PENGEMBANGAN ANTIVIRUS HUMAN PAPILLOMA VIRUS BERBASIS MOLEKUL KECIL

AbstrakSekalipun telah ada program skrining deteksi dini infeksi HPV maupun kanker servis sertaadanya dua vaksin yang telah berlisensi, sekarang ini belum ada obat antivirus yang efektif.Prospek pengembangan molekul kecil inhibitor sebagai antivirus HPV sangat menjanjikan.Modulasi interaksi diantara protein-protein virus atau protein virus dengan protein hospesmenjadi strategi dalam upaya pengembangan molekul inhibitor sebagai antiviral HPV. Halini didukung oleh kemajuan pengetahuan mengenai fungsi protein HPV yang terlibat dalamsiklus hidupnya diantaranya yaitu protein E1, E2, E6 dan E7. Beberapa kandidat antivirustelah ditemukan dan masih dalam penelitian lebih lanjut untuk mendapatkan senyawa turunandengan aktivitas yang lebih tinggi diantaranya asam bifenil sulfonasetat (inhibitor ATPase E1).Indandione dan repaglinide (inhibitor interaksi E1-E2) dan senyawa-senyawa lainnya.AbstractEventhough there has been screening programs for HPV infection and cervical canceras well as the two vaccines that have been licensed, currently there is no effective cure forHPV. The prospects of the development of small molecule inhibitors as HPV antiviral is verypromising. Development strategy was based on the modulation of interactions between viralproteins or viral proteins with host proteins. This is supported by the advances in knowledgeabout HPV’s protein functions involved in their life cycle such as E1, E2, E6 and E7 proteins.Some antiviral molecule candidates have been found and need further studies to obtainderivatives with higher activity including acid biphenyl sulfonasetat (inhibitor ATPase E1),Indandione & repaglinide (inhibitor interaction E1-E2), etc

RESPON IMUN SELULER DAN HUMORAL MENCIT YANG DIIMUNISASI KANDIDAT VAKSIN DNA DENGUE BERBASIS GEN preM-E SEROTIPE 4 STRAIN INDONESIA

AbstrakInfeksi virus dengue (DENV) terkadang tanpa gejala atau dapat menunjukkan gejala klinis yang luas, berkisar dari sindrom flu ringan (dengue fever/DF), dengue haemorrhagic fever (DHF), hingga syok hipovolemik (dengue shock syndrome/DSS). Hipotesis yang berkaitan dengan tingkat keparahan infeksi DENV meliputi mekanisme antibody-dependent enhancement (ADE) dan keterlibatan sitokin. Hingga kini, belum ada obat antiviral yang efektif untuk mengeradikasi dan mencegah infeksi DENV, sehingga pencegahan berupa vaksin perlu dikembangkan. Kandidat vaksin DNA berbasis gen preM-E serotipe 4 strain Indonesia yang dikembangkan pada penelitian terdahulu disuntikkan ke mencit ddY, kemudian diuji tantang dengan DENV. Pada hari ke-4 dan ke-21 pascauji tantang, keberadaan sitokin IL-2 dalam serum dideteksi dengan metode ELISA. Serum hari ke-21 digunakan dalam uji ADE menggunakan sel K562. Sel limpa diambil pada hari ke-21 pascauji tantang, kemudian keberadaan IL-2 dan antibodi in vitro dideteksi dengan metode ELISA. Tingkat IL-2 tertinggi terdapat pada serum hari ke-4 pada kelompok mencit yang tidak diimunisasi namun diuji tantang, yaitu sebesar 69,83 pg/ml. Konsentrasi IL-2 terendah ditunjukkan oleh kelompok mencit yang diimunisasi namun tidak diuji tantang, yaitu 0 pg/ml. Pengukuran IL-2 pada serum dan supernatan sel limpa hari ke-21 tidak mendapatkan konsentrasi IL-2. Titer antibodi tertinggi terdapat pada kelompok sel limpa mencit yang diimunisasi, diuji tantang, dan diinduksi in vitro dengan DENV. Hasil uji ADE menunjukkan tingkat pengenceran serum berpengaruh terhadap jumlah sel yang terinfeksi oleh DENV, namun tidak ditemukan kondisi netralisasi dan enhancing. Berdasarkan metode yang digunakan, kandidat vaksin DNA tersebut dapat memicu respon imun seluler dan humoral.AbstractDengue virus (DENV) infection can be asymptomatic or cause wide range of clinical symptoms, from mild febrille ilness (dengue fever/DF), dengue haemorrhagic fever (DHF), to hipovolemic shock (dengue shock syndrome/DSS). Hypotheses related to the severity of DENV infection mechanisms including antibody-dependent enhancement (ADE) and cytokines involvement. Until now, there are no effective antiviral drugs can eradicate and prevent DENV infection, therefore the development of vaccines is the alternative. DNA vaccine candidate preM-E serotype 4 strain of Indonesia which was developed in previous studies injected into ddY mice, then challenge with DENV. At day 4 and 21 post-challenge, serum was taken to detect the presence of cytokines IL-2 using ELISA method. Day 21 serum used in the antibody-dependent enhancement (ADE) assay using K562 cell line. Splenocytes were taken at day 21 post-challenge to measure the presence of IL-2 and in vitro antibody using ELISA method. Measurement of IL-2 on day 4 serum produced the highest levels of IL-2 (69.83 pg/ml) in the group of non-immunized, challenged mice, whereas the lowest concentration (0 pg/ml) shown by the group of immunized, non-challenged mice. Measurement of IL-2 in serum and splenocytes day 21 did not get the concentration of IL-2. The highest result of in vitro antibody measurements shown by the group of splenocytes from immunized, challenged mice then in vitro induced with DENV. ADE assay results showed that level of serum dilution has effect on the number of dengue-infected cells, but netralization and enhancing condition were not found in this assay. Based on this methods, the DNA vaccine candidate can trigger cellular and humoral immune responses

