ERUDITIO Vol.
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ERUDITIO Vol.
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Optimization and Validation of Analytical Method for Detection of Shigella sp.
in Oral Preparations of Quasi-Drugs in the Form of Lozenges Desty Herawati a,1,*.
Eko Apriantob,2.
Eni Cahyaningsiha,3.
Nur Miftahurrohmahc,4 Indonesian Food and Drug Authority.
Jl.
Percetakan Negara No.
Jakarta Pusat, 10560.
Indonesia Indonesian Food and Drug Authority Regional Office in Banda Aceh.
Indonesian Food and Drug Authority.
Jl.
Daud Beureueh No.
Banda Aceh, 23126.
Indonesia Microbiology Laboratory.
Faculty of Pharmacy.
Universitas Pancasila.
Srengseng Sawah.
Jagakarsa.
Jakarta Selatan, 12640.
Indonesia herawati@pom.
2 eko.
aprianto@pom.
cahyaningsih@pom.
4nur_miftahurrohmah@univpancasila.
*corresponding author
ARTICLE
INFO
Article history Received:
October 31, 2024 Revised:
September 21.
Accepted:
October 02, 2024 DOI:
https://doi.
org/10 54384/eruditio.
ABSTRACT / ABSTRAK
Lozenges, a quasi-drug used to relieve sore throats, must meet the safety and quality requirements according to Indonesian FDA Regulation No.
7 of 2023 concerning the criteria and procedures for quasi-drug registration, which stipulates that oral preparations of quasi-drugs must be free from Shigella sppAi microbial contamination.
Contamination by Shigella bacteria can cause diseases such as shigellosis, characterized by symptoms such as diarrhoea, vomiting, and This study aims to validate the Shigella sppAidetection method in quasidrug lozenges using the WHO (Quality Control Methods for Herbal Material.
reference method.
Validation was carried out to ensure that this method can be used for routine analysis in the Indonesian FDA laboratory.
The guidelines used to validate the microbiological method are the Singapore Accreditation Council (SAC) Guidance Notes C&B AND ENV 002 of 2019 and refer to The study results showed that the WHO (Quality Control Methods for Herbal Material.
method in detecting Shigella sp.
has a sensitivity and specificity of 100%, with false positive and false negative rates of 0% each.
The detection limit obtained was 3 cfu/g, which meets the requirements .
elow 10 cfu/.
The method suitability test .
showed that the initial solvent used for sample homogenization in Shigella detection was sufficient using Tryptone Soya Broth .
without additional neutralization or modification procedures at the sample homogenization stage.
Based on these results, the Shigella spp.
The WHO reference (Quality Control Methods for Herbal Material.
detection method is accurate.
It can be applied for routine microbial contamination testing analysis in the Food and Drug Supervisory Agency laboratories.
Tablet hisap obat kuasi yang digunakan untuk meredakan sakit tenggorokan harus memenuhi persyaratan keamanan dan mutu sesuai Peraturan Badan POM No.
7 Tahun 2023 tentang kriteria dan tata laksana registrasi obat kuasi, yang menetapkan bahwa sediaan oral obat kuasi harus bebas dari cemaran mikroba Shigella spp.
Kontaminasi oleh bakteri Shigella dapat menyebabkan penyakit seperti shigellosis yang ditandai dengan gejala seperti diare, muntah, dan Penelitian ini bertujuan untuk memvalidasi metode deteksi Shigella spp.
pada tablet hisap obat kuasi menggunakan metode acuan WHO (Quality Control Methods for Herbal Material.
Validasi dilakukan untuk memastikan metode ini dapat digunakan untuk analisis rutin di laboratorium BPOM.
Pedoman yang digunakan untuk memvalidasi metode mikrobiologi adalah Singapore Accreditation Council (SAC) Guidance Notes C&B AND ENV 002 Tahun 2019 serta farmakope.
Hasil penelitian menunjukkan bahwa metode WHO (Quality Desty Herawati.
Eko Aprianto.
Eni Cahyaningsih.
Nur Miftahurrohmah Optimization and Validation of Analytical Method for Detection of Shigella sp.
in Oral Preparations of Quasi-Drugs in the Form of Lozenges ERUDITIO Vol.
