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Multiplex nested polymerase chain reaction for Treponema pallidum using blood is more sensitive than using serum Effendi, Ida; Rosana, Yeva; Yasmon, Andi; Indriatmi, Wresti
Universa Medicina Vol 37, No 1 (2018)
Publisher : Faculty of Medicine, Trisakti University

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.18051/UnivMed.2018.v37.75-84

Abstract

BackgroundSyphilis is a multistage disease transmitted primarily through sexual intercourse. Nowadays, the polymerase chain reaction (PCR) test for Treponema pallidum has been widely used and is expected to overcome problems in diagnostic tests for syphilis. The Treponema pallidum PCR is influenced by type of specimens, PCR methods and target genes. This study aimed to assess the use of blood and serum in multiplex nested PCR for Treponema pallidum, targeting the 23S rRNA.MethodsA cross-sectional study was conducted from April 2015 - April 2016. Sampling was carried out consecutively among patients with clinical features of secondary syphilis who came to Sexually Transmitted Disease (STD) clinics in Jakarta. All sera were also tested with Rapid Plasma Reagin (RPR) and Treponema pallidum Hemagglutination Assay (TPHA) assay, which was considered as the gold standard for this study. We determined the sensitivity and specificity of the multiplex nested PCR for Treponema pallidum using blood and serum.ResultsPCR test was performed on 122 clinical specimens (61 blood and 61 serum). 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%. The sensitivity of Treponema pallidum multiplex nested PCR on blood was 30.95% compared to serum 9.52% (p=0.006). PCR test on blood is able to detect 3.25 times higher than serum. ConclusionThe use of blood has a higher proportion of positives compared to serum in Treponema pallidum multiplex nested PCR using 23S rRNA target gene.
DETEKSI GEN MSG UNTUK PENINGKATKAN KETEPATAN DIAGNOSIS PNEUMOCYSTIS JIROVECII PADA PASIEN HIV DENGAN PNEUMONIA Tjampakasari, Conny Riana; Yasmon, Andi; Sudarmo, Fitrahwati
Indonesian Journal of Biotechnology and Biodiversity Vol 2, No 1 (2018): Indonesian Journal of Biotechnology and Biodiversity
Publisher : Universitas Esa Unggul

Show Abstract | Download Original | Original Source | Check in Google Scholar

Abstract

AbstractPneumocystis jirovecii is the cause of opportunistic infections in the lower respiratory tract of immunocompromised patients, especiallyin human immunodeficiencyvirus (HIV). Until now the case of P. jirovecii in Indonesia is not known with certainty because the data obtained only based on patient’s clinical condition. Culture is still on going research while microscopic as a gold standard has some limitation, among others, takes a long time in the process, requires special kills to work and interpret results. The diagnosis of P.jirovecii infection in Indonesia is still based on clinical and microscopic examination, which takes a long time, is less sensitive and specific. Based on this reason then developed rPCR that  more sensitive and specific. In addtionthe results obtained are less sensitive and spesific. Based on that, in this study developed real-time PCR (rPCR) molecular test that more sensitive and apesific using gene MajorSurface Glicoprotein (MSG). MSG is detection target of its presence in the fungus cell wall is very abundant and has a sustainable sequence. The rPCR was successfully optimized with a minimum DNA detection capability of 6.55 cop / μl and did not cross-react with other microorganisms tested in this study. Obtauned 50 clinical sampels consisting of sputum and sputum induction. The result of comparison between microscopic test and rPCR show that rPCR increase positive results up to 20%. The rPCR test can detect P.jirovecii on clinical samples of sputum and induced sputum from HIV patients with pneumonia with CD4 +> 200 or ≤ 200 cells.  Keywords : real time PCR, HIV, pneumocystis jirovecii. AbstrakPneumocystisjiroveciiadalah penyebab infeksi oportunistik di saluran pernapasan bawah pada individu dengan sistem kekebalan tubuh yang lemah, terutama pada pasien HIV. Pemeriksaan infeksi P. jirovecii di Indonesia masih berdasarkan pemeriksaan klinis dan mikroskopis, yang memerlukan waktu yang cukup lama, kurang sensitif dan spesifik. Berdasarkan hal tersebut maka dalam penelitian ini dikembangkan uji molekuler real time PCR (rPCR) yang lebih sensitif dan spesifik. Uji rPCR telah berhasil dioptimasi dengan kemampuan deteksi minimum DNA 6,55 copy/μl dan tidak bereaksi silang dengan mikroorganisme yang diuji pada penelitian ini. Dibandingkan dengan uji mikroskopis, uji rPCR memberikan hasil positif 20% lebih tinggi daripada uji mikroskopis. Uji rPCR dapat mendeteksi P.jiroveciipada sampel klinis sputum dan sputum induksi dari pasien HIV dengan pneumonia denganjumlah sel CD4+> 200 maupun ≤ 200. Oleh karena itu, uji rPCR yang telah dioptimasi dalam studi ini dapat mendeteksi P.jirovecii pada sampel klinis sputum dan sputum induksi dari pasien HIV dengan pneumonia dengan jumlah sel CD4+> 200 maupun ≤ 200. Kata Kunci : real time PCR, HIV, pneumocystis jirovecii.
Microscopic examination using negative staining for rapid diagnosis of syphilis Rosana, Yeva; Effendi, Ida; Indriatmi, Wresti; Yasmon, Andi
Universa Medicina Vol. 41 No. 1 (2022)
Publisher : Faculty of Medicine, Universitas Trisakti

