Konfirmasi laboratorium kasus difteri dilakukan dengan isolasi dan tes toksigenisitas bakteri penyebab difteri menggunakan metode konvensional berbasis kultur. Metode konvensional memiliki keterbatasan dalam hal sensitivitas pemeriksaan. Penelitian ini bertujuan untuk menguji penggunaan darah domba + telurit sebagai enrichment-selective medium untuk meningkatkan sensitivitas pemeriksaan laboratorium difteri dengan metode konvensional. Sebanyak 18 spesimen klinis (swab tenggorok) penderita difteri digunakan sebagai sampel penelitian. Swab tenggorok tersebut sebelumnya telah digunakan untuk pemeriksaan Polymerase Chain Reaction (PCR) sehingga proses ekstraksi DNA menyebabkan jumlah sel bakteri yang tertinggal menjadi sangat terbatas. Sel bakteri tersebut ditumbuhkan menggunakan 2 cara yang berbeda, yaitu inokulasi langsung dan inokulasi yang didahului dengan penggunaan enrichment-selective medium. Hasil identifikasi bakteri penyebab difteri dibandingkan antara keduanya. Hasil penelitian menunjukkan bahwa inokulasi secara langsung hanya berhasil mengisolasi dan mengidentifikasi bakteri penyebab difteri (Corynebacterium diphtheriae) pada 3 dari 18 sampel yang diperiksa. Sebaliknya dengan penggunaan enrichment-selective medium, bakteri penyebab difteri berhasil diisolasi dan diidentifikasi pada 9 dari 18 sampel yang diperiksa. Oleh karena itu, disimpulkan bahwa enrichment-selective medium (darah domba + telurit) dapat digunakan untuk meningkatkan sensitivitas pemeriksaan laboratorium difteri. Laboratory confirmation for diphtheria cases are performed by using conventional culture-based method for isolation/identification and toxigenicity of the bacteria causing diphtheria. This method has limitation in its sensitivity. This study aimed to examine the sensitivity of sheep blood + tellurite as the enrichment selective medium to improve the sensitivity of the conventional method. The samples were 18 clinical specimens (throat swabs) obtained from diphtheria patient, which had been used for Polymerase Chain Reaction (PCR) assay, therefore the DNA extraction process caused the number of bacteria cells to be very limited. The samples were cultured by two different methods, directly on the agar medium and indirectly through enrichment selective medium previously. The result showed that the directly inoculation could isolate C. diphtheriae as many as 3 out of 18 samples, whereas indirectly method by using enrichment selective medium could isolate and identify 9 out of 18 samples. In conclusion, enrichment selective medium (sheep blood + tellurite) may improve the examination sensitivity of bacteria causing diphtheria identification in the laboratory.
Diarrheal diseases are the second cause of the high morbidity and mortality in children under five years old. According to the Basic Health Survey 2018 conducted by the Ministry of Health, the prevalence of diarrheal diseases among children under five years old that were diagnosed by healthcare workers was 11.0%. The aim of this study was to describe the enteric pathogen isolated from children with diarrhea. The study was conducted in five cities in Indonesia: Jakarta, Serang, Denpasar, Makassar, and Mataram. The Inclusion criteria were children aged one month to five years old, with diarrhea that was diagnosed by a healthcare worker. The rectal swabs were sent to the Centre for Research and Development for Biomedical and Basic Health Technology, National Institute of Health Research and Development, Ministry of Health in Jakarta. Virus and Enterotoxigenic Escherichia coli (ETEC) identification by using multiplex PCR from Seegene, meanwhile bacteria identified by conventional method. As many as 2626 children under five years old participated in this study. The highest viral pathogen that causes diarrhea is viral 1.807 (68,81%) and 486 (18,56%). The virus etiology was Rotavirus 982 (54,34%) cases, followed by Adenovirus 916 (50.69) cases, Norovirus II 444 (24,57%) cases, meanwhile the bacteria pathogen were Enterotoxigenic Escherichia coli detected in 262 (9,98%) followed by Campylobacter jejuni and Shigella spp. This study described Rotavirus is the prevalence etiology of diarrhea among children under five years old followed by Adenovirus and Norovirus, some other cases reported the cause of diarrhea were bacteria ETEC E. coli followed Campylobacter jejuni, Shigella spp, etc.
