Puspa Wardhani

Performance of commercial dengue NS1 ELISA and molecular analysis of NS1 gene of dengue viruses obtained during surveillance in Indonesia

BackgroundEarly diagnosis of dengue infection is crucial for better management of the disease. Diagnostic tests based on the detection of dengue virus (DENV) Non Structural Protein 1 (NS1) antigen are commercially available with different sensitivities and specificities observed in various settings. Dengue is endemic in Indonesia and clinicians are increasingly using the NS1 detection for dengue confirmation. This study described the performance of Panbio Dengue Early NS1 and IgM Capture ELISA assays for dengue detection during our surveillance in eight cities in Indonesia as well as the genetic diversity of DENV NS1 genes and its relationship with the NS1 detection.MethodsThe NS1 and IgM/IgG ELISA assays were used for screening and confirmation of dengue infection during surveillance in 2010–2012. Collected serum samples (n = 440) were subjected to RT-PCR and virus isolation, in which 188 samples were confirmed for dengue infection. The positivity of the ELISA assays were correlated with the RT-PCR results to obtain the sensitivity of the assays. The NS1 genes of 48 Indonesian virus isolates were sequenced and their genetic characteristics were studied.ResultsUsing molecular data as gold standard, the sensitivity of NS1 ELISA assay for samples from Indonesia was 56.4% while IgM ELISA was 73.7%. When both NS1 and IgM results were combined, the sensitivity increased to 89.4%. The NS1 sensitivity varied when correlated with city/geographical origins and DENV serotype, in which the lowest sensitivity was observed for DENV-4 (19.0%). NS1 sensitivity was higher in primary (67.6%) compared to secondary infection (48.2%). The specificity of NS1 assay for non-dengue samples were 100%. The NS1 gene sequence analysis of 48 isolates revealed the presence of polymorphisms of the NS1 genes which apparently did not influence the NS1 sensitivity.ConclusionsWe observed a relatively low sensitivity of NS1 ELISA for dengue detection on RT-PCR-positive dengue samples. The detection rate increased significantly when NS1 data was combined with IgM. In our study, the low sensitivity of NS1 antigen detection did not relate to NS1 genetic diversity. Rather, the performance of the NS1 antigen test was affected by the infection status of patients and geographical origin of samples.

Performance of Simplexa Dengue Molecular Assay Compared to Conventional and SYBR Green RT-PCR for Detection of Dengue Infection in Indonesia

Diagnostic tests based on detection of dengue virus (DENV) genome are available with varying sensitivities and specificities. The Simplexa Dengue assay (Focus Diagnostics) is a newly developed real-time RT-PCR method designed to detect and serotype DENV simultaneously. To assess the performance of the Simplexa Dengue assay, we performed comparison with conventional RT-PCR and SYBR Green real-time RT-PCR on patients sera isolated from eight cities across Indonesia, a dengue endemic country. A total of 184 sera that were confirmed using NS1 and/or IgM and IgG ELISA were examined. Using conventional and SYBR Green real-time RT-PCR, we detected DENV in 53 (28.8%) and 81 (44.0%) out of 184 sera, respectively. When the Simplexa Dengue assay was employed, the detection rate was increased to 76.6% (141 out of 184 samples). When tested in 40 sera that were confirmed by virus isolation as the gold standard, the conventional RT-PCR yielded 95% sensitivity while the sensitivity of SYBR Green real-time RT-PCR and Simplexa Dengue assay reached 97.5% and 100%, respectively. The specificities of all methods were 100% when tested in 43 non-dengue illness and 20 healthy human samples. Altogether, our data showed the higher detection rate of Simplexa Dengue compared to conventional and SYBR Green real-time RT-PCR in field/surveillance setting. In conclusion, Simplexa Dengue offers rapid and accurate detection and typing of dengue infection and is suitable for both routine diagnostic and surveillance.