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.18051/UnivMed.2022.v41.64-70

Abstract

BACKGROUNDSyphilis is a global health problem, especially in developing countries including Indonesia. Treponema pallidum, the etiologic agent of syphilis, cannot be cultured in vitro. Syphilis has several clinical manifestations, making laboratory testing a very important aspect of diagnosis. Microscopic examination may support the diagnosis but is rarely used in Indonesia. The aim of this study was to evaluate negative staining using the light microscope to detect T. pallidum in syphilitic lesions. METHODSA cross-sectional study was conducted involving 27 subjects who came to several dermato-venereology clinics in Jakarta. Exudates were collected from genital ulcers, condylomata lata, and dry mucocutaneous rash on palms and soles of syphilis patients. Negative staining using one drop of Indian ink was used to examine for treponemas under the light microscope at 10x100 magnification. RESULTSMicroscopic examination using negative staining showed a few clusters of small and spiral shaped bacteria. Of the 39 specimens from 27 subjects, microscopic examinations were successfully done on 10 specimens. Observations could only be conducted on 5 specimens, 3 (60.0%) of which showed the morphology of spirochetes. This examination is the easiest method for detecting the bacteria. Moreover, the bacteria that were isolated from painless genital ulcers could be observed more clearly than those from erythematous maculopapular lesions. CONCLUSIONTreponema pallidum was successfully detected by microscopic examination in all moist lesions, but was difficult to detect in dry lesions. Negative staining under the light microscope appears to be simple, affordable, and available in most microbiology laboratories in Indonesia.
Comparison of RT-PCR-Dot Blot Hybridization Based on Radioisotope 32P with Conventional RT-PCR and Commericial ELISA Assays for Blood Screening of HIV-1 Maria Lina Rosilawati; Andi Yasmon
Jurnal Ilmiah Aplikasi Isotop dan Radiasi Vol 7, No 2 (2011): Desember 2011
Publisher : BATAN

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (209.623 KB) | DOI: 10.17146/jair.2011.7.2.90