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Control Methods for Herbal Material.
dalam mendeteksi Shigella sp.
sensitivitas dan spesifisitas sebesar 100%, dengan tingkat positif palsu dan negatif palsu masing-masing 0%.
Limit deteksi yang diperoleh adalah 3 cfu/g, yang memenuhi persyaratan .
i bawah 10 cfu/.
Dari uji kesesuaian metode .
yang dilakukan menunjukkan bahwa pelarut awal yang digunakan untuk homogenisasi sampel pada deteksi Shigella cukup menggunakan Tryptone Soya Broth .
tanpa perlu prosedur penetralan atau modifikasi tambahan pada tahapan homogenisasi sampel.
Berdasarkan hasil ini, metode deteksi Shigella spp.
menggunakan metode acuan WHO (Quality Control Methods for Herbal Material.
dinyatakan akurat dan dapat diterapkan untuk analisis rutin pengujian cemaran mikroba di laboratorium-laboratorium Badan Pengawas Obat dan Makanan.
Keywords: Method validation.
Shigella, quasi-drug, lozenges, microbiological examination of non-sterile product.
SAC-Singlas Kata Kunci: Validasi metode.
Shigella, obat kuasi, tablet hisap, pengujian mikrobiologi produk nonsteril.
SAC-Singlas Introduction According to Indonesian FDA regulation No.
7 of 2023, quasi-drugs are preparations containing active ingredients with pharmacological effects that are non-systemic or local to treat minor complaints.
Quasi-drug preparations consist of 2 types, namely topical and oral.
Various kinds of quasi-medicinal products are circulating in Indonesia, so monitoring these products' physical, chemical and biological safety aspects is necessary.
Quasi-drugs circulating in Indonesia must meet the safety and quality requirements of finished quasi-drug products listed in the latest Indonesian FDA regulation No.
7 of 2023 concerning criteria and procedures for registration of quasi-drugs.
One of the requirements in the regulation states that oral quasi-medicinal products must not contain Shigella contamination.
Based on the website .
ttps://cekbpom.
id/obat_kuasi, accessed on October 23, 2.
, the Indonesian FDA has issued 12 quasi-medicinal products in the form of lozenges.
Adults and children widely use quasi-medicinal lozenges to treat minor throat complaints (Rathod et al.
, 2.
Quasi-medicinal lozenges commonly circulated in the market are generally complex candy types, also known as lozenges.
Hard candy-type lozenges have hygroscopic properties, so if not packaged properly, they will increase the water content in the lozenges, which can potentially cause the growth of microorganisms (Choursiya & Andheriya, 2.
Microbial contamination in quasi-medicinal products can potentially reduce or even activate therapeutic activity and harm one's health (Aini et al.
, 2.
Shigella bacteria are capable of producing toxins and causing diseases such as dysentery, diarrhoea, and even Shigella is a gram-negative bacterium in the Enterobacteriaceae family that causes Shigellosis (Muzembo et al.
, 2.
Shigellosis is a disease caused by Shigella species bacteria that infect the intestinal tract and rectum (Kotloff et al.
, 2.
In a study in East Africa.
Salmonella and Shigella were the main pathogenic bacteria most commonly reported as contaminants in traditional medicines (Walusansa et al.
, 2.
Shigella was a bacterial contaminant in traditional medicine samples studied in Nairobi.
Kenya (Korir et al.
, 2.
Shigellae are highly infectious.
the infective dose is about 103 organisms compared to 105108 for Salmonella and Vibrio (Brooks et al.
, 2.
Quasi-medicinal products before circulating to the market and those that have circulated in the market must meet safety, efficacy and quality requirements.
One safety requirement Desty Herawati.
Eko Aprianto.
Eni Cahyaningsih.
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Detection ofEko Shigella in Oral of Quasi-Drugs in the Form of Desty Aprianto.
EniPreparations Cahyaningsih.
Nur Miftahurrohmah Lozenges Optimization and Validation of Analytical Method for Detection of Shigella in Oral Preparations of Quasi-Drugs in the Form of Lozenges ERUDITIO Vol.
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that oral quasi-drugs must meet is that they must not contain Shigella microbial this can be proven through Shigella detection testing in the laboratory.
Therefore, the laboratory needs a validated analytical method to detect microbial Method validation is an essential factor in obtaining analytical results that are valid, reliable, and can be scientifically accounted for by the intended use (Faridah et al.
