Platelet and Monocyte Microvesicles as Potential Biomarkers of COVID-19 Severity: A Cross-Sectional Analysis.

Background: Coronavirus disease (COVID-19) induces inflammation, coagulopathy following platelet and monocyte activation, and fibrinolysis, resulting in elevated D-dimer levels. Activated platelets and monocytes produce microvesicles (MVs). We analyzed the differences in platelet and monocyte MV counts in mild, moderate, and severe COVID-19, as well as their correlation with D-dimer levels. Methods: In this cross-sectional study, blood specimens were collected from 90 COVID-19 patients and analyzed for D-dimers using SYSMEX CS-2500. Platelet MVs (PMVs; PMVCD42b+ and PMVCD41a+), monocyte MVs (MMVs; MMVCD14+), and phosphatidylserine-binding annexin V (PS, AnnV+) were analyzed using a BD FACSCalibur instrument. Results: PMV and MMV counts were significantly increased in COVID-19 patients. AnnV+ PMVCD42b+ and AnnV+ PMVCD41a+ cell counts were higher in patients with severe COVID-19 than in those with moderate clinical symptoms. The median (range) of AnnV+ PMVCD42b+ (MV/µL) in mild, moderate, and severe COVID-19 was 1,118.3 (328.1-1,910.5), 937.4 (311.4-2,909.5), and 1,298.8 (458.2-9,703.5), respectively (P =0.009). The median (range) for AnnV+ PMVCD41a+ (MV/µL) in mild, moderate, and severe disease was 885.5 (346.3-1,682.7), 663.5 (233.8-2,081.5), and 1,146.3 (333.3-10,296.6), respectively (P =0.007). D-dimer levels (ng/mL) weak correlated with AnnV+ PMVCD41a+ (P =0.047, r=0.258). Conclusions: PMV PMVCD42b+ and PMVCD41a+ counts were significantly increased in patients with severe clinical symptoms, and PMVCD41a+ counts correlated with D-dimer levels. Therefore, MV counts can be used as a potential biomarker of COVID-19 severity.

Comparison of SARS-CoV-2 virus detection using the Xpert Xpress rapid molecular test on Abbott M2000 Real-Time System: a cross-sectional study.

Emergency use of molecular rapid test kits approved by the Food and Drug Administration (FDA) includes the Xpert Xpress SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) kit. The rapid molecular test is one of the examinations using the reverse transcription-polymerase chain reaction (RT-PCR) method. Compared to conventional PCR, the examination time is faster, so it is suitable for diagnostic purposes. Objectives: Determining the diagnostic capabilities of the Xpert Xpress SARS-CoV-2 rapid molecular test in detecting the SARS-CoV-2 virus in the Indonesian population. Methods: A cross-sectional study was conducted with consecutive sampling, in which participants were diagnosed with coronavirus disease 2019 (COVID-19) infection using the RT-PCR Abbott M2000 SARS-CoV-2 System. A molecular rapid test examination was carried out using the Xpert Xpress SARS-CoV-2 kit. Assessing the correlation between the cycle threshold (CT) value of Xpert Xpress SARS-CoV-2 and the Abbott M2000 SARS-CoV-2 System using the Pearson and Spearmen test with P<0.05. Results: Molecular rapid test using Xpert Xpress has a compatibility of 100% with RT-PCR using Abbott M2000 SARS-CoV-2 and a sensitivity and specificity value of 100%. The Xpert Xpress SARS-CoV-2 CT value had a significant correlation with the Abbott M2000 SARS-CoV-2 System CT value, with moderate correlation strength for the CT protein E value (r=0.444; P=0.007) and robust correlation for CT value of protein N2 (r=0.829; P<0.001). The negative predictive and positive predictive values were 100% each. Conclusion: The Xpert Xpress SARS-CoV-2 molecular rapid test has a sensitivity and specificity of 100% and can be recommended for diagnosing COVID-19.

The Comparison of Monocyte Human Leukocyte Antigen-D-Related (mHLA-DR) Expression Levels Between Corona Virus Disease 2019 (COVID-19) Patients and Healthy Subjects.

