UChicago CMSC 23320 - The Best Commit Messages of 2024
04_Prof Lia_Metode Uji Mikrobiologi untuk Obat, Obat tradisional F.pptx
1. Perkembangan Metode Uji Mikrobiologi untuk Obat,
Obat Tradisional, Kosmetik dan Suplemen Kesehatan
New Technology and Recent Update in Microbiology Testing
Marlia Singgih Wibowo
Sekolah Farmasi ITB
25 Juni 2020
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2. Metode uji mikrobiologi di bidang Farmasi
Untuk Obat : Berdasarkan Farmakope Indonesia edisi 5 tahun 2014, USP, BP, EP edisi
terbaru
Untuk Obat Tradisional : Peraturan Ka BPOM RI no.12 thn 2014, no.32 thn 2019
Untuk Suplemen Kesehatan : mulai USP 40 ttg Dietary Supplement Chapter
2021,2022,2023 ; perka bpom no.17 thn 2019
Untuk Kosmetika : Peraturan Menteri Kesehatan no. 1175 thn 2010, perka bpom
no.34 thn 2013, no.23 thn 2019
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3. Uji uji dan Informasi yang berhubungan dengan Mikrobiologi dalam
Farmakope Indonesia edisi V 2014
• <51> Uji Batas Mikroba
• <61> Uji Efektivitas Pengawet
• <65> Uji Kinerja Resistensi Indikator Biologi
• <71> Uji Sterilitas
• <81> Desain dan Analisis Penetapan Hayati
• <91> Penetapan Aktivitas Vitamin B12
• <121> Penetapan Kadar Kalsium Pantotenat
• <131> Penetapan Potensi Antibiotik Secara Mikrobiologi
• <201> Endotoksin bakteri
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4. • <1321> Indikator biologik untuk Sterilisasi
• <1352> Praktek Laboratorium Mikrobiologi yang baik
• <1371> Sterilisasi dan Jaminan Sterilitas pada Suatu Sediaan
• <1381> Validasi prosedur dalam Farmakope
• <1382> Verifikasi Prosedur dalam Farmakope
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Uji uji dan Informasi yang berhubungan dengan Mikrobiologi
dalam Farmakope Indonesia edisi V 2014
5. Persyaratan mikrobiologi umum produk obat, obat
tradisional, suplemen kesehatan, kosmetik
• Batas ALT (Angka Lempeng Total/TAMC)
• Batas AKK (Angka Kapang Khamir/TYMC)
• Batas mikroorganisme tertentu (E.coli, Staphylococcus, Pseudomonas,Candida,
Salmonella, Shigella (OT), dll)
• Angka mikroba tertentu (Enterobacteriaceae, dll)
• Batas cemaran toksin dari mikroba tertentu (mis.Aflatoksin utk OT)
• Parameter tertentu untuk produk farmasi steril (sterilitas, batas endotoksin, pirogen,
endotoksin, dll)
• Identifikasi mikroorganisme tertentu (mis.utk produk SK mengandung probiotik)
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6. Mengapa diperlukan “rapid method” dalam Microbiological Assay?
Namun untuk obat (bahan obat, eksipien, produk intermediate dan produk akhir)
persyaratan harus berdasarkan Farmakope Indonesia dan atau acuan kompendial lainnya.
“Rapid Method” diperlukan untuk tujuan konfirmasi, misalnya melalui new technology dalam
proses identifikasi
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Beberapa keuntungan dari metode yang sudah ada antara lain : dari segi
kemudahan pengerjaan, kemudahan perhitungan, tidak memerlukan
peralatan yang mahal
Kerugian /kekurangan Traditional microbiological methods untuk deteksi,
enumerasi, dan identifikasi yang menggunakan metode kultur seringkali time-
consuming dan labor-intensive, kurang akurat, range acceptance yang lebar
7. Hal yang perlu diperhatikan untuk Rapid method untuk identifikasi bakteri
(1) Apakah rapid identification for bacteria sangat diperlukan?
(2) Kebutuhan akan deteksi atau identifikasi cepat: identifikasi fenotype atau genotype?
perlukah molecular identification?
(3) Membandingkan keunggulan dan kekurangan dari culture-based dibandingkan
dengan molecular methods.
(4) User friendly techniques dalam proses uji
(5) Penggunaan metode baru yang sederhana misalnya : Chromogenic and fluorogenic
enzyme substrates for bacterial detection.