In 2023, the Food and Drug Administration did not have a test method for detecting Shigella quasi-drugs in lozenges.
Indonesian FDA only has a validated Shigella detection analysis method (MA PPOMN No.
52/MI/.
for testing traditional medicine samples because there were no regulations requiring microbial contamination limits for finished quasi-medicine products.
According to ISO 17025:2017, the laboratory must validate the reference method used if it is used outside the intended scope, outside its designation, or In this study, the WHO issued the reference method for Shigella detection analysis on quality control methods for herbal materials (World Health Organization, 2.
The method issued by WHO is intended for herbal/traditional medicine samples, so if the laboratory wants to apply the technique to quasi-medicine samples, validation testing of the method is needed before use.
This study focuses on validating Shigella sp detection methods in quasi-medicinal products in the form of lozenges using conventional culture-based methods based on the methods recommended by WHO.
The selection of this method considers various factors such as cost, equipment, reagents/media, human resources, as well as accommodation and environmental conditions in the Indonesian FDA Provincial Offices throughout Indonesia.
In addition, based on the United States Pharmacopeia Convention (USP) 2022 in appendix <1223>, if there are differences in test results between alternative and conventional microbiological methods, the traditional/culture method is still used as a gold standard However, recent developments have shown that several alternative molecular based (DNA) methods offer higher speed and accuracy in detecting Shigella.
For example, a study by Yang et al.
compared the performance of conventional PCR, real-time PCR, and droplet digital PCR .
dPCR) in detecting Shigella in food, where ddPCR showed the highest sensitivity with a detection limit as small as 0.
1 cfu/mL.
Another rapid method is real-time Loop-Mediated Isothermal Amplification (LAMP), which, according to a study by Liew et .
, can detect Shigella within 90 minutes.
Still, it has a relatively large detection limit 9 x 10^5 cfu/mL.
In this study, the culture-based WHO method will be validated to prove that the process meets the requirements of the predetermined validation parameters.
This study aims to produce a valid analytical method to detect Shigella contamination in quasi-oral drug samples in the form of lozenges.
A valid analytical process is expected to protect the public from products that pose health risks, and this method can later be used routinely in testing Methodology Time and Place of Research This research was conducted at the Microbiology and Molecular Biology Laboratory.
National Food and Drug Testing Development Center.
Indonesian FDA.
The study was conducted from February to March 2023.
Desty Herawati.
Eko Aprianto.
Eni Cahyaningsih.
Nur Miftahurrohmah of Eni Analytical Method for Nur Detection of Shigella sp.
in Oral Preparations of Quasi-Drugs in the Form of DestyOptimization Herawati, and EkoValidation Aprianto.
Cahyaningsih.
Miftahurrohmah Optimization and Validation of Analytical Method for Detection of Shigella sp.
in Oral Preparations of Quasi-Drugs in the Form of Lozenges Lozenges ERUDITIO Vol.
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Materials and Research Instruments The materials used in this study include Tryptone Soy Broth (TSB).
Enterobacteriaceae Enrichment Broth Mossel (EEB-Mosse.
Mac Conkey Agar (MCA).
Xylose Lysine Desoxycholate Agar (XLD).
Kligler Iron Agar (KIA).
Tryptone Soy Agar (TSA).
Buffered Sodium Chloride-Pepton Solution pH 7.
0, physiological NaCl solution 0, 9%.
Mc Farland standard.
GN VITEKA card, quasi oral lozenges that have Indonesian FDA marketing license with the same batch number, raw microbes Shigella sonnei ATCC 9290.
Shigella dysentriae ATCC 13313.
Shigella flexneri ATCC 9199.
Escherichia coli ATCC 8739 and Salmonella typhimurium ATCC 14028.
The tools used in this study include a set of glassware, oven, autoclave sterilization and deconstruction, incubator, microscope, pH meter, top loading scale, stomacher.
Ose needle.
Ose incinerator.
Biosafety Cabinet (BSC), vortex mixer, micropipette and pipette tipsAi biochemical identification of bacteria using VITEKA 2 System instrument.
Preparation of Raw Microbial Cultures Standard commercially available microbes such as ATCC (American Type Culture Collectio.
or other collection cultures are used.