Pertussis cases have been reported most frequently in developed countries, but they are predicted to be the most prevalent in developing countries. Indonesia, a developing country, routinely conducts case-based surveillance for pertussis. We reviewed the data on pertussis cases and close contacts based on clinical sample documents examined in the National Reference Laboratory for pertussis, Indonesia (2016–2020). Our objective was to analyze the laboratory and epidemiological aspects of pertussis cases and close contacts, particularly to evaluate the implementation of a 5-year case-based surveillance of pertussis in Indonesia. Data were collected from sample documents and annual laboratory reports between January 2016 and December 2020. We analyzed the proportion of pertussis cases and close contacts by geographic region, year, age, and sex. We used the χ2 test to correlate the laboratory and epidemiological data. In total, 274 clinical cases of pertussis and 491 close contacts were recorded in 15 provinces. The peak number of cases occurred in 2019, with a positivity rate (percentage of laboratory-confirmed cases) of 41.23% (47/114). Clinical cases were dominated by infants aged <1 year (55.5%), and 52.9% of them were aged <6 months. Similarly, 72.3% (68/94) of the laboratory-confirmed cases were infants. Both clinical cases and positivity rates tended to be higher in females (155 cases, 38.1%) than in males (119 cases, 29.4%). No confirmed cases were found in children aged ≥10 years, although positive results still occurred in close contact. Age-group and laboratory-confirmed cases were correlated (p = 0.00). Clinical and confirmed cases of pertussis occurred mostly in the early age group and may be lower in those aged ≥10 years, especially in confirmed cases. New policies are needed for pertussis prevention at an early age, as well as the application of serology tests to increase laboratory-confirmed cases in children aged ≥10 years.
Cerebral malaria is a complication of Plasmodium falciparum infection and can cause death in humans. Plasmodium berghei ANKA (PbA) infection in C57BL/6 strain is widely used for cerebral malaria research. However, research to assess differences in brain histopathology, TNFα levels and the degree of parasitemia in Swiss websters infected with PbA are still limited. Therefore an infection with P. berghei ANKA strain was carried out on Swiss webster mice and C57BL/6 as a model of cerebral malaria. This research is a laboratory experiment with a post-test only control group design. Each experimental animal was divided into 3 groups namely PbA group (infected and given aquades), DHP (infected and given Dihidroartemisinin piperakuin), and healthy (mice not infected with PbA called aquades). Animal testing tried to examine parasitemia by microscopic counts on thin blood smear, calculation of serum TNFα levels by ELISA method and histopathology of the brain and slide microscopic examination with Haematoxylin Eosin staining conducted at the Experimental Animal Laboratory, Parasite Laboratory, Puslitbang BTDK, NIHRD; and Balitvet Pathology Laboratory by the research team. The results showed a peak percentage of parasitemia in the PbA group on day 4 for the Swiss webster strain (68.8%) and on the 5th day for the C57BL/6 strain (43.7%). The percentage of parasitemia was higher in the Swiss webster strain than in the C57BL/6 strain. TNFα levels in the Swiss webster strain (3.6 pg/ml) were higher than TNFα levels in the C57BL/6 strain (0.18 pg/ml). Histopathological changes in the brain consisting of lymphocyte cells, infiltration, macrophages, gliosis, necrosis, vacuolization and malaria parasitemia were found in both strains. The results showed that Swiss webster mice can be used as a model of cerebral malaria when seen from the clinical picture, the percentage of parasitemia, serum TNFα levels, macroscopic and microscopic brains that have similarities to the C57BL/6 strain. Keywords: Cerebral malaria, Plasmodium berghei ANKA, Swiss webster, C57BL/6. Abstrak Malaria serebral merupakan salah satu komplikasi infeksi Plasmodium falciparum dan dapat menyebabkan kematian pada