Clinical and virological characteristics of dengue in Surabaya, Indonesia

Dengue disease is still a major health problem in Indonesia. Surabaya, the second largest city in the country, is endemic for dengue. We report here on dengue disease in Surabaya, investigating the clinical manifestations, the distribution of dengue virus (DENV) serotypes, and the relationships between clinical manifestations and the genetic characteristics of DENV. A total of 148 patients suspected of having dengue were recruited during February-August 2012. One hundred one (68%) of them were children, and 47 (32%) were adults. Dengue fever (DF) and Dengue hemorrhagic fever (DHF) were equally manifested in all of the patients. We performed DENV serotyping on all of the samples using real-time RT-PCR. Of 148, 79 (53%) samples were detected as DENV positive, with DENV-1 as the predominant serotype (73%), followed by DENV-2 (8%), DENV-4 (8%), and DENV-3 (6%), while 5% were mixed infections. Based on the Envelope gene sequences, we performed phylogenetic analyses of 24 isolates to genotype the DENV circulating in Surabaya in 2012, and the analysis revealed that DENV-1 consisted of Genotypes I and IV, DENV-2 was of the Cosmopolitan genotype, the DENV-3 viruses were of Genotype I, and DENV-4 was detected as Genotype II. We correlated the infecting DENV serotypes with clinical manifestations and laboratory parameters; however, no significant correlations were found. Amino acid analysis of Envelope protein did not find any unique mutations related to disease severity.

Production of recombinant dengue non-structural 1 (NS1) proteins from clinical virus isolates

Dengue is a febrile disease caused by infection of dengue virus (DENV). Early diagnosis of dengue infection is important for better management of the disease. The DENV Non-Structural Protein 1 (NS1) antigen has been routinely used for the early dengue detection. In dengue epidemic countries such as Indonesia, clinicians are increasingly relying on the NS1 detection for confirmation of dengue infection. Various NS1 diagnostic tests are commercially available, however different sensitivities and specificities were observed in various settings. This study was aimed to generate dengue NS1 recombinant protein for the development of dengue diagnostic tests. Four Indonesian DENV isolates were used as the source of the NS1 gene cloning, expression, and purification in bacterial expression system. Recombinant NS1 proteins were successfully purified and their antigenicities were assessed. Immunization of mice with recombinant proteins observed the immunogenicity of the NS1 protein. The generated recombinant proteins can be potentially used in the development of NS1 diagnostic test. With minimal modifications, this method can be used for producing NS1 recombinant proteins from isolates obtained from other geographical regions.

Genomic analysis of dengue virus serotype 1 (DENV-1) genotypes from Surabaya, Indonesia

Dengue has caused a significant public health impact globally. With the diverse genetic of the causative viruses, analysis of dengue virus (DENV) genomes is important to supplement epidemiological data with information that can be used to reconstruct the history of epidemics in time and space. We have reported the clinical and virological characteristics of dengue in Surabaya, Indonesia and revealed the presence of all four DENV serotypes and the predominance of DENV-1. The further classification of Surabaya DENV-1 into two different genotypes warrants in-depth genomic analysis to study the dynamics of both genotypes and their contribution to virus evolution, virus transmission, and disease. We performed full-length genome sequencing to nine isolates’ representatives from DENV-1 Genotype I and Genotype IV. Phylogenetic and evolutionary analyses suggested the more recent introduction of Genotype I viruses compared to the more endemic Genotype IV. Comparative analysis of Surabaya DENV-1 genomes and other sequences available publicly revealed that the majority of the DENV-1 codons were under strong purifying selection, while seven codon sites identified to be under positive selection. We highlight a unique codon site under the positive pressure in the NS1 gene of DENV-1. Our results provide additional genomic data of DENV from Indonesia that may contribute to the better understanding of dengue disease dynamics.

IDENTIFICATION OF DENGUE VIRUS SEROTYPES AT THE DR. SOETOMO HOSPITAL SURABAYA IN 2016 AND ITS CORRELATION WITH NS1 ANTIGEN DETECTION