BACKGROUND: SARS-CoV-2 can trigger a dysfunctional immune response in COVID-19 patients and lead to immunosuppression. HLA-DR molecule expressed on the surface of monocytes, known as mHLA-DR, has been widely used as a reliable marker of immunosuppression. Downregulation of mHLA-DR reflects an immunosuppressed state. This study aimed to compare the expression level of mHLA-DR between COVID-19 patients and healthy subjects concerning immune system dysregulation that can be triggered by SARS-CoV-2 and lead to immunosuppression. METHODS: This was an analytic observational study with a cross-sectional design that measured the mHLA-DR expression in EDTA blood samples from 34 COVID-19 patients and 15 healthy subjects using the BD FACSLyricTM Flow Cytometry System. The mHLA-DR examination results were expressed in AB/C (antibodies bound per cell) that were quantified using a standard curve constructed with Quantibrite phycoerythrin beads (BD Biosciences). RESULTS: Expression of mHLA-DR in COVID-19 patients (n = 34) were 21,201 [2,646-92,384] AB/C, with 40,543.5 [9,797-92,384] AB/C mild cases (n = 22), 21,201 [9,831-31,930] AB/C moderate cases (n = 6), and 7,496 [2,646-13,674] AB/C severe to critical cases (n= 6). Expression of mHLA-DR in healthy subjects (n = 15) was 43,161 [25,147-89,846] AB/C. Based on the Mann-Whitney U test, the mHLA-DR expression in COVID-19 patients significantly differed from the mHLA-DR expression in healthy subjects (p = 0.010). CONCLUSION: The level of mHLA-DR expression in COVID-19 patients was lower and significantly different from healthy subjects. Moreover, immunosuppression could be indicated by the decrease of mHLA-DR expression, which was below the reference range found in severe to critically ill COVID-19 patients.

Los factores que influyen en el proceso de adaptación 6 meses después de un ictus: un análisis de trayectoria / The factors that influence the adaptation process 6months after a stroke: A path analysis

Objetivo: El propósito de este estudio fue identificar los factores determinantes que influyen en el proceso de adaptación y calidad de vida después de un ictus. Métodos: Este estudio es un estudio observacional utilizando un diseño transversal. Se encuestaron pacientes 6 meses después de su alta tras un accidente cerebrovascular no hemorrágico y sus familiares cuidadores. La información sobre los encuestados se obtuvo de los datos de registros médicos en dos hospitales generales regionales en la provincia de Kalimantan Occidental, Indonesia. Se seleccionó un total de 80 pacientes mediante un método de muestreo consecutivo. Los modelos teóricos de los factores del paciente y del cuidador que influyen en las respuestas de adaptación y la calidad de vida posterior al accidente cerebrovascular se probaron mediante análisis de ruta. Resultados: El afrontamiento, la autoeficacia y la aceptación de la enfermedad del cuidador tuvieron un efecto directo en la respuesta de adaptación psicosocial posterior al ictus en un 58,1%, siendo la autoeficacia la que más contribuyó (β=0,668, p<0,0001). La autoeficacia, la aceptación de la enfermedad y el comportamiento saludable tuvieron un efecto directo en la respuesta de adaptación fisiológica en un 24,3%, donde la autoeficacia también contribuyó en mayor medida (β=0,272, p<0,014). La adaptación psicosocial y la adaptación fisiológica tuvieron un efecto directo en la calidad de vida del 54,6%, donde la adaptación psicosocial presentó la mayor contribución (β=0,63, p<0,0001). Conclusión: La autoeficacia contribuye más a las adaptaciones psicosociales y fisiológicas 6 meses después del accidente cerebrovascular. La adaptación psicosocial y la autoeficacia han demostrado ser los factores determinantes que más contribuyen a la calidad de vida de los pacientes 6 meses después del ictus.(AU)