manusia. Infeksi Plasmodium berghei ANKA (PbA) pada mencit galur C57BL/6 banyak digunakan untuk penelitian malaria serebral. Di Indonesia, mencit Swiss webster banyak digunakan sebagai hewan coba untuk malaria, namun penelitian untuk menilai perbedaaan histopatologi otak, kadar TNFα dan derajat parasitemia pada Swiss webster yang diinfeksi PbA masih terbatas. Oleh sebab itu dilakukan infeksi P. berghei galur ANKA pada mencit Swiss webster dan C57BL/6 sebagai model malaria serebral. Penelitian ini merupakan eksperimen laboratorium dengan desain post-test only control group. Masing-masing galur hewan coba dibagi menjadi 3 kelompok yaitu kelompok PbA (diinfeksi dan diberi akuades), DHP (diinfeksi dan diberi Dihidroartemisinin piperakuin), dan sehat (mencit yang tidak diinfeksi PbA yang diberi akuades). Pengujian pada hewan coba meliputi pemeriksaan parasitemia dengan penghitungan kepadatan parasit secara mikroskopis pada ulas darah tipis, penghitungan kadar TNFα serum dengan metode ELISA serta gambaran histopatologi sediaan otak dengan pewarnaan Haematoksilin Eosin dilakukan di Laboratorium Hewan Coba, Laboratorium Parasit Puslitbang BTDK, Badan Litbangkes; dan Laboratorium Patologi Balitvet oleh tim peneliti. Hasil penelitian menunjukkan puncak persentase parasitemia kelompok PbA pada hari ke- 4 untuk galur Swiss webster (68.8%) sementara untuk galur C57BL/6 (43.7%) pada hari ke-5. Hal ini berarti bahwa puncak parasitemia lebih cepat dan tinggi terjadi pada galur Swiss webster dibandingkan pada galur C57BL/6. Demikian juga untuk kadar TNFα didapatkan bahwa pada galur Swiss webster (3.6 pg/ml) lebih tinggi dibandingkan galur C57BL/6 (0.18 pg/ml). Perubahan histopatologik otak berupa infiltrasi sel limfosit, makrofag, gliosis, nekrosis, vakuolisasi dan parasitemia malaria ditemukan pada kedua galur. Hasil penelitian menunjukkan bahwa mencit galur Swiss webster dapat dijadikan sebagai model malaria serebral. Kata kunci: Malaria serebral, Plasmodium berghei ANKA, Swiss webster, C57BL/6.
Diphtheria is a vaccine-preventable disease. The clinical features and complications of diphtheria are associated with toxins produced by the causative bacteria. Diphtheria toxin synthesis is encoded by tox gene. This study aimed to provide an overview of the DNA sequences of the tox gene of Corynebacterium diphtheriae causing diphtheria in several region of Indonesia. A total of 65 Corynebacterium diphtheriae isolated from several provinces in Indonesia (2010-2017) were used as samples. Isolates recultured on blood agar medium (BA), incubated at 37 0 C overnight. DNA extraction conducted using the QiaAmp DNA Mini Kit. The DNA sequencing was carried out using the Whole Genome Sequencing (WGS) approach. The data conversion and analysis conducted using U-gene and BioEdit programs. Examination of 65 isolate C. diphtheriae with 1683 bp of tox gene sequences showed that there are 3 patterns of gene sequences with 3 mutation site. All mutations were silent mutation. The mutation sites were also not commonly used as 3’end binding site of the PCR primer. We concluded that tox gene of C. diphtheriae that causes diphtheria in some provinces in Indonesia have limited variations and these variations do not encode amino acid changes. This indicates that the vaccines used in Indonesia are still in accordance with the variations in circulating bacteria and PCR can be used for screening and predicting the toxigenicity of diphtheria-causing bacteria. Keywords: C. diphtheriae, gene tox, diphtheria, Indonesia Abstrak Difteri merupakan salah satu penyakit yang dapat dicegah dengan imunisasi (PD3I). Gambaran klinis dan komplikasi difteri dikaitkan dengan toksin yang diproduksi oleh bakteri penyebab. Sintesis toksin difteri dikode oleh gen tox. Penelitian ini bertujuan untuk memberikan gambaran sekuens DNA gen tox Corynebacterium diphtheriae penyebab difteri di beberapa wilayah Indonesia. Sebanyak 65 isolat C. diphtheriae tersimpan milik Badan Litbangkes yang diisolasi dari beberapa wilayah Indonesia tahun 2010- 2017 dijadikan sebagai sampel. Rekultur dilakukan pada medium agar darah (BA), diinkubasi pada suhu 37 o C selama sehari