Standard microbes used for validation should not exceed 5 passages (United States Pharmacopeia Convention, 2.
The standard microbes Shigella sonnei.
Salmonella typhimurium and Escherichia coli used in this study used the National Food and Drug Testing Development Center standard microbial lyophilization derived from the master MicrobiologicsA ATCC pasase-2 culture.
Observation of conformity and Selective Media incubation 35-370 C MASTER CULTURE a, b (Passage Ae .
BHIB
Incubation 35-370 C
TSA
(Passage-.
Selective Media ( ) 3 mL of sterile 0,9% NaCl incubation 35-370 C Observation of conformity and TSA LYOPHYLIZATE c,d,e (Passage-.
LYOPHILIZATION (Freeze Dr.
(Standard Microbial Stock Cultur.
(Passage-.
Shigella dysentriae ATCC 13313, b Shigella flexneri ATCC 9199, c Shigella sonnei ATCC 9290, d Escherichia coli ATCC 8739, e Salmonella TyphimuriumATCC 1402 Figure 1.
Schematic of Raw Microbial Culture Preparation Desty Herawati.
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Nur Miftahurrohmah Optimization and Validation of Analytical Method for Detection of Shigella sp.
in Oral Preparations of Quasi-Drugs in the Form of Desty Herawati.
Eko Aprianto.
Eni Cahyaningsih.
Nur Miftahurrohmah Lozenges Optimization and Validation of Analytical Method for Detection of Shigella sp.
in Oral Preparations of Quasi-Drugs in the Form of Lozenges ERUDITIO Vol.
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The preparation of standard microbial cultures that will be used for validation is illustrated in Figure 1.
Indonesian FDA standard microbial lyophilization .
was dissolved by adding 0.
9% physiological NaCl and then waited for 10-15 minutes.
It was then streaked onto several TSA and selective media, such as MCA and XLD, to observe its Colonies that grow on TSA media .
can be used as raw microbial cultures for spiking.
Sample Preparation The samples used were complex candy-type lozenges with Indonesian FDA marketing authorization on the market.
The lozenges were aseptically crushed using a sterile mortar and pestle until smooth, then weighed 10 g into a sterile stomacher bag.
Shigella Detection Analysis Using WHO Reference Method Shigella detection was performed using the reference method from WHO .
Quality Control Methods for Herbal Materials listed in Chapter 18: Determination of Microorganisms.
The test began with sample preparation, followed by adding 90 mL of TSB as an enrichment medium, then homogenized using a stomacher and incubated at 30-37AC for 2-5 hours .
re-enrichment stag.
From the pre-enrichment suspension, 10 mL was pipetted into 90 mL of EEB-Mossel, then incubated at 35-37AC for 18-24 hours.
One ose of the enrichment results (EEB-Mosse.
was inoculated on the surface of XLD and MCA media and then incubated at 35-37AC for 18-24 hours.
Observations were made of the specific colonies that grew.
Cultures were considered Shigella positive if there were colonies with the following characteristics:
- On XLD: small round, red, 1-2 mm in diameter.
- On MCA: convex round, colourless, 2-3 mm diameter.
If no colonies grow on MCA and XLD, then Shigella is negative.
Specific colonies were propagated by inoculating 3 colonies onto TSA/NA slant, incubated at 35-37AC for 24 hours for a confirmation test.
The growing colonies were confirmed through Gram staining.
KIA/TSIA biochemical media, and automatic bacterial identification using VITEK 2 Compact with GN reagent cards.
The following Shigella detection analysis procedure in schematic form is presented in Figure 2.
Desty Herawati.
Eko Aprianto.
Eni Cahyaningsih.
Nur Miftahurrohmah of Eni Analytical Method for Nur Detection of Shigella sp.
in Oral Preparations of Quasi-Drugs in the Form of DestyOptimization Herawati, and EkoValidation Aprianto.
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Figure 2.
Shigella Detection Analysis Test Scheme (World Health Organization, 2.
Preparation of contaminant inoculum Preparation of bacterial suspensions for contamination is based on the calculation of the number of target bacteria (Shigella sonnei.
Shigella dysentriae.
Shigella flexner.
using the Total Plate Numbers method with 1 McFarland standard or UV-Vis Spectrophotometer (% Transmittanc.