Abstract

There are many commercial ELISA and rapid test kits that have been used for blood screening; however, the kits can give false positive and negative results. Therefore, RT-PCR (Reverse Transcription Polymerase Chain Reaction) - Dot Blot Hybridization based on radioisotope 32P (RDBR) method was developed in this research, to compare the method with the conventional RT-PCR and commercial ELISA Enzyme-Linked Immunosorbent Assay) kit. This method is efficient for screening of large blood specimens and surveillance study. Eighty seven samples were used and serum of the samples were tested by ELISA to detect HIV-1. The HIV-1 RNA genome was extracted from plasma samples and tested using the RT-PCR and RDBR methods. Of 87 samples that were tested, the rates of positive testing of the RT-PCR, the RDBR, and the ELISA were 71.26%, 74.71%, and 80.46%, respectively. The RDBR (a combination of RT-PCR and dot blot hybridization) was more sensitive than conventional RT-PCR by showing 3.45% in increase number of positive specimens. The results showed that of 9 samples (10.34%) were negative RDBR and positive ELISA, while 4 samples (4.60%) werenegative ELISA and positive RDBR. The two methods showed slightly difference in the results but further validation is still needed. However, RDBR has highpotential as an alternative method for screening of blood in large quantities when compared to method of conventional RT-PCR and ELISA.
DETEKSI MUTASI GEN KATG MYCOBACTERIUM TUBERCULOSIS DENGAN METODE PCR (POLYMERASE CHAIN REACTION) - HIBRIDISASI DOT BLOT MENGGUNAKAN PELACAK OLIGONUKLEOTIDA BERTANDA 32 Maria Lina R.; Budiman Bela; Andi Yasmon
Jurnal Ilmiah Aplikasi Isotop dan Radiasi Vol 5, No 1 (2009): Juni 2009
Publisher : BATAN

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.17146/jair.2009.5.1.525

Abstract

Penanganan dan pengendalian penyakit tuberkulosis (TB), penyakit yang disebabkan kuman M. tuberculosis, menjadi semakin sulit dengan meningkatnya kasus resistensi kuman penyebab terhadap obat anti tuberkulosis (oat) sepertiisoniazid. Resistensi dapat terjadi karena penggunaan obat yang tidak tepat dan tidak teratur, sehingga menimbulkan mutasi pada gen yang mengkode/menyandi target oatseperti gen katG (katalase peroxidase G) untuk isoniazid. Teknik biologi molekuler seperti PCR dilanjutkan dengan hibridisasi dot blot dengan pelacak oligonukleotida berlabel radioisotop merupakan teknik deteksi cepat adanya mutasi pada gen tersebut. Percobaan ini bertujuan menerapkan teknik PCR-hibridisasi dot blot dengan pelacak oligonukleotida bertanda radioisotop (32P) untuk mendeteksi adanya mutasigen katG pada kodon 315 yang menyebabkan M. tuberculosis resisten terhadap isoniazid. Dalam penelitian ini digunakan 89 contoh sputum dan kuman standar M. tuberculosis H37Rv. Ekstraksi DNA dilakukan dengan menggunakan metode BOOMuntuk contoh sputum dan metode fenol kloroform untuk kuman standar. Primer oligonukleotida yang dipakai untuk proses PCR adalah Pt8 & Pt9 untuk mendeteksi Mycobacterium penyebab tuberkulosis dan RTB 59 & RTB36 untuk mendeteksi keberadaan gen katG, yang masing-masing dirancang dari daerah pada sekuens sisipan IS6110 dan gen katG M. tuberculosis. Hasil PCR dideteksi dengan teknik elektroforesis gel agarosa. Metode hibridisasi dot blot dengan pelacak oligonukleotida 315 mu bertanda radioisotop 32P, dilaksanakan untuk mengetahui adanya mutasi gen katG dari hasil PCR dengan primer RTB59 & RTB36. Proses PCR dengan primer Pt8dan Pt9 menunjukkan hasil positif untuk semua contoh sputum yang menyatakan sputum mengandung Mycobacterium penyebab tuberkulosis. Hasil proses hibridisasi dot blot dengan pelacak oligonukleotida 315 mu bertanda 32P, memperlihatkan, 11 strain dari 89 strain M. tuberculosis pada contoh sputum, mengalami mutasi pada kodon 315 dari gen katG. Berdasarkan hasil tersebut, dapat dinyatakan bahwa teknik PCR-hibridisasi dot blot dengan pelacak oligonukleotida bertanda 32P adalah metode yang cepat, spesifik, dan sensitif untuk mendeteksi adanya mutasi gen katG M. tuberculosis yang berkaitan dengan resistensinya terhadap isoniazid.
The effect of dadih in BALB/c mice on pro-inflammatory and anti-inflammatory cytokine productions Ria Kodariah; Hadits Lissentiya Armal; Heri Wibowo; Andi Yasmon
Journal of the Medical Sciences (Berkala Ilmu Kedokteran) Vol 51, No 4 (2019)
Publisher : Journal of the Medical Sciences (Berkala Ilmu Kedokteran)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (266.193 KB) | DOI: 10.19106/JMedSci005104201902