Objective: The purpose of this study was to identify the determinant factors that influence the adaptation process and quality of life after a stroke. Methods: This study is an observational study using a cross-sectional design. Respondents were patients who were 6 months post-discharge after non-hemorrhagic stroke and their family caregivers. Information about respondents was taken from medical record data at two regional general hospitals in West Kalimantan Province, Indonesia. A total of 80 patients were selected using a consecutive sampling method. Theoretical models of patient and caregiver factors that influence adaptation responses and post-stroke quality of life were tested using path analysis. Results: Caregiver coping, self-efficacy, and illness acceptance had a direct effect on the post-stroke psychosocial adaptation response by 58.1%, with self-efficacy contributing the most (β=0.668, p<0.0001). Self-efficacy, illness acceptance, and healthy behavior had a direct effect on the physiological adaptation response by 24.3%, where self-efficacy also contributed the most (β=0.272, p<0.014). Psychosocial adaptation and physiological adaptation had a direct effect on the quality of life by 54.6%, where psychosocial adaptation showed the largest contribution (β=0.63, p<0.0001). Conclusion: Self-efficacy contributes the most to both psychosocial and physiological adaptations 6 months after stroke. Psychosocial adaptation and self-efficacy have been proven to be the determinant factors that contribute the most to the quality of life of patients 6 months after stroke.(AU)

The factors that influence the adaptation process 6 months after a stroke: A path analysis.

OBJECTIVE: The purpose of this study was to identify the determinant factors that influence the adaptation process and quality of life after a stroke. METHODS: This study is an observational study using a cross-sectional design. Respondents were patients who were 6 months post-discharge after non-hemorrhagic strokae and their family caregivers. Information about respondents was taken from medical record data at two regional general hospitals in West Kalimantan Province, Indonesia. A total of 80 patients were selected using a consecutive sampling method. Theoretical models of patient and caregiver factors that influence adaptation responses and post-stroke quality of life were tested using path analysis. RESULT: Caregiver coping, self-efficacy, and illness acceptance had a direct effect on the post-stroke psychosocial adaptation response by 58.1%, with self-efficacy contributing the most (ß = 0.668, P < .0001). Self-efficacy, illness acceptance, and healthy behavior had a direct effect on the physiological adaptation response by 24.3%, where self-efficacy also contributed the most (ß = 0.272, P < .014). Psychosocial adaptation and physiological adaptation had a direct effect on the quality of life by 54.6%, where psychosocial adaptation showed the largest contribution (ß = 0.63, P < .0001). CONCLUSION: Self-efficacy contributes the most to both psychosocial and physiological adaptations 6 months after stroke. Psychosocial adaptation and self-efficacy have been proven to be the determinant factors that contribute the most to the quality of life of patients 6 months after stroke.

Genetic characterization of dengue virus 4 complete genomes from East Java, Indonesia.

Dengue is an endemic arboviral disease with continuous transmission in Indonesia for more than five decades. A recent outbreak in Jember, East Java province, demonstrated the predominance of DENV-4, a serotype known for its low global spread and limited transmission. While epidemiological factors such as new serotype introduction and lacking herd immunity may explain its predominance, viral factors may also contribute. Using next-generation sequencing, we generated 13 representative complete genomes of DENV-4 responsible for the outbreak. Phylogenetic and evolutionary analyses on complete genomes were performed to understand the spatial and temporal dynamics of the viruses. Further analyses were done to study amino acid variations in DENV genes, as well as the potential events of recombination and selection pressure within the genomes. We revealed the DENV-4 genetic factors that may lead to its predominance in the 2019 Jember dengue outbreak. A combination of selection pressure and mutational genetic changes may contribute to the DENV-4 predominance in East Java, Indonesia. The possible intra-serotype recombination events involving the non-structural protein 5 (NS5) gene were also observed. Altogether, these genetic factors may act as additional factors behind the complex dengue outbreak mechanism.

Non-structural protein 1 and hematology parameters as predictors of dengue virus infection severity in Indonesia.

Dengue virus infection (DVI) remains a significant health challenge, and diagnosis must still be considered. Non-structural protein 1 (NS1) is a potential marker of the dengue virus that can help diagnose DVI. The study aimed to assess the role of NS1 as a predictor of the severity of DVI. We utilized Dengue PCR-confirmed samples and employed semi-quantitative NS1Ag ELISA for NS1 examination, adhering to the World Health Organization South-East Asia Region (WHO-SEARO) 2011 criteria for DVI. We included DVI patients from Indonesia aged 1-65 years. Secondary infections had more severe clinical conditions than primary infections. Leukocyte and platelet levels had a more significant effect on NS1 positivity (6.19 (1.9-30.2); p<0.001; 190 (11-417); p=0.015; respectively). Multivariate analysis revealed leukocytes as a more significant predictor of NS1 values than platelets, with an odds ratio of 5.38 contributing to 30.5% of the NS1 value variation. The NS1 value could distinguish undifferentiated fever and dengue fever in the children group with a sensitivity of 76.0% and specificity of 87.5% (p=0.015). The number of NS1(-) in the severe dengue hemorrhagic fever (DHF) group was higher than NS1(+). DENV-4 type and primary infection were dominant in this study, although they did not significantly differ from the NS1 value. NS1 value can be used as a predictor to determine the severity of DVI in children but not in the adult group. The levels of leukocytes and platelets influenced the NS1 value.