580 nm.
One Ose of standardized microbial culture was inoculated onto TSA media and incubated at 37AC for 24 hours.
The growing colonies were suspended in 9% physiological NaCl solution until the turbidity reached 1 McFarland.
The number of colonies was counted using the Total Plate Count technique.
This standardized microbial suspension was inoculated into the sample preparation with a volume according to the required concentration level.
Desty Herawati.
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Nur Miftahurrohmah Optimization and Validation of Analytical Method forHerawati.
Detection ofEko Shigella in Oral of Quasi-Drugs in the Form of Desty Aprianto.
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Method Conformance Test (Optimizatio.
The suitability test will be conducted for each new product or sample using microbiological methods.
This test aims to ensure that the product or sample does not interfere with detecting the analysis results.
The test is performed by analyzing negative samples .
amples without contaminatio.
, positive samples .
amples contaminated with Shigella sp.
standard microbe.
, and positive controls .
olvents without samples contaminated with Shigella sp.
standard microbe.
Positive samples and positive controls are contaminated with a standardized microbial inoculum of no more than 100 cfu and then tested according to the reference analytical method with the shortest incubation period.
The positive samples' and controls' test results must be positively detected according to the indicative reaction described in the reference method.
The suitability test is declared successful if the positive sample and positive control can grow Shigella sp.
microbes while the negative sample shows no growth of Shigella sp.
Measurement or Data Collection Validation of the Shigella test method refers to the microbiology method validation guide (Singapore Accredited Council, 2.
The laboratory must meet acceptance parameters, including 100% sensitivity and specificity and 0% false positive and negative rates.
addition, the method's detection limit (LOD) must be below 100 cfu.
Determination of Limit of Detection The detection limit was determined by analyzing samples spike at low concentrations of A1, 3, 9 cfu/g.
Each concentration level was tested for 6x replicates using the reference The limit of detection was determined by observing the response that gave all positive results from 6x replicates at the slightest concentration.
The Singapore Accredited Council validation guidelines, 2019, only mention that it must be detectable below 100 cfu, which means that if the sample weighing is 10 g, the lowest concentration will be below 10 cfu/g.
The most minor concentration is 1 cfu/g, while 3 and 9 are multiples of 3.
Determination of Sensitivity.
Specificity.
False Positive & Negative Rate Determination of sensitivity, specificity, false positive and false negative rates was carried out according to validation guidelines (Singapore Accredited Council, 2.
by analyzing negative samples, samples spike and positive controls.
As shown in Figure 3, the test scheme for sensitivity, specificity, and false positive and false negative rates was carried out by contaminating samples spike and positive controls using microbial inoculum according to the concentration obtained from determining the limit of detection.
Samples spike and positive controls were contaminated with three and two non-target microbial Tests were performed using the reference analytical method, with six replicates for samples and two for controls.
Negative samples containing only crushed lozenges and diluted with TSB without microbial inoculum were tested six times.
- Negative sample = sample without the addition of any contaminants - Sample positive spike = sample contaminated with target standard microbes (Shigella sonnei.
Shigella dysentriae.
Shigella flexner.
and non-target standard microbes (Escherichia coli.
Salmonella Typhimuriu.
- Positive control = Solvent media contaminated with target standard microbes (Shigella sonnei.
Shigella dysentriae.
Shigella flexner.
and non-target standard microbes (Escherichia coli.
Salmonella Typhimuriu.
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Miftahurrohmah Optimization and Validation of Analytical Method for Detection of Shigella sp.
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Negative control = media blank a o a C Description: The difference in treating negative samples, approving samples, and positive controls to determine sensitivity, specificity, and false positive and negative favourable rates lies in the sample homogenization or initial dilution stage.
Negative sample = 10 g sample 90 mL TSB .
x replicate.
Target Sample Spike = 10 g sample 90 mL TSB target microbial inoculum (Shigella sonnei.
Shigella dysentriae.
Shigella flexner.
x replicate.
Non-Target Sample Spike = 10 g sample 90 mL TSB non-target microbial inoculum (Escherichia coli and Salmonella Typhimuriu.
x replicate.
Target Positive Control = 100 mL TSB target microbial inoculum (Shigella sonnei.
Shigella dysentriae.
Shigella flexner.
x replicate.
