Direct PCR: Alternative Diagnostic Method for Diagnosis of Diphtheria Rapidly, Easily and Cost Effective

Some diseases require immediate and appropriate treatment to decrease the fatality risk patients incident, for example diphtheria. Time to help patients is very crucial since delay of therapy may increase the mortality cases up to 20 times. In other hands, conventional diagnostic methods (the gold standard) for diagnosis of diphtheria is time consuming and laborious. Therefore, an alternative diagnostic method which is rapid, easy and inexpensive is needed. In this case, direct PCR has been proved to reduce time and cost in laboratory examination. This study aimed to develop direct PCR as alternative diagnostic method for diagnosis of diphtheria rapidly, easily, and inexpensive. Fifteen samples include 10 isolates of Corynebacterium diphtheriae (toxigenic) and 3 isolates of Corynebacterium non- diphtheriae (nontoxigenic) and 2 clinical specimens (throat swab) was examined by performing direct PCR method and a standard PCR method was used for optimizing the protocols. Result showed that direct PCR can be used to amplify target genes correctly as well as standard PCR. All of C. diphtheriae samples showed bands at 168 bp (dtxR gene marker) and 551 bp (tox gene marker) while no band appeared in others. Direct PCR detected at least 71 CFU/uL of bacterial cells in samples. We concluded that direct PCR can be used for alternative diagnostic method for diagnosis of diphtheria which is rapid, easy and cost effective

Metode Cepat Ekstraksi Dna Corynebacterium Diphtheriae Untuk Pemeriksaan Pcr

Diagnosis of diphtheria caused byCorynebacterium diphtheriaeshould be done immediately since delay of therapy may cause 20-fold increase rate of death. One method of rapid diagnostic to identify diphtheria is by using polymerase chain reaction (PCR). The fundamental issue of this method depends on the DNA, either its quality or quantity. The simple DNA extraction method, which is using mechanical/physical principles with a little of chemical reagents (such as boiling method and the use of sodium hydroxide (NAOH)), will have some benefits, such as easy to be performed, low cost, fast, and environmentally friendly. This study aimed to evaluate effectivity and efficiency of boiling method with NaOH to extract DNA of C. diphtheriae compared to the use of a commercial diagnostic kit for PCR assay. We used C. diphtheriae toxygenic(NCTC 10648) isolates, which are grown in blood agar plates. We then prepared the suspensions of cell/colony in aquadest with several dilutions. Each dilution was extracted using boiling method, NaOH and controlled with the use of a commercial diagnostic kit (QiAmp DNA Minikit). The results were evaluated quantitatively with spectrophotometer and qualitatively with gel electrophoresis. The results showed that the extracted DNA from boiling method with NaOH has an adequate quality and quantity for PCR assay (up to 9 CFU/uL cell/reaction). Therefore, it can be summarized that boiling method with NaOH is effective and efficient to be applied in PCR assay forC. diphtheriae.Key words: boiling extraction method, NaOH, C.diphtheriae, PCRAbstrakKematian kasus difteri yang disebabkan oleh Corynebacterium diphtheriaedapat meningkat 20 kali lipat karena keterlambatan pengobatan sehingga penegakan diagnosis harus dilakukan sesegera mungkin. Salah satu metode diagnostik yang cukup cepat untuk mendeteksi penyakit difteri adalah pemeriksaan polymerase chain reaction(PCR). Keberhasilan pemeriksaan PCR dipengaruhi oleh kualitas dan kuantitas DNA. Metode ekstraksi DNA sederhana menggunakan prinsip mekanik/fisika dengan reagen kimia minimalis, seperti metode pemanasan (boiling)dan penggunaan (NAOH) memiliki beberapa kelebihan, diantaranya mudah dilakukan, biaya murah, waktu yang dibutuhkan singkat dan lebih ramah lingkungan. Penelitian ini bertujuan untuk menilai efektifitas dan efisiensi metode boilingdan NAOH untuk ekstraksi DNA C.diphtheriae dibandingkan dengan penggunaan kit komersial dalam pemeriksaan PCR. Sampel berupa isolat C.diphtheriae toksigenik (NCTC 10648), ditumbuhkan dalam blood agar, kemudian dibuat suspensi sel/koloni dalam aquadest dengan beberapa kali pengenceran. Masing-masing pengenceran diekstraksi dengan metode boilingdan NaOH yang terkontrol dengan kit komersial (QiAmp DNA Minikit). Hasil ekstraksi dinilai secara kuantitatif dengan spektrofotometer dan secara kualitatif dengan gel elektroforesis. Hasil penelitian menunjukkan bahwa DNA hasil ekstraksi dengan metode boilingdan NaOH mempunyai kualitas dan kuantitas yang cukup untuk pemeriksaan PCR hingga 9 CFU/uL sel bakteri/reaksi sehingga dapat disimpulkan bahwa metode boiling dan NaOH cukup efektif dan efisien untuk diaplikasikan pada metode PCR untuk C.diphtheriae.Kata Kunci: boiling, NaOH, C.diphtheriae, PC

Validity and Reliability of the Patient Assessment of Constipation: Symptoms (PAC-SYM ©) in the Indonesian Language