semalam. Ekstraksi DNA menggunakan kit QiaAmp DNA Minikit. Sekuensing DNA dilakukan dengan pendekatan Whole Genome Sequencing (WGS). Konversi dan analisis data menggunakan program U-gene dan BioEdit.Pemeriksaan 65 isolat C. diphtheriae dengan 1683 bp sekuens gen tox menunjukkan ada 3 pola sekuens gen dengan 3 lokasi mutasi. Seluruh mutasi bersifat silent mutation. Lokasi mutasi juga bukan merupakan tempat penempelan ujung 3’ primer PCR yang umum digunakan. Berdasarkan hasil penelitian dapat disimpulkan bahwa variasi gen tox yang ditemukan pada C. diphtheriae penyebab difteri di Indonesia memiliki variasi yang terbatas dan mutasi yang ada tidak mengkode perubahan asam amino. Hal ini mengindikasikan bahwa vaksin yang digunakan di Indonesia masih sesuai dengan variasi bakteri yang bersirkulasi. Hasil penelitian juga mengindikasikan bahwa PCR dapat digunakan untuk skrining dan memprediksi toksigenisitas bakteri penyebab difteri. Kata kunci : C. diphtheriae, gen tox, difteri, Indonesia
Diphtheria is a vaccine-preventable disease. The clinical features and complications of diphtheria are associated with toxins produced by the causative bacteria. Diphtheria toxin synthesis is encoded by tox gene. This study aimed to provide an overview of the DNA sequences of the tox gene of Corynebacterium diphtheriae causing diphtheria in several region of Indonesia. A total of 65 Corynebacterium diphtheriae isolated from several provinces in Indonesia (2010-2017) were used as samples. Isolates recultured on blood agar medium (BA), incubated at 37 0 C overnight. DNA extraction conducted using the QiaAmp DNA Mini Kit. The DNA sequencing was carried out using the Whole Genome Sequencing (WGS) approach. The data conversion and analysis conducted using U-gene and BioEdit programs. Examination of 65 isolate C. diphtheriae with 1683 bp of tox gene sequences showed that there are 3 patterns of gene sequences with 3 mutation site. All mutations were silent mutation. The mutation sites were also not commonly used as 3’end binding site of the PCR primer. We concluded that tox gene of C. diphtheriae that causes diphtheria in some provinces in Indonesia have limited variations and these variations do not encode amino acid changes. This indicates that the vaccines used in Indonesia are still in accordance with the variations in circulating bacteria and PCR can be used for screening and predicting the toxigenicity of diphtheria-causing bacteria. Keywords: C. diphtheriae, gene tox, diphtheria, Indonesia Abstrak Difteri merupakan salah satu penyakit yang dapat dicegah dengan imunisasi (PD3I). Gambaran klinis dan komplikasi difteri dikaitkan dengan toksin yang diproduksi oleh bakteri penyebab. Sintesis toksin difteri dikode oleh gen tox. Penelitian ini bertujuan untuk memberikan gambaran sekuens DNA gen tox Corynebacterium diphtheriae penyebab difteri di beberapa wilayah Indonesia. Sebanyak 65 isolat C. diphtheriae tersimpan milik Badan Litbangkes yang diisolasi dari beberapa wilayah Indonesia tahun 2010- 2017 dijadikan sebagai sampel. Rekultur dilakukan pada medium agar darah (BA), diinkubasi pada suhu 37 o C selama sehari semalam. Ekstraksi DNA menggunakan kit QiaAmp DNA Minikit. Sekuensing DNA dilakukan dengan pendekatan Whole Genome Sequencing (WGS). Konversi dan analisis data menggunakan program U-gene dan BioEdit.Pemeriksaan 65 isolat C. diphtheriae dengan 1683 bp sekuens gen tox menunjukkan ada 3 pola sekuens gen dengan 3 lokasi mutasi. Seluruh mutasi bersifat silent mutation. Lokasi mutasi juga bukan merupakan tempat penempelan ujung 3’ primer PCR yang umum digunakan. Berdasarkan hasil penelitian dapat disimpulkan bahwa variasi gen tox yang ditemukan pada C. diphtheriae penyebab difteri di Indonesia memiliki variasi yang terbatas dan mutasi yang ada tidak mengkode perubahan asam amino. Hal ini mengindikasikan bahwa vaksin yang digunakan di Indonesia masih sesuai dengan variasi bakteri yang bersirkulasi. Hasil penelitian juga mengindikasikan bahwa PCR dapat digunakan untuk skrining dan memprediksi toksigenisitas bakteri penyebab difteri. Kata kunci : C. diphtheriae, gen tox, difteri, Indonesia