Non-target Positive Control = 100 mL TSB non-target microbial inoculum (Escherichia coli and Salmonella Typhimuriu.
x replicate.
Figure 3.
Test Scheme of Sensitivity.
Specificity.
False Positive and Negative Rate 8 Data Analysis Data analysis was performed using descriptive statistical analysis.
A descriptiveanalytical method is a method that serves to describe or give an overview of the object under study through data or samples that have been collected as is without analyzing and making conclusions that apply to the public (Sugiyono, 2.
Validation categories and calculation formulas refer to Tables 1 and 2 (Singapore Accredited Council, 2.
Table 1.
Validation Categories (Singapore Accredited Council, 2.
Number of test Confirmed ( ) Confirmed (-) ( ) Presumptive (-) Description: n = total number of tests Category a = number of confirmed positive presumptive positives .
rue positive.
Category b = number of presumptive negatives confirmed positive .
alse negative.
Category c = number of presumptive positive confirmed negatives .
alse positive.
Category d = number of confirmed negative presumptive negatives .
rue negative.
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After calculating the number of categories a, b, c, and d, input the category using the calculation formula according to Table 2.
to determine whether the method validation we have done meets the acceptance requirements.
Table 2.
Calculation Formula Parameter Calculation Formula Sensitivity a/.
x 100% Specificity d/.
x 100% False positive rate c/.
x 100% False negative rate d/.
x 100% Results and Discussion Method suitability test .
The method suitability test is carried out to ensure that no product/sample effect will obscure the analysis results (United States Pharmacopeia Convention, 2.
The results of the method suitability test for Shigella detection analysis using TSB solvent media .
with a concentration of 50 cfu/g are presented in Table 3.
Table 1.
Method Conformance Test Results Test Microbe Inoculum Level Shigella sonnei Shigella flexneri Shigella dysentriae 50 cfu/g 50 cfu/g 50 cfu/g Positive Control Positive Sample Negative Sample Description:
( ) = there is growth (-) = no growth The results of the method suitability test in Table 3.
show that all Shigella sp.
microbes, namely Shigella sonnei.
Shigella flexneri, and Shigella dysenteriae with an inoculum level of 50 cfu/g .
elow 100 cfu/.
, showed growth ( ) in the positive control and positive samples.
This indicates that the method used can detect the presence of these microbes under the specified conditions.
In the negative sample, there was no microbial growth (-), meaning there was no contamination or unwanted microbial growth under supposedly sterile conditions.
This indicates that the test method works well and is reliable in detecting or identifying the tested microbes as intended.
Thus, the analysis of Shigella detection in lozenge samples analyzed using the WHO method with the initial solvent TSB .
met the method's suitability requirements without needing prior neutralization/modification procedures for the sample.
Microbiological method suitability requirements, as listed in the USP pharmacopoeia guidelines, usually cover several essential aspects, including the ability of the method to detect the growth of target microorganisms under appropriate conditions without interference from external factors such as the physicochemical properties of the sample.
In this context, the starting Desty Herawati.
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solvent used was TSB at a ratio of 1:10, a commonly used enrichment medium in microbiology to support the growth of various microorganisms, including Shigella.
This method is said to meet the suitability requirements of the technique because:
- Appropriate Growth (Recover.
: One of the main criteria of microbiological method validation is the growth of the target microorganism in positive controls and positive In this case, the growth of Shigella in both the control and sample indicates that there are no inhibiting factors from the sample that affect the detection results.
- No Neutralization or Modification Required: Usually, in some cases, samples with specific physicochemical properties may inhibit the growth of microorganisms, so neutralization or modification of the procedure is required.
However, in this case, the lozenge sample does not require any additional adjustments, which means that the standard method used is optimal for detecting Shigella.
- Proven Method Suitability: Validation was performed by ensuring that the method could detect Shigella in positive controls .
ithout the influence of the sampl.
and positive samples .
ontaining both the sample and the target microorganis.
The method was qualified without additional modification when consistent growth was observed in both Therefore, this validation showed that the WHO method effectively detected Shigella in lozenge samples and met the requirements per international standards.
Limit of Detection (LOD) Determination A test with a limit of detection was conducted to determine the sensitivity of the test The detection limit was determined by analyzing positive samples from various low inoculum concentrations of 1, 3, and 9 cfu per gram.