Rapid diagnosis of dengue viremia by reverse transcriptase-polymerase chain reaction using 3\u27-noncoding region universal primers

A reverse transcriptase-polymerase chain reaction (RT-PCR) method was developed as a rapid diagnostic test of dengue viremia. To detect dengue viruses in serum or plasma specimens, a pair of universal primers was designed for use in the RT-PCR. Using these primers, the 3\u27-noncoding region of dengue virus types 1, 2, 3, and 4 could be amplified, but not those of other flaviviruses, such as West Nile virus, Japanese encephalitis virus, and yellow fever virus, or the alphavirus Sindbis virus. The sensitivity of the RT-PCR assay was similar to that of a quantitative fluorescent focus assay of dengue viruses in cell culture. Combining a silica method for RNA isolation and RT-PCR dengue virus could be detected in a 6-hr assay. In a preliminary study using this method, we detected dengue virus in 38 of 39 plasma specimens from which dengue virus had been isolated by mosquito inoculation. We then applied this method for detecting dengue viremia to 117 plasma samples from 62 children with acute febrile illnesses in a dengue-endemic area. We detected dengue viremia in 19 of 20 samples obtained on the day of presentation, which had been confirmed as acute dengue infection by mosquito inoculation and antibody responses. The overall sensitivity of this method was 91.4% (32 of 35; 95% confidence interval [CI] = 82.2-100%). The results from testing plasma samples from febrile nondengue patients showed a specificity of 95.4% (42 of 44; 95% CI = 89.3-100%)

Incubation of Denatured Samples Increased Reproducibility and Quality of Proteomic Profile of SELDI-TOF MS

Protein profiling with high-throughput proteomic technology, SELDI-TOF, is a new potential tool for diagnosis of human diseases. This advanced technique has increasingly been used for the detection of disease biomarker. However, analytical reproducibility is a significant challenge in SELDI-TOF profiling in order to have confidence in the results.  Here, we showed a simple step to improve its analytical performance. Incubation of denaturated samples overnight at 4oC increased significantly reproducibility and quality of proteomic profile of SELDI-TOF MS for IMAC30-Cu ProteinChip. This strategy could be concerned and apply to address reproducibility issue in this system.&nbsp

Incubation of Denatured Samples Increased Reproducibility and Quality of Proteomic Profile of SELDI-TOF MS

Protein profiling with high-throughput proteomic technology, SELDI-TOF, is a new potential tool for diagnosis of human diseases. This advanced technique has increasingly been used for the detection of disease biomarker. However, analytical reproducibility is a significant challenge in SELDI-TOF profiling in order to have confidence in the results. Here, we showed a simple step to improve its analytical performance. Incubation of denaturated samples overnight at 4o C increased significantly reproducibility and quality of proteomic profile of SELDI-TOF MS for IMAC30-Cu ProteinChip. This strategy could be concerned and apply to address reproducibility issue in this system

