Aryati

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.

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.

PERBANDINGAN NILAI DIAGNOSTIK IGE SPESIFIK TUNGAU DEBU RUMAH, METODE ELISA DAN IMUNOBLOT PADA RINITIS ALERGI

The detection of allergen types is very helpful in allergic rhinitis (AR) management. Some methods had been performed to examine the specific IgE due to HDM such as ELISA and immunoblot methods. The aim of this research is to know the difference of specific IgE diagnostic value due to HDM between ELISA and immunoblot in allergic rhinitis method which is expected to be used as in vitro alternative method which is safe, fast, effective, with a high sensitivity and specificity by provement. The samples were allergic rhinitis and non-allergic rhinitis patients at ENT of Head and Neck Out patients Clinic of Dr. Soetomo Hospital Surabaya. The sera was examined for specific IgE due to HDM by ELISA and immunoblot methods and then analyzed for its diagnostic value using the 2 x 2 table with a 95% confidence interval. The comparation between both methods were analyzed with Wilcoxon test. The diagnostic value of the specific HDM IgE with immunoblot method showed sensitivity of 90% and 80% specificity, positive predictive value 90% and the negative 80% and diagnostic efficiency 86.67%. The positive likelihood ratio 4.5 and the negative one 0.125. The diagnostic value of the specific IgE HDM/D.p with ELISA showed a sensitivity of 75% and specificity 75%, the positive predictive value 85.71% and the negative one 0% and diagnostic efficiency 75%. The positive likelihood ratio was 3 and the negative one 0.33. The diagnostic value of the specific IgE HDM with immunoblot showed a sensitivity of 90% and specificity 80%, the positive predictive value 90% and the negative one 80% and the diagnostic efficiency 86.67%. The positive likelihood ratio was 4.5 and the negative one 0.125. The difference of diagnostic value in both methods revealed that the p value was 0.013. It can be concluded in this study that there was a significant difference of specific IgE due to HDM between ELISA and immunoblot methods in allergic rhinitis.

SPECIFIC IGE IMMUNOBLOT METHOD IN ALLERGIC RHINITIS (IgE Spesifik Menurut Metode Imunoblot di Rinitis Alergi)

Rinitis alergi merupakan penyakit bukan akibat non-infeksi yang ditemukan antara 10−30% penduduk dewasa dunia dan dapat menyebabkan penurunan mutu kehidupan seseorang. Rinitis alergi merupakan manifestasi alergi tipe 1 atau tipe cepat yang dimediasi oleh IgE. Pemeriksaan utama rinitis alergi adalah Skin Prick Test (SPT) dan IgE spesifik. Pemeriksaan IgE spesifik mempunyai kepekaan dan kekhasan yang menyerupai SPT, tidak memerlukan tenaga terlatih dan menyebabkan anafilaktik. Penelitian ini untuk mengetahui adakah kesesuaian nilai diagnostik IgE spesifik menurut metode imunoblot dengan SPT di pasien rinitis alergi dengan mengujinya. Rancangan penelitian adalah potong lintang yang dilakukan terhadap pasien yang datang di Unit Rawat Jalan THT-KL RSUD Dr. Soetomo pada bulan Mei sampai dengan Oktober 2014. Pasien dikelompokkan berdasarkan diagnosis rinitis alergi dan yang nonalergi dan non-infeksi serta ditetapkan secara klinis, ada riwayat alergi, pemeriksaan fisik, serta tingkat jumlah keseluruhan IgE serum dan atau eosinofil darah. Pemeriksaan SPT dilakukan dengan memakai ekstrak alergen dari Stallergens dan IgE spesifik menurut metode imunoblot memakai Foresight®. Dalam kajian ini didapatkan empat puluh tiga pasien didiagnosis rinitis akibat alergi. Hasil IgE spesifik menurut metode imunoblot positif terdapat di 36 (84%) pasien dengan pola alergen terbanyak D1/D2 29 (67%). Kepekaan dan kekhasan diagnostik IgE spesifik menurut metode imunoblot berturut-turut adalah 72,34% dan 46,15%. Kesesuaian nilai diagnostik IgE spesifik menurut metode imunoblot dengan SPT mempunyai koefisien kappa 0,158. Didasari telitian ini tidak didapatkan kesesuaian antara IgE spesifik menurut metode imunoblot dengan SPT. Di ketahui pula bahwa IgE spesifik menurut metode imunoblot dapat digunakan bersama-sama dengan SPT dalam mendiagnosis rinitis akibat alergi.

ANALISIS FILOGENETIK DENGUE DI INDONESIA

Molecular epidemiology is needed to solve the problem for endemic Dengue Hemorrhagic Fever in Indonesia. This research has been carried out consisting of 525 Dengue Hemorrhagic Fever sera, according to the WHO criteria. These sera were collected from 19 cities in Indonesia comprising the islands of Sumatera, Batam, Kalimantan, Sulawesi, Papua, Java, Bali and Lombok from 2003 until 2005. The immune response profile was as follows 57.14% (300/525) secondary infection, 12.57% (66/525) primary infection, 4.20% (22/525) equivocal and 26.09% (137/525) negative. From 192 PCR samples, 100 (52%) sera were positive, consisting of 65% DEN-2, 15% DEN-3, 12% DEN-4 and 8% DEN-1. Homology analysis showed nucleotide differences in capsid region DEN-2 serotypes, while DEN-3 serotypes were relatively consistent. Phylogenetic analysis using envelope (E) gene revealed that the Cosmopolitan genotype from Gorontalo in 2005, is currently circulating locally, with the potential to cause a severe hemorrhagic disease. Members of this genotype were closely related to viruses from Malaysia, Singapore, Thailand, Philippines and Australia. The isolate from Jakarta, 2003 showed DEN-3 with I genotype. This genotype was similar to the isolate from Indonesia 1978, 1985, and also from Thailand 1992, Philippines 1997, and Fiji 1992. These results showed Cosmopolitan genotype from DEN-2 was similar to Southeast Asia countries. It was also revealed that genotype-I from DEN-3 showed no change over the years since 1978.

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.

NILAI DIAGNOSTIK IgA ANTIVCA ANTIBODI EPSTEIN-BARR DI KARSINOMA NASOFARING

Early diagnosis of nasopharyngeal carcinoma (NPC) is difficult, so most patients arrived already in an advanced stage. The biopsy as the gold standard for the diagnosis of NPC at an early stage also have limitations. Epstein-Barr virus as the cause of NPC is paving the way for early diagnosis was through serological method. The purpose of this study is to know the diagnostic value of IgA antiviral capsid antigen (VCA) Epstein-Barr antibody for NPC by analyzing it. The samples were NPC patients and others whome have head-neck malignancies arrived in the Oncology Outpatient Clinic, Dr. Soetomo Hospital. Their sera were examined for IgA antiVCA Epstein-Barr antibody using ELISA method and then analyzed for its diagnostic value using the 2x2 table with a 95% confidence interval. IgA antiVCA cutoff was determined by ROC. The results show that the diagnostic value of IgA antiVCA Epstein-Barr antibody have the sensitivity and specificity around 93.3% and 93.8%, respectively. Positive predict value was 96.6%% and the negative one was 88.2%, while the diagnostic efficiency was 93.5%. The positive likelihood ratio was 14.9 times and the negative was only 0.07. The cut off value of IgA antiVCA according ROC was 13.45 U/mL with AUC 97.9%. Based on this study, can be concluded that IgA antiVCA Epstein-Barr antibody showed an excellent validity in supporting the diagnosis of NPC. However, the researchers needed further research to know the obtainable early stage of NPC.