Serotipe virus dengue yang beredar terus mengalami perubahan dan berbeda di setiap daerah. Pergeseran serotipe maupun genotipe di dalamnya, mempengaruhi terjadinya wabah dengue di berbagai negara. Perbedaan serotipe diduga bernasab dengan deteksi antigen (Ag) non-structural 1 (NS1), namun belum banyak penelitian yang mendukung hal tersebut. Penelitian potong lintang dikerjakan sejak Februari-Agustus 2016 dan didapatkan 60 subjek infeksi virus dengue (IVD) dan 25 non-IVD. Ribonucleic acid (RNA) virus dengue diperiksa di semua subjek menggunakan Simplexa Dengue Real-Time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) termasuk identifikasi serotipe virus dengue dan pemeriksaan NS1 menggunakan uji cepat NS1 Panbio. Perbedaan perbandingan variabel kategorikal dianalisis dengan uji Fisher Exact. Kenasaban antara serotipe dengan deteksi Ag NS1 dianalisis dengan Chi-Kuadrat. RNA virus dengue terdeteksi di 43 dari 60 subjek IVD (71,7%). Serotipe terbanyak adalah DENV-3 (62,8%). Pergeseran dominasi serotipe telah terjadi di Surabaya, sebelumnya dari DENV-2 ke DENV-1 dan sekarang DENV-3, kemungkinan akibat mobilitas pejamu, transpor virus dan faktor geografis. Kepekaan uji cepat NS1 75% dan kekhasan 100%. Persentase deteksi NS1 antar serotipe berbeda bermakna (p=0,002). Deteksi NS1 lebih rendah pada DENV-1 dibandingkan DENV-2 (p=0,007) ataupun DENV-3 (p=0,003). Serotipe virus dengue bernasab dengan deteksi NS1 (p=0,005). Ciri serotipe maupun genotipe virus dengue kemungkinan mempengaruhi sekresi NS1. Telah terjadi pergeseran serotipe virus dengue di pasien IVD di Surabaya sehingga diperlukan surveillance berkesinambungan untuk memperkirakan terjadinya wabah. Serotipe bernasab dengan deteksi NS1. Salah satu penyebab hasil negatif palsu NS1 adalah perbedaan serotipe.

APPLICATION OF DNA METHYLATION ON URINE SAMPLE FOR AGE ESTIMATION (Penggunaan Metilasi DNA dalam Perkiraan Umur Individu di Sampel Air Kemih)

Perkiraan umur sangat penting dalam analisis forensik. Umur individu lebih sering diperkirakan dengan menggunakan tulang dan gigi. Namun, hanya terbatas pada kasus tertentu yang berhubungan dengan kerangka manusia. Metilasi DNA merupakan salah satu cara dalam memperkirakan umur di sampel biologis. Perkiraan umur menggunakan metode metilasi DNA dengan penggunaan sampel air kemih hingga saat ini belum pernah ada yang melakukan, oleh sebab itu penelitian ini akan mengunakan isolasi metilasi DNA dalam memperkirakan umur individu di sampel airkemih. Di penelitian ini digunakan 6 sampel air kemih yang didapatkan dari pendonor sehat. Tahap pertama adalah isolasi DNA dengan menggunakan DNAzol dan kloroform setelah itu, dikonversi bisulfit dengan kit DNA metilasi. Hasil isolasi kemudian di amplifikasi dengan metode PCR dan di elektroforesis dengan gel agarosa. Hasil elektroforesis dapat sebagai acuan panjang pita yang di sekuensing. Hasil sekuensing dianalisis persen metilasinya dan korelasinya dengan perkiraan umur. Hasil dari pembacaan aplikasi dan perhitungan tersebut di sampel 001 menunjukkan 64,99%, sampel 002 menunjukkan 69,45%, sampel 003 menunjukkan 57,52%, sampel 4 menunjukkan 58,61%, sampel 005 menunjukkan 63,66% dan sampel 006 menunjukkan 61,19%. Berdasarkan hasil penelitian ini merupakan langkah awal dalam penggunaan metilasi DNA dalam perkiraan umur individu terutama di sampel air kemih.