Abstract

The normal microflora formed as commensal bacteria have roles in maintaining homeostasis in the intestine tract. The reduction in the amount and on the diversity of the commensal bacteria lead to gastrointestinal dysbiosis which increase number of pathogens, induce inflammatory and can drive to colorectal cancer. Probiotics can be used to prevent, regulate, and modulate immune response by triggering the development of pathogen-specific memory. Currently, many foreign probiotic products are available in the market that cause the domestic products are less well known. Dadih is an original probiotic’s products originally from West Sumatra, Indonesia. It is made from fermented buffalo milk containing lactic acid bacteria (LAB). The objective of the study was to investigate the effect of dadih pro-inflammatory and anti-inflammatory cytokine production. The study was conducted using male BALB/c mice aged 6-8 weeks with body weight (BW) 20-30 g. Mice were given dadih at doses of 112 mg/20g BW for eight weeks. The results indicated that LAB bacteria in dadih are coccus, Gram-positive bacteria with 3x107 colony-forming units (CFU) and dominated by Lactococcus lactis subsp. lactis. In addition, the increase of both the anti-inflammatory cytokines (IL-10) and pro-inflammatory cytokines (TNF-α and IL-1β) was observed. In conclusion, the dadih can be used to maintain the immune system of mice.
Untranslated region-5' and viral protein 1-based genetic stability analysis of bulk polio in Indonesia 2010-2019 Andi Yasmon; Normasari Normasari; Fithriyah Fithriyah; Fadilah Fadilah
Journal of the Medical Sciences (Berkala Ilmu Kedokteran) Vol 53, No 4 (2021)
Publisher : Journal of the Medical Sciences (Berkala Ilmu Kedokteran)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.19106/JMedSci005304202106

Abstract

Cases of vaccine-associated paralytic poliomyelitis (VAPP) continued increasingly from 2010-2019 in the world. Oral polio vaccine (OPV) is the live attenuated virus-based vaccine that could genetically revert to neurovirulent during the vaccine production process or when the virus replicates in the human body. The poliovirus neurovirulence is determined by the UTR-5' region and VP1 coding region. UTR-5' played a role in protein translation and VP1 was responsible for the immunogenicity of the virus. Some reported mutations in UTR-5' and VP1 could affect the neurovirulence of poliovirus. In this study, we analyzed the genetic stability of the UTR-5' and VP1 in the bulk of OPV types -1 and -3 produced in 2010 - 2019. The results of the analysis of UTR-5' sequences in Sabin strain types-1 and -3 and VP1 sequences on Sabin virus type 1 did not show any mutations. Meanwhile, the VP1 sequences in Sabin strain type 3 showed nucleotide mutation C2493U that caused the substitution amino acid Thr6Ile amino acid in all samples of the type 3 bulk polio test. Based on the results of in silico analysis, this mutation in VP1 did not contribute significantly to the neurovirulence of the virus.
Development of a SYBR Green real-time PCR-based assay system for detection of Neisseria gonorrhoeae Andi Yasmon; Rela Febriani; Louisa Ivana Utami; Fithriyah Fithriyah; Yeva Rosana; Fera Ibrahim; Pratiwi Sudarmono
Journal of the Medical Sciences (Berkala Ilmu Kedokteran) Vol 54, No 1 (2022)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.19106/JMedSci005401202201