The LOD value was determined based on the lowest concentration of test microbial inoculum in the sample, which could still be detected in all replicates.
Table 2.
Detection Limit Determination Test Results Contaminant Concentration Sample S.
sonnei 9 cfu/g Sample S.
sonnei 3 cfu/g Sample S.
sonnei 1 cfu/g Number of positive results / Number of tests Positive Response 83,3 % Based on Table 4, samples contaminated with Shigella sonnei at concentrations of 9 and 3 cfu/g showed positive results in all 6 test samples .
%).
However, at a concentration of 1 cfu/g, only 5 out of 6 test samples .
3%) gave positive results, while 1 other sample did not detect the presence of Shigella sp.
Desty Herawati.
Eko Aprianto.
Eni Cahyaningsih.
Nur Miftahurrohmah Optimization and Validation of Analytical Method forHerawati.
Detection ofEko Shigella in Oral of Quasi-Drugs in the Form of Desty Aprianto.
EniPreparations Cahyaningsih.
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Positive response (%) Determination of Shigella Detection Limit
LOD
Microbial concentration.
Figure 1.
Determination of Limit of Detection Based on the detection limit concentration determination test (Figure .
, the LOD value of the Shigella spp.
The detection method was obtained at 3 cfu/g sample.
This concentration is the lowest, giving 100% of the test microbes detected.
According to the Indonesian Pharmacopeia .
, microbial detection methods must be able to detect microbes with numbers below 100 colonies per gram of sample.
Still, the actual detection limit has never been determined quantitatively because many variables can affect microbial recovery in The results of the limit of detection determination can be used as a reference for the LOD value for contaminant inoculum concentration in the validation and verification of an analytical method.
Method Validation Results Data from the sensitivity, specificity, false positive rate and false negative rate tests on the validation of the Shigella detection analysis method are presented in Table 5 and the sum of the results in Table 6.
Table 3.
Method Validation Testing Results Analysis Repeat Presumptive Confirmation Category Negative Sample Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Positive Sample sonnei .
arget microb.
Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Control S.
arget microb.
Positive Positive Positive Positive Desty Herawati.
Eko Aprianto.
Eni Cahyaningsih.
Nur Miftahurrohmah of Eni Analytical Method for Nur Detection of Shigella sp.
in Oral Preparations of Quasi-Drugs in the Form of DestyOptimization Herawati, and EkoValidation Aprianto.
Cahyaningsih.
Miftahurrohmah Optimization and Validation of Analytical Method for Detection of Shigella sp.
in Oral Preparations of Quasi-Drugs in the Form of Lozenges Lozenges ERUDITIO Vol.
No.
Juni 2024
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Analysis Repeat Presumptive Confirmation Category Positive Sample S.
arget microb.
Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Control S.
arget microb.
Positive Positive Positive Positive Positive Sample S.
arget microb.
Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Control S.
arget microb.
Positive Positive Positive Positive Positive Sample coli .
on-target microbe.
Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Positive Control E.
ontarget microb.
Negative Negative Negative Negative Positive Sample Typhimurium .
on-target Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Positive Control Typhimurium .
on-target Negative Negative Negative Negative Based on Table 5, the presumptive determination of Shigella sp.
is as follows:
In negative samples, the presumptive result is negative.
The presumptive result was positive in positive samples and positive controls contaminated with target microbes (Shigella sonnei.
Shigella flexneri and Shigella - The presumptive results were negative in positive samples and positive controls contaminated with non-target microbes (E.
coli and Salmonella Typhimuriu.
Negative samples did not show colony growth, so the confirmation result was negative.
In contrast, a complete confirmation test (MCH scratch stain.
Gram stain, and automatic identification with VITEK .
was performed to approve samples and positive controls and Desty Herawati.
Eko Aprianto.
Eni Cahyaningsih.
Nur Miftahurrohmah Optimization and Validation of Analytical Method forHerawati.
Detection ofEko Shigella in Oral of Quasi-Drugs in the Form of Desty Aprianto.
Eni Preparations Cahyaningsih.
Nur Miftahurrohmah Lozenges Optimization and Validation of Analytical Method for Detection of Shigella in Oral Preparations of Quasi-Drugs in the Form of Lozenges ERUDITIO Vol.