Introduction: Constipation is a very subjective symptom experienced by patients. Although ROME-IV could be used for diagnosis of functional constipation, it could not assess treatment response. The Patient Assessment of Constipation – Symptoms (PAC-SYM©) questionnaire was developed for this purpose. However, the PAC-SYM©had not been translated and adapted into Indonesian language. Methods: The original PAC-SYM© questionnaire was translated and adapted based on the available guidelines. The final version of the translation was then used to perform validity and reliability analysis. A total of 64 patients with chronic constipation from the local community in Jakarta, Indonesia was used for the psychometric evaluation. Principal component analysis and structural equation modeling were also attempted. Results: Most of the study subjects were female. The Cronbach’s alpha for the overall average score was 0.869 which showed good internal consistency. The intraclass correlation coefficient for the overall score was 0.743 which showed good test-retest reliability. Content validity was considered to be sufficient by experts. Each domain correlated strongly with the total score. PAC-SYM© had moderate correlation with PAC-QOL which showed concurrent validity. The multi-trait analysis showed scaling success. Question 7 showed a very high floor effect (84.4%), and therefore omitted from the factor analysis. The principal component analysis revealed a new ‘two-factors’ structure, with question from the original ‘rectal’ domain combined into the ‘stool’ domain. The structural equation modeling indicated good model fit. Conclusion: The Indonesian version of PAC-SYM© was valid and reliable to be used in clinical settings. Keywords: constipation severity, symptoms, Indonesia, validity, reliabilit

Potential of Neuraminidase from Pasteurella multocida for Inhibiting Avian Influenza Virus Subtype H9N2 Replication In Ovo

In recent decades, neuraminidase/sialidase-based antivirals have been produced to suppress respiratory viral infections, including avian influenza, which relies on sialic acid as the entry point for viruses into cells. While neuraminidase has been extensively studied as an antiviral agent, numerous neuraminidases still have not been evaluated for their antiviral activities. Among these is NanB neuraminidase derived from Pasteurella multocida, which has received limited research attention. This study aimed to assess the potential of NanB neuraminidase in inhibiting H9N2 avian influenza virus infection in ovo. The research commenced with the molecular re-identification of the H9N2 A/Layer/Indonesia/WestJava-04/17 virus isolate, followed by determining the EID50 through Rapid HA test results. The toxicity of NanB neuraminidase was assessed by administering various doses to embryonated chicken eggs (ECE). The antiviral activity of NanB neuraminidase on ECE was evaluated through challenge tests, including treatment before, during, and after the challenge. The assessment involved monitoring the time of embryo death, virus titer through HA test, and viral copy number via RT-qPCR. The results indicated that the H9N2 virus titers capable of infecting 50% of ECE amounted to 108.83 EID50/mL. A dose of 0.258 U/mL of NanB neuraminidase was found to be toxic, leading to embryo mortality after 48 hours of incubation at 37 ℃, while a non-toxic dose was determined to be 0.129 U/mL. The post-challenge treatment group exhibited the most significant reduction in virus titer in ECE. Notably, NanB neuraminidase derived from P. multocida demonstrated the ability to inhibit H9N2 avian influenza virus infection in the ovo model, with the optimal dosage of 0.129 U/mL. The observed decrease in virus titers in the hemagglutination assay and viral copy number assays suggests that NanB neuraminidase holds promise as a potential antiviral candidate for therapeutic approach

Prevalence and epidemiological characteristics of COVID-19 after one year of pandemic in Jakarta and neighbouring areas, Indonesia: A single center study

We determined the prevalence and epidemiological characteristics of COVID-19 in Jakarta and neighboring areas, Indonesia from March 2020 to February 2021, based on nasopharyngeal/oropharyngeal (NP/OP) swab specimens that were tested at the Eijkman Institute for Molecular Biology, Jakarta. NP/OP swab specimens were collected from COVID-19 suspects or individuals in contact tracing programs from primary healthcare centers (PHC) and hospitals. The specimens were screened for the SARS-CoV-2 by qRT-PCR. Demography data and clinical symptoms were collected using national standardized laboratory form. Of 64,364 specimens, 10,130 (15.7%) were confirmed positive for SARS-CoV-2, with the peak prevalence of infection in March 2020 (26.3%) follow by in January 2021 (23.9%) and February 2021 (21.8%). We found that the positivity rate of the specimens from Jakarta, West Java, and Banten was 16.3%, 13.3%, and 16.8%, respectively. Positivity rate was higher in specimens from hospitals (16.9%) than PHC (9.4%). Of the positive specimens, 29.6% were from individuals aged >60 years old, followed by individuals aged 41–60 years old (24.2%). Among symptomatic cases of SARS-CoV-2, the most common symptoms were cough, fever, and a combination of both cough & fever. In conclusion, this study illustrates the prevalence and epidemiological characteristics from one COVID-19 diagnostic center in Jakarta and neighbouring areas in Indonesia

Zika Virus Seropositivity in 1–4-Year-Old Children, Indonesia, 2014

We assessed Zika virus seroprevalence among healthy 1–4-year-old children using a serum sample collection assembled in 2014 representing 30 urban sites across Indonesia. Of 662 samples, 9.1% were Zika virus seropositive, suggesting widespread recent Zika virus transmission and immunity. Larger studies are needed to better determine endemicity in Indonesia