Some epitopes conservation in non structural 3 protein dengue virus serotype 4

AbstrakLatar belakang: Protein Non Struktural 3 (NS3) virus dengue menginduksi respon antibodi netralisasidan respon sel T CD4+ dan CD8+, serta berperan dalam replikasi virus. Protein NS3 memiliki epitopepitopsel T dan B yang terdapat perbedaan kelestarian pada berbagai strain virus dengue serotipe 4(DENV-4). Penelitian ini bertujuan untuk mengetahui kelestarian epitop sel T dan B pada protein NS3DENV-4 strain-strain dunia dan keempat serotipe virus dengue strain Indonesia.Metode: Penelitian ini dilakukan di Departemen Mikrobiologi Fakultas Kedokteran UI sejak Juni 2013 - April2014. Sekuens asam amino NS3 DENV-4 strain 081 didapatkan setelah produk PCR gen NS3 DENV-4 081disekuensing. Epitop-epitop sel T dan sel B protein NS3 DENV-4 081 dianalisis dan dibandingkan dengansekuens asam amino protein NS3 dari 124 strain DENV-4 di dunia dan keempat serotipe DENV strain Indonesia.Strain-strain dunia merupakan strain yang ada di benua Amerika (Venezuela, Colombia, dll) dan Asia (Cina,Singapura, dll). Referensi posisi epitop sel T dan B protein NS3 diperoleh dari laporan penelitian terdahulu.Hasil: Delapan epitop sel T dan 2 epitop sel B dari protein NS3 DENV-4 081 ternyata identik dan lestaripada protein NS3 dari 124 strain DENV-4 dunia. Epitop sel B di posisi asam amino 537-544 pada proteinNS3 DENV-4 081 ternyata identik dan lestari dengan epitop sel B protein NS3 dari keempat serotipeDENV strain Indonesia.Kesimpulan: Kelestarian yang luas dari epitop sel T dan B pada hampir seluruh strain DENV-4 dunia danserotipe-serotipe DENV strain Indonesia. (Health Science Journal of Indonesia 2015;6:126-31)Kata kunci: virus dengue, protein NS3, epitop sel T, epitop sel B AbstractBackground: Non Structural 3 (NS3) protein of dengue virus (DENV) is known to induce antibody, CD4+and CD8+ T cell responses, and playing role in viral replication. NS3 protein has T and B cell epitopes,which has conservation difference between DENV-4 strains. This study aimed to identify conservation ofT and B cell epitope in NS3 protein among DENV-4 strains and four serotypes DENV of Indonesia strains.Methods: Research was held at the Department of Microbiology, Faculty of Medicine, UniversitasIndonesia, June 2013 to April 2014. NS3 amino acid sequence of DENV-4 081 strain was obtained afterNS3 gene of DENV-4 081 PCR products were sequenced. T and B cell epitopes of NS3 protein of DENV-4081 strain were analysed and compared to NS3 proteins of 124 DENV-4 strains around the world and fourserotypes of Indonesia strains. World strains were isolated from America (i.e. Venezuela, Colombia, etc.)and Asia (i.e. China, Singapore, etc.). For the comparison, T and B cell epitope positions of NS3 proteinwere obtained from published report.Results: Eight positions of T cell epitopes and two positions of B cell epitopes of NS3 DENV-4 081 wereidentical and conserved to NS3 protein of 124 DENV-4 strains around the world. B cell epitope of NS3 DENV-4 081 protein at aa 537-544 was found identical and conserved to four serotypes DENV of Indonesia strains.Conclusion: This wide conservation of T and B epitopes in almost all DENV-4 strains around the worldand all serotypes of Indonesia strains. (Health Science Journal of Indonesia 2015;6:126-31)Keywords: dengue virus, NS3 protein, T cell epitope, B cell epitop

Multidrug Resistance and Extensively Drug-Resistance in Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus haemolyticus

Antimicrobial resistance in bacteria has become a leading global public health issue. Staphylococcus sp. has an efficient mechanism to deal with antimicrobial agents that make them hard to treat in hospital-acquired and community-acquired infections. This study was conducted due to limited data about multidrug resistance and extensively drug resistance in Staphylococcus sp. in Indonesia. This study was a descriptive retrospective study using a cross-sectional design to get the prevalence and antimicrobial susceptibility of S. haemolyticus, S. aureus, and S. epidermidis. The data was secondary data extracted from WHONET 2022 software. This study’s data were from bacteria from samples sent to UKK LMK FKUI, Jakarta from 2017 to 2021 for routine diagnostic. In this study, we found that the prevalence of methicillin-resistant S.aureus was 24,9%, methicillin-resistant S.epidermidis was 65,5%, and methicillin-resistant S.haemolyticus was 86,8%. The prevalence of MDR S.aureus is less than S.epidermidis and S.haemolyticus, respectively. MDR S.haemolyticus was consistently above 85% each year, while S.epidermidis was above 50% and S.aureus was below 50%. XDR Staphylococcus was only found in S.aureus and S.haemolyticus, i.e. three and seven XDR isolates of S.aureus and S.haemolyticus respectively during 2017-2021. Although we could not find any pan-resistant isolates from all samples, we found methicillin-resistant S.aureus and S.haemolyticus isolates that were also resistant to vancomycin and linezolid. S.haemolyticus dan S. epidermidis were an important coagulase-negative Staphylococcus species that can’t be neglected due to the high percentage of MDR and the discoveries of XDR in S.haemolyticus so that they have the potential to disseminate resistance plasmids to the more virulent bacteria. Therefore we need to control the use of antimicrobial agent to prevent this resistance