No.
Juni 2024, pp.
P-ISSN: 2580-7722 | E-ISSN: 2807-6222
ERUDITIO Vol.
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Juni 2024
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determine whether the colonies growing on MCA and XLD selective media were positive or negative.
After obtaining the presumptive-confirmation test results from each treatment, the results were grouped as follows: Negative presumptive results that were confirmed negative were categorized as AudAy.
Positive presumptive results that were confirmed positive were classified as AuaAy.
No test results fell into categories AubAy or AucAy.
Table 4.
Number of Validation Test Result Categories Presumptive Number of test
( )
Confirmed ( ) a = 24
Confirmed (-) d = 22
n = 46
(-)
After grouping and categorizing the test results, as shown in Table 6.
there are 24 for Category A and 22 for Category D, with 46 tests.
Tabel 5.
Calculation of Sensitivity.
Specificity.
False Positive and Negative Rates Parameter Validation Calculation Conclusion Sensitivity Qualified Qualified Qualified False Positive Rate Qualified False Negative Rate Qualified In the calculation based on Table 7.
the sensitivity and specificity values obtained the results of 100%.
These results indicate that the method used meets the requirements for detecting Shigella sp.
microbes, specifically in the presence of other components in the sample matrix.
According to the Singapore Accredited Council .
, the difference between sensitivity and specificity is that sensitivity is the ability of the test method to give positive results on samples that have been contaminated with target microbes (Shigella sonnei.
Shigella flexneri and Shigella dysentria.
while specificity is the ability of the test method to give negative results on samples that do not contain target microbes or samples contaminated with non-target microbes (Escherichia coli and Salmonella typhimuriu.
The test results show that the false positive and false negative rate is 0%.
All samples that should be positive gave positive results, and all samples that should be negative gave negative results.
Thus, the Shigella spp.
The detection method tested using the WHO method is reliable.
The false positive rate test was performed to determine the possibility of a negative sample giving a positive result due to the influence of reagents, media used, or contamination due to non-aseptic procedures.
Conversely, a false negative test is performed to determine the possibility of a positive sample giving a negative result due to the influence of the sample matrix or reagent (Ismail, 2.
This method qualifies validation parameters, including sensitivity, specificity, and false positive and negative rates (World Health Organization, 2.
Therefore, the WHO method Desty Herawati.
Eko Aprianto.
Eni Cahyaningsih.
Nur Miftahurrohmah of Eni Analytical Method for Nur Detection of Shigella sp.
in Oral Preparations of Quasi-Drugs in the Form of DestyOptimization Herawati, and EkoValidation Aprianto.
Cahyaningsih.
Miftahurrohmah Optimization and Validation of Analytical Method for Detection of Shigella sp.
in Oral Preparations of Quasi-Drugs in the Form of Lozenges Lozenges ERUDITIO Vol.
No.
Juni 2024
ERUDITIO
Vol.
No.
Juni 2024.
P-ISSN:
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is very applicable in testing laboratories throughout the POM Agency.
With a sensitivity result of 100% and a detection limit of 3 cfu/g, this method allows for routine analysis.
The WHO analytical method (Quality control methods for herbal material.
for the detection of Shigella in quasi-medicinal lozenges that have been validated in this study can be proposed as an analytical method text (MA-pOMN) and formalized in the MApOMN discussion session.
This method can then be officially used in all Indonesian FDA laboratories in Indonesia to monitor Shigella microbial contamination in quasi-medicines Conclusion This study aims to validate the method used for Shigella detection analysis in quasimedicinal lozenges using the WHO reference method (Quality control methods for herbal The results showed that the technique had a sensitivity and specificity of 100% and a false positive and false negative rate of 0%.
The limit of detection obtained was 3 cfu/g, which meets the acceptability requirements of microbiological analysis methods .
elow 100 cfu/.
With these validation results, the process is declared accurate and can be applied for routine Shigella sppAitesting analysis in the Food and Drug Administration Acknowledgements The authors would like to thank the leadership and staff of the National Food and Drug Testing Development Center.
Indonesian FDA, for all the contributions made so that this research can be carried out correctly.
Our gratitude also goes to Mr Decky Indrawan Junaedi.
Env.
Ph.
, who is the supervisor of this journal writing.
References