For different bacterial preservation techniques, there is no single method applicable for all bacteria. This study aimed to assess the viability of seven species/species groups of clinical bacteria isolates on the long-term storage (more than 5 years) by using Tryptic Soy Broth with 15% glycerol in the deep freezer (-70 to -80°C). A total 10,654 clinical bacteria isolates used as samples in this study. The isolates consisted of seven species/species groups (i.e. Escherichia coli, Campylobacter spp, Shigella spp, Vibrio spp, Salmonella spp, Aeromonas hydrophila, and Neisseria gonorhoeae). The isolates were collected from some previous studies and preserved in the Tryptic Soy Broth (TSB) with 15% glycerol and stored in the deep freezer (-70 to -80°C) for more than five years. The samples were revived on the suitable medium to evaluate the viability of bacteria. Identification conducted by microscopic examination, biochemical test, and latex agglutination. The study showed that the viability of Salmonella spp, Shigella spp, Aeromonas hydrophila and E. coli was 100%, while Campylobacter spp, Vibrio spp, and N. gonorhoeae were 66.7%, 66.4%, and 52.5% respectively. We concluded that viability of Salmonella spp, Shigella spp, A. hydrophila, and E. coli was optimum thus better than Campylobacter spp, Vibrio spp, and N. gonorhoeae for more than 5 years storage by using TSB with 15% glycerol in the deep freezer (-70 to -80 °C).
Identification of ESBL-E.coli from environment without selective medium will be challenging to do considering that E.coli mixes with various other microorganisms in the environment. This study aimed to determine the performance of TBX Agar supplemented with Cefotaxime as a selective medium for ESBL-E. coli screening from 138 water samples of environmental sampling obtained from rivers, open sewers in the market, poultry slaughterhouses and hospital waste water inlets and outlets around Jakarta. Laboratory examinations were carried out through the filtration stage, inoculation on the TBX Agar supplemented with Cefotaxime medium as well as species confirmation and ESBL with the indol test and double-disk test. The results showed that 87.08% (40-100%) of suspect colonies growing on TBX Agar supplemented with Cefotaxime medium were confirmed as E.coli and 82.51% (12-100%) were confirmed as ESBL-E.coli. However, there was no correlation between TBX Agar supplemented with Cefotaxime performance and sampling locations. Based on the results of the study, it can be concluded that the TBX supplemented with Cefotaxime medium can be used for ESBL-E.coli screening in the environment, but further confirmation is needed using the indole and double-disk tests. Keywords: Escherichia coli, ESBL, TBX Agar suplemented with Cefotaxime Abstrak Identifikasi ESBL-E.coli tanpa medium selektif akan sangat sulit dilakukan pada sampel lingkungan mengingat ESBL-E.coli bercampur dengan berbagai mikroorganisme lainnya. Tujuan penelitian ini yaitu untuk mengetahui performa TBX Agar yang disuplementasi Cefotaxime sebagai medium selektif untuk skrining ESBL-E.coli. Sampel penelitian sebanyak 138 sampel air lingkungan yang diambil dari sungai, saluran pembuangan terbuka di pasar, rumah pemotongan hewan unggas (RPHU) serta inlet dan outlet limbah rumah sakit di sekitar Jakarta. Pemeriksaan laboratorium melalui tahapan filtrasi, inokulasi pada medium TBX Agar dengan suplementasi Cefotaxime serta konfirmasi spesies dan ESBL dengan uji indol dan double-disk test. Hasil menunjukkan bahwa koloni tersangka yang tumbuh pada medium TBX Agar dengan suplementasi Cefotaxime sebanyak 87,08% (40-100%) terkonfirmasi sebagai E.coli dan 82,51% (12-100%) terkonfirmasi sebagai ESBL-E.coli. Namun, tidak ada hubungan antara performa TBX Agar dengan suplementasi Cefotaxime dengan lokasi pengambilan sampel. Berdasarkan hasil penelitian dapat disimpulkan bahwa medium TBX Agar dengan suplementasi Cefotaxime cocok digunakan untuk skrining ESBL-E.coli di lingkungan namun tetap diperlukan konfirmasi lanjut menggunakan uji indol dan double disk test. Kata kunci: Escherichia coli, ESBL, TBX Agar suplemented with Cefotaxime
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