STUDI PEMETAAN AWAL DNA Mycobacterium tuberculosis COMPLEX SECARA Spoligotyping PADA HASIL ISOLASI DAHAK PASIEN TUBERKULOSIS PARU DARI 10 IBU KOTA PROPINSI (BAGIAN I)

Abstract. Mapping TB genotype of Mycobacterium tuberculosis (Mtb) is an important study to identify their distribution or characteristic and also may lead to improve control of the disease. This study conducted a preliminary mapping of the tubercle bacilli which had been circulating in Indonesia. Cultures of DNA isolated from TB patients at urban areas in 16 provinces in Indonesia, are chosen based on TB Case Detection Rate (CDR) 2006 from Indonesia Directorate General of Communicable Disease Control and Environment Health (Ministry of Health), were analyzed by spoligotyping for strain differentiation. In this first part, the analyzed result only came from urban areas in 10 provinces, i.e. Palembang, Bandar Lampung, Serang, Jakarta, Bandung, Surabaya, Banjarmasin, Makassar, Pontianak and Ambon. Sample were 269 DNA from 294 isolates collected from sputum of suspect TB patients with sputum-smear positive (SS+) and age above 15 years old. All samples were obtained from peripheral health laboratory in each province. The procedure collection is accordance to Indonesia DOTs guidelines (AFB smears) and samples were transported from those 10 areas to Bacteriology Laboratory at CBPRD. Sputum was taken for culture in liquid media MGIT Bactec 960 and solid media Lowenstein Jensen. The DNAs from positive liquid media MGIT Bactec 960 were isolated and analyzed by spoligotyping to identify the spoligo pattern. The spoligotyping results converted into octal format within Words & Excel spreadsheets and compared to International Spoligotype-database (SpolDB4). The previous study (Parwati et.al.) found some differences geographical distribution between Beijing genotype strain of tubercle bacilli in West Indonesia compare to East Indonesia, and the same pattern was also found in this study. Furthermore, the results in this study showed the differences in spoligo pattern of Mtb complex at 10 urban areas in West, Middle and East Indonesia. The percentage of Beijing strain family in the samples from West Indonesia showed around 26.61% (31.48% in Sumatra, 28.83% in Java and 16.98% in Kalimantan); from the Middle Indonesia around 25.93%; and none were found in samples from the East Indonesia. The SpolDB4 pattern also showed that the majority of isolates belonged to major clades of M.tuberculosis, i.e. the East African-Indian (EAI); Haarlem (H), Latin American-Mediterranean (LAM), the Central and Middle Eastern Asian (CAS); U = undefined; T = T family; and the MANU/ Manu family. We also found some isolates of Mycobacterium bovis. There were no significant association showed between genotype families and gender, but strong significant association found with ethnics and geography. Further confirmation of the results is still ongoing (k value; RFLP and MIRU-VNTR). As conclusion, this study constituted a first attempt to describe the preliminary mapping of genetic population structure of the tubercle bacilli circulating in Indonesia.   Key words: preliminary mapping, Mtb complex, spoligotyping, Beijing genotype strain, SpolDB

Microplate-reverse hybridization method to determine dengue virus serotype

A reverse transcriptase-polymerase chain reaction (RT-PCR) and microplate-reverse hybridization method were developed to detect and type dengue viruses in patients plasma specimens. A silica method was used to isolate RNA; and 3\u27-noncoding region universal primers were used to amplify dengue virus RNA. Using RT-PCR and ethidium bromide staining we could detect dengue virus in serum spiked with serially diluted dengue virus with a level of sensitivity similar to that of a quantitative fluorescent focus assay of dengue viruses in cell culture, i.e. 1.4 fluorescent focus units per reaction. Applying this assay to 14 dengue-positive plasma samples and 13 dengue-negative samples, dengue viremia was detectable by RT-PCR with a sensitivity comparable to mosquito inoculation. To determine the serotypes, digoxigenin-labeled PCR products from plasma samples and six laboratory adapted dengue viruses were hybridized in stringent conditions to serotype-specific DNA probes immobilized on microplates, and the hybridized product was detected with a colorimetric assay. Serotypes of dengue viruses, in cell culture and in patient plasma specimens, were identified using this method