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Indonesia
8. Why we need rapid method for identification
• Diperlukan hasil uji yang cepat tetapi juga tepat
• Meningkatnya prevalensi mikroorganisme yang resisten terhadap
antibiotik, sehingga diperlukan metode analisis yang rapid dan akurat
• Menjamin produk akhir yang aman, berkualitas, berkhasiat (safety,
quality,efficacy)
• The majority of nosocomial infections are caused by the ESKAPE
(Enterococcus faecium, Staphylococcus aureus, Klebsiella
pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa,
and Enterobacter species) organisms, (Chemical Society Review, issue 16, 2017)
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9. Kelebihan dan kekurangan rapid method
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Kelebihan
• Dapat mendeteksi kontaminan lebih awal, reject lebih awal dan lebih
cepat mengambil keputusan utk CAPA
• Improve sensitivity dan selectivity
• Meningkatkan kontrol dan konsistensi pada proses produksi
Kekurangan
• Investasi Peralatan yang “mahal”
• Kerumitan dalam validasi
• Beberapa metode cukup kompleks
10. Area yang memungkinkan implementasi rapid
microbiological method (RMM)
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• Environmental monitoring on clean rooms
• Microbiological control of raw materials
• Water testing
• finished non-sterile product testing
• sterility test
• antimicrobial effectiveness tests
• RMM applications regarding environmental monitoring consist of air
monitoring, isolator integrity and surfaces monitoring
11. How to improve method for identifying bacteria
• using a combination of morphological features (e.g. colony size and
colour), microscopy (e.g. by Gram stain) and rapid biochemical tests
(e.g. for catalase and/or oxidase activity) using simple reagents.
• Identification to species level (e.g. S. aureus) is then performed using
targeted biochemical or serological tests (e.g. latex agglutination
tests), because the different species within a genus can have very
different pathogenicities and resistance profiles
• One approach to high specificity is, for example, to use DNA
detection to provide genomic data in molecular diagnostic
techniques). The use of PCR (polymerase chain reaction) to amplify
the DNA present in a sample has transformed gene-based assays and
overcomes the major challenge of rapid testing of samples
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12. Contoh identifikasi mikroba dengan media selektif
Salmonella sp
Salmonella sp
Staphylococcus sp
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13. Penggunaan media kromogenik utk identifikasi Pseudomonas aeruginosa
β-alanyl pentylresorufamine
resorufamine
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16. Syarat Rapid method in Microbiology
• Rapid
• Sensitive
• Broad spectrum detection
• Potential for specificity
• Identification
• Viability assessment
• Simple
• Potential for automation
• Reproducible
• Compatible with sample matrices
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17. USP general chapter [2] introduces four alternatives to the demonstration
of equivalence of rapid microbiological method (RMM) :
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(i) acceptable procedures, where a standard inoculum of a specific
microorganism is used,
(ii) equivalence of performance, where validation parameters are
compared with the compendial methods,
(iii) equivalence of results, that deals with comparison of numerical
results or correlation with results obtained by compendial
methods, and
(iv) decision of equivalence, for qualitative methods, where the result
absence or presence of bacteria is compared.
18. Contoh new method in Microbiological Assay
• Fluorescent labeling : Stain microorganisms using a viability-indicating
fluorophore; direct enumeration, usually after filter capture, by light
excitation（epifiuorescent microscopy or laser scanning）and image
analysis
• ATP bioluminescence : Light emission from microbial ATP by
luciferin/luciferase reaction. Amenable to amplification by intracellular
adenylate kinase
• Carbon dioxide detection : Monitoring of microbial metabolism using
14C-radiolabelled substrate to produce 14C-labelled carbon dioxide.
Infrared CO2 detection offers a more acceptable substitute
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20. ATP Assay
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ATP-bioluminescence -
Adenosine triphosphate (ATP)
bioluminescence is a well
established rapid method for
assessing contamination levels
in pharmaceutical products and
raw materials.
21. Chromatographic analysis : Detection of microbial metabolites and cellular
components; gas chromatographic analysis of microbial fatty acid has been employed
in identification
Dye reduction : Monitoring microbial metabolism of specified substrates by color
changes in redox dyes; can form the basis of identification profile
Electrical resistance : Measurement of electrical changes (conductance, impedance )
in specialized media due to microbial growth;
Enzyme monitoring : Detection of microbial enzymes. By using appropriate substrates
can form the basis of identification profiles
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22. Cont’d
Limulus amoebocyte lysate : Detection of endotoxin from Gram-negative bacterial
lipopolysaccharide by gelation or colorimetric reaction
Nucleic acid probes : Labeled DNA or RNA probe hybridization to specific target
sequences. Amplification of target by the polymerase chain reaction(PCR) increases
sensitivity; competitive quantitative PCR offers enumeration
Phage-interaction technology : Host-specific bacteriophage infects target cells
leading to phage DNA replication. Detection by expression of new protein (using
recombinant phage) or cell lysis
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29. Matrix assisted laser desorption/ionization-time of
flight mass spectrometry (MALDI-TOF MS)
In MALDI-TOF MS the sample is treated with a matrix, which absorbs energy from a laser,
resulting in rapid heating, vaporization, and the ionization of the analytes; the ions are
then separated on the basis of the time they take to reach the detector, as all ions of the
same charge are given the same kinetic energy.
In practice, the majority of bacterial molecules observed by MALDI-TOF MS are ribosomal
proteins, with characteristic masses for the proteins of particular bacteria giving rise to
‘fingerprint’ MS spectra (a peptide mass fingerprint [PMF]) that can be compared to an
online MS database to identify specific genera and species.
The identification of subspecies and strains depends upon the availability of extensive
databases, while the use of whole/intact cells has resulted in improvements to
reproducibility in comparison to earlier methods, which required protein extraction and
analysis to be performed under standardized conditions.
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