakan aluminium, didapatkan waktu jenuh untuk memanaskan reagen selama 32 detik dan 35 detik untuk mendinginkan. Pada PCR TCP desain 2 (a) yang langsung menggunakan PDMS, didapatkan waktu untuk jenuh memanaskan reagen adalah 3,2 detik dan 4,3 detik untuk mendinginkan, sedangkan pada TCP desain 2 (b) didapatkan waktu untuk memanaskan reagen selama 4,3 detik dan 4,6 detik untuk mendinginkan.

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Medical diagnosis is the initial stage in solving a person's condition and disease. One method that is widely used now is the Polymerase Chain Reaction (PCR). The use of thermal cycler device material in the PCR reaction affects the time required in the process. With the advancement of microfluidic technology, thermal cycler chips with microfluidic systems have been developed to increase the speed of PCR reactions. This study discusses several materials and geometries used as PCR thermal cycler reactions. COMSOL Multiphysics 5.3 application used to simulate two PCR designs, block-type thermal cycles (TCP design 1), and microfluidic type thermal cycles (TCP design 2). The results obtained are the saturation time for TCP design 1 using aluminum material for 29 seconds for heating and 26 seconds for safety, copper for 37 seconds for heating and 35 seconds for cooling, nickel for 51 seconds for heating and 53 seconds for heating, silver for 26 seconds for heating and cooling, and PDMS for 1480 seconds for heating and cooling. In TCP design 1, when aluminum was used, saturation time is obtained to heat the reagent for 32 seconds and 35 seconds to cool. In TCP PCR design 2 (a) which directly uses PDMS, obtained time to saturate the reagent heating is 3.2 seconds and 4.3 seconds to cool, whereas in TCP design 2 (b) it takes time to heat the reagent for 4.3 seconds and 4.6 seconds to cool down.

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"Malaria merupakan penyakit yang masih menimbulkan masalab kesehatan Masyarakat Indonesia. Prevalensi malaria di beberapa daerah cukup tinggi dan menjadikan daerah tersebut endemik malaria. Diagnosis malaria ditegakkan melalui pemariksaan gejala ktJnis dan penemuan parasit pada pemariksaan darah seoara mikroskopik. Pemariksaan mikroskopik masih merupakan "Gold Standard", tetapi masih terdapat beberapa kendala dalam sensitivitas dan akurat. Oleh karena itu penelitian ini bertujuan untuk mengemhangkan diagnosis altematif pemariksaan malaria yang lebih sensitif dan akurat. Teknik PCR pada sampel urin tems dikembangkan sehagai altematif diagnosis malaria. Penelitian ini dileknkan pada 58 sampel urin yang diambil pada orang yang tlnggal di daerah endemik malaria dan dipariksa dengan teknik PCR dengan menggunakan primer ssu rRNA, didapatkan 42 sampel positif dengan sensitivitas 98 % dan spesilisitas 94 %. Uji diagnostik mikroskopik pada sampel darah dan PCR pada sampel untuk P falclparum didapatkan 18 posltif dengan sensitivitas 94% dan spesifisitas 94%, sedangkan untuk P. vlvax didapatkan 25 sampel positlf dengan sensitivitas 96% dan spesifisitas 94%. Teknik PCR dengan sampel urin dapat digunakan sebagai alat diagnostik malaria untuk menggantikan pemeriksaan mikroskopik darah karena memilild sensitivitas dan spesifisitas yang tinggi (lebih dari 90% ).

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Malaria is an infectious disease which is still causing a public health problem in many parts of Indonesia. There are many endemic areas where the prevalence of malaria is high . The diagnosis of malaria is commonly done by clinical examination and parasite finding at microscopic examination of blood sample. Microscopic examination is still used as a gold standard for malaria diagnosis, however this method is less sensitivity and accuracy especially in low parasitemia.

Therefore, it is a need to develop an alternative method which is more sensitive and accurate fur Malaria diagnosis. PCR method for urine sample is being developed as an alternative diagnosis for Malaria. A total of 58 individuals living in malaria endemic areas participated in blood and urine collections. The presence of malaria parasites in blood samples were detected by microscopic examination whereas the DNA of mrdarial parasites, P. falciparum and P. vivax, in urine samples were detected by PCR method using ssu rRNA primers. Positive results of both malarial parasites were found in 42 samples with 98% sensitivity and 94 % specificity.

Diagnostic test of microscopic examination of blood samples and PCR of urine samples showed that 18 samples were P. falciparum positive with 94% sensitivity and 94% specificity whereas 25 samples were positive for P. vivax with 96% sensitivity and 94% specificity. This study revealed that PCR method can be used as an alternative diagnostic tool for malaria because it has high sensitivity and specificity (more than 90 %)."

"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: 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.

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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."