Abstract

Diagnosis of Neisseria gonorrhoeae infection is needed for patient therapy and for reducing this bacterial transmission in the population. The culture method is a gold standard method for N. gonorrhoeae detection, however it has low sensitivity. Among molecular methods with high sensitivity and specificity, SYBR Green real-time PCR is the potential method for N. gonorrhoeae detection. In this study, we developed an SYBR Green real-time PCR-based system assay for N. gonorrhoeae detection. Several PCR conditions were optimized and analyzed including primer annealing temperature, DNA template volume, the limit of detection (LoD), cross-reaction with others (bacteria, viruses, fungus, protozoa), and quality assurance. The results showed that the annealing temperature and DNA template volume were 60oC and 5 µL, respectively. The LoD was 29 DNA copies corresponding to 3 bacterial cells per reaction. No cross-reaction was detected for other bacteria, viruses, fungus and protozoa. The external quality assurances enrolled in 2019 and 2021 showed 100% concordance. The preliminary testing for clinical samples was also 100% concordance. In conclusion, the SYBR Green real-time PCR-based system assay developed in this study is promising for application in clinical laboratories.
Five Unique Amino Acid Residues of Hemagglutinin (HA) Proteins of Swine Influenza A (H1N1) Detected in 2009 in Jakarta, Indonesia ANDI YASMON; YULIANTY MUHAYAR; VIVI SETIAWATY; BETI ERNAWATI DEWI; BUDIMAN BELA; FERA IBRAHIM
Microbiology Indonesia Vol. 6 No. 2 (2012): June 2012
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (669.535 KB) | DOI: 10.5454/mi.6.2.3

Abstract

Nine HA genes of influenza A (H1N1) viruses originating from swine which were detected in 2009 in Jakarta, Indonesia, were characterized in this study. Nasopharyngeal and/or pharyngeal samples were extracted to obtain viral RNA genomes. Amplification of the HA segment was performed by using the reverse transcription-polymerase chain reaction (RT-PCR), and followed by nested PCR in cases of RT-PCR negative. DNA sequencing was performed by using eight overlapping primers. All the Jakarta strains were closely related to vaccine strain A/California/07/2009. Nine amino acid changes were found in the Jakarta strains, and 5 (P100S, S220T, G239D, R240Q, and I338V) of those were unique to all Jakarta strains with respect to strain A/California/07/2009 used to produce vaccine. An I338V substitution was detected in a cleavage site of HA and no amino acid changes were detected in potential sites for N-linked glycosylation. For seven sites (positions 131, 158, 160, 183, 187, 222, and 227) playing an important role in viral attachment to host receptor, none of the expected amino acid changes was detected; however, a S220T substitution close to amino acid 222 was found in all the Jakarta strains. All amino acid changes potentially affect the pathogenicity of the viruses and the efficacy of strain A/California/07/2009 in neutralizing the Jakarta strains.
Gene Families of AmpC-producing Enterobacteriaceae Present in the Intensive Care Unit of Cipto Mangunkusumo Hospital Jakarta Lucky Hartati Moehario; Thomas Robertus; Anis Karuniawati; Rudyanto Sedono; Delly Chipta Lestari; Andi Yasmon
The Indonesian Biomedical Journal Vol 11, No 1 (2019)
Publisher : The Prodia Education and Research Institute (PERI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.18585/inabj.v11i1.552