ANALYSIS OF DENGUE SPECIFIC IMMUNE RESPONSE BASED ON SEROTYPE, TYPE AND SEVERITY OF DENGUE INFECTION (Analisis Respons Imun Spesifik Dengue terhadap Serotipe, Jenis dan Derajat Infeksi Virus Dengue)

Infeksi Virus Dengue (IVD) menimbulkan derajat klinis beragam dari DD hingga DBD/SSD. Respons imun spesifik dengue berupa IgM dan IgG anti dengue masih merupakan perdebatan untuk patogenesis DBD di samping faktor virulensi virus dan jenis infeksi. Penelitian ini bertujuan untuk menganalisis respons imun spesifik dengue terhadap serotipe, jenis dan derajat IVD di Surabaya. Subjek adalah pasien IVD yang dirawat di Ruang Tropik Infeksi Penyakit Dalam RSUD Dr. Soetomo dengan hasil penyaringan uji cepat NS1 (SD Bioline Dengue Duo) dan/atau PCR (Simplexa Dengue) positif. Pemeriksaan IgM dan IgG anti dengue kuantitatif dengan metode ELISA (Panbio Dengue Duo IgM and IgG Capture). Penelitian dilakukan Maret–Agustus 2016 dan didapatkan 61 pasien dengan hasil NSI dan/ atau PCR dengue positif. Identifikasi serotipe didominasi DEN-3, namun serotipe yang lebih virulen ditunjukkan DEN-1 yaitu semua pasien bermanifestasi sebagai infeksi sekunder dan DBD. Jenis infeksi primer sebanyak 19 (31,1%) dan infeksi sekunder 42 (68,9%). Derajat IVD meliputi DD 10 (16,4%), DBD 47 (77%) dan SSD 4 (6,56%). Nilai indeks rerata IgM dan IgG anti dengue di kelompok infeksi serotipe DEN-1 (5,140 dan 5,774), DEN-2 (2,971 dan 2,222), DEN-3 (1,863 dan 2,792); kelompok jenis infeksi primer (1,478 dan 0,746), sekunder (4,028 dan 4,864) dan kelompok derajat DD (1,170 dan 1,492), DBD I (3,370 dan 3,651), DBD II (3,924 dan 4,439) dan DBD III (4,164 dan 4,243). Sebagai simpulan respons imun spesifik dengue didapatkan lebih tinggi bermakna di kelompok infeksi serotipe DEN-1, kelompok jenis infeksi sekunder dan kelompok DBD/SSD.

ANTI DENGUE IGG/IGM RATIO FOR SECONDARY ADULT DENGUE INFECTION IN SURABAYA

Infeksi Virus Dengue (IVD) dibedakan menjadi infeksi primer dan sekunder berdasarkan respons antibodi yang dihasilkan. Infeksi sekunder perlu dibedakan dari infeksi primer karena umumnya menimbulkan manifestasi klinis yang berat. Uji hemaglutinasi inhibisi sebagai baku emas untuk menentukan infeksi primer atau sekunder dirasa tidak praktis karena membutuhkan sepasang sera dengan selang waktu waktu yang cukup lama. Penelitian ini bertujuan mengetahui cut-off rasio IgG/IgM anti dengue untuk infeksi dengue sekunder dewasa di Surabaya. Subjek adalah pasien IVD dengan hasil NS1 dan/atau PCR dengue positif. Rasio IgG/IgM anti-dengue diperoleh dari pembagian nilai indeks IgG dan IgM metode ELISA. Nilai cut-off rasio ditentukan berdasarkan kurva ROC. Berdasarkan pola reaktivitas IgM dan IgG ELISA, 19 (31,1%) pasien dikelompokkan sebagai infeksi primer dan 42 (68,9%) infeksi sekunder. Hasil PCR didominasi DEN-3. Nilai cut-off optimal rasio IgG/IgM ≥0,927 sebagai peramal infeksi sekunder memiliki kepekaan 66,7% dan kekhasan 63,2%. Dianalisis pula nilai cut-off optimal IgM dan IgG anti dengue, yaitu IgM ≥1,515 dan IgG ≥2,034 sebagai peramal infeksi sekunder memiliki kepekaan dan kekhasans masing-masing 85,7% dan 84,2%; 100% dan 100%. Disimpulkan bahwa rasio IgG/IgM ≥0,927 tidak dapat digunakan sebagai tolok ukur tunggal peramal infeksi sekunder sedangkan cut-off IgG ≥2,034 dapat dipertimbangkan sebagai peramal infeksi sekunder.