Abstract

BACKGROUND: Antibiotic resistance has become a worldwide problem. Among Asia countries, Indonesia has high prevalence of multi-drug resistant organisms mainly due to Gram-negative bacilli Enterobacteriaceae. This study aimed to find out whether gene family of AmpC and AmpC/ESBL-producing Enterobacteriaceae were present in the Intensive Care Unit (ICU) of Cipto Mangunkusumo Hospital, Jakarta, Indonesia.METHODS: Specimens were obtained from several body sites of adult patients with infection hospitalised in ICU of Cipto Mangunkusumo Hospital. VITEK®2 was used to identify the microorganisms. Antibiotic susceptibility tests were conducted using VITEK®2 and disc diffusion technique according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Double disc synergy (DDS) test method was employed to detect AmpC activity. Gene families of ampC were identified using multiplex polymerase chain reaction (PCR).RESULTS: Forty five isolates were identified as putative AmpC, extended-Spectrum β-lactamases (ESBL) and AmpC/ESBL-producing Enterobacteriaceae. Klebsiella pneumoniae (n=32) were predominant, followed by Escherichia coli (n=6), Enterobacter cloacae (n=5) and Enterobacter aerogenes (n=2). AmpC activity was detected in 9 isolates, in which 4 isolates were AmpC producing and 5 isolates were AmpC/ESBL. In vitro, AmpC-producing Enterobacteriaceae showed good susceptibility to many antibiotic tested, while those of AmpC/ESBL-producing only to Amikacin. The gene families of ampC were DHA, EBC and CIT identified from 6 isolates.CONCLUSION: DHA, EBC and CIT gene families were identified from AmpC and AmpC/ESBL-producing Enterobacteriaceae in the ICU of Cipto Mangunkusumo Hospital. While the AmpC-producing was still susceptible to almost all antibiotics tested, the AmpC/ESBL-producing showed resistant except for Amikacin.KEYWORDS: Enterobacteriaceae, β-lactamases, AmpC, ESBL
Co-Authors . Andriansjah Abror Irsan Ade P.R. Simaremare Agustini, Riani Anis Anis Anis Karuniawati Ardiana Kusumaningrum Ariyani Kiranasari ASRI SULFIANTI Beti Ernawati Dewi Budiman Bela Burhanuddin Burhanuddin Burhanuddin Burhanuddin Delly Chipta Lestari Delly Chipta Lestari Dewi Murniati Diah Dwi Utami Diana Natalia Diana Natalia Efendi, Ida Effendi, Ida Effiana Effiana Effiana, Effiana Elisabeth D. Harahap Elisna Syahruddin Erlina, Linda Fadilah Fadilah Fadilah FERA IBIRAHIM Fera Ibrahim Fithriyah Fithriyah Fithriyah Fithriyah Fithriyah Fithriyah Gultom, Desy A. Heri Wibowo Ibnu Agus Ariyanto Ika Ningsih Junita Indarti Ketut Tuti Parwati Linosefa Linosefa Lisnawati Rachmadi Lola Febriana Dewi Louisa Ivana Utami Lucky H Moehario Mardhia, Mardhia Mardhia, Mardhia Mardiastuti H Wahid Maria L. Rosilawati Maria L. Rosilawati Maria Lina R. Maria Lina Rosilawati Maya Ulfah Moehario, Lucky Hartati Murti, Anissa Wari Ni Made Adi Tarini Ni Nengah Dwi Fatmawati Normasari Normasari Oktania Sandra Puspita Paramita, Rafika Indah Pramita G. Dwipoerwantoro Pratiwi Pujilestari Sudarmono Purba, Hastuti Handayani S Purnomosidi, Muhammad Abhi Rahayu, Ratih Ratnoglik, Suratno Lulut Rela Febriani Ria Kodariah Rifdah Hanifah Rosa, Yulia Rudyanto Sedono Safari, Dodi Salsabila, Korrie Samsuridjal Djauzi Sari Rahmayanti Simon Yosonegoro Liem SJATHA, FITHRIYAH Sudarmo, Fitrahwati Tafroji, Wisnu Teguh Sarry Hartono Thomas Robertus Tjampakasari, Conny Riana Utami, Diah Dwi VIVI SETIAWATY Winarti, Yayah Wresti Indriatmi B. Makes Wulandari, I Gusti Ayu Inten Yeva Rosana Yulia Rosa Saharman YULIANTY MUHAYAR Yusmaniar Yusmaniar Zaenab