Suppressive effect of goat bile in Plasmodium berghei ANKA infection in mice.

BACKGROUND AND AIM: Some individuals in Indonesia consume intact goat gallbladder to prevent and treat malaria. The acute and subacute toxicity tests of goat bile (GB) have shown mild diarrhea in mice. Therefore, this study aimed to evaluate the suppressive effect of GB on parasitemia, splenomegaly, hepatomegaly, and blood biochemistry to assess liver and kidney function in BALB/c mice infected with Plasmodium berghei ANKA. MATERIALS AND METHODS: Fifty healthy mice were infected with P. berghei ANKA and divided into five groups. Mice in three groups were administered 0.5 mL of 25%, 50%, or 100% of GB by gavage. Animals in Group 4 were administered 187.2 mg/kg BW of dihydroartemisinin-piperaquine phosphate as a positive control (POS Group). Mice in fifth group were administered sterile water as negative (NEG) controls. Further, 30 uninfected mice were divided into groups 6-8 and administered GB as were mice in the first three groups. Group 9 included 10 uninfected and untreated animals as healthy controls. Treatments were administered in a 4-day suppressive test followed by daily observation of Giemsa-stained blood smears. On day 7, mice were sacrificed to measure the length and weight of spleens and livers, plasma levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine. RESULTS: GB suppressed parasitemia but did not affect the size and weight of spleens or livers or plasma levels of AST and ALT compared to uninfected GB-treated and healthy control animals. Conversely, plasma levels of BUN and creatinine were suppressed and remained in the normal range in all groups of mice. CONCLUSION: GB suppresses parasitemia with no significant impact on hepatic enzymes in GB-treated infected mice. Liver dysfunction in GB-treated infected mice was due to P. berghei rather than GB treatment.

Performance comparison of two malaria rapid diagnostic test with real time polymerase chain reaction and gold standard of microscopy detection method.

Background: The diagnostic test for malaria is mostly based on Rapid Diagnostic Test (RDT) and detection by microscopy. Polymerase Chain Reaction (PCR) is also a sensitive detection method that can be considered as a diagnostic tool. The outcome of malaria microscopy detection depends on the examiner's ability and experience. Some RDT has been distributed in Indonesia, which needs to be evaluated for their results. Objective: This study aimed to compare the performance of RightSign RDT and ScreenPlus RDT for detection of Plasmodium in human blood. We used specific real-time polymerase chain reaction abTESTMMalaria qPCRII) and gold standard of microscopy detection method to measure diagnostic efficiency. Methods: Blood specimens were evaluated using RightSign RDT, ScreenPlus RDT, Microscopy detection, and RT-PCR as the protocol described. The differences on specificity (Sp), sensitivity (Sn), positive predictive value (PPV), and negative predictive value (NPV) were analyzed using McNemar and Kruskal Wallis analysis. Results: A total of 105 subjects were recruited. Based on microscopy test, RightSign RDT had sensitivity, Specificity, PPV, NPV, 100%, 98%, 98.2%, 100%, respectively. ScreenPlus showed 100% sensitivity, 98% specificity, 98.2% PPV, 100% NPV. The sensitivity of both RDTs became lower (75%) and the specificity higher (100 %) when using real-time PCR. Both RDTs showed a 100% agreement. RTPCR detected higher mix infection when compared to microscopy and RDTs. Conclusion: RightSign and ScreenPlus RDT have excellent performance when using microscopy detection as a gold standard. Real-time PCR method can be considered as a confirmation tool for malaria diagnosis.

Dengue Virus Serotype 4 Is Responsible for the Outbreak of Dengue in East Java City of Jember, Indonesia.

Outbreaks of dengue virus (DENV) in Indonesia have been mainly caused by the DENV serotype-1; -2; or -3. The DENV-4 was the least-reported serotype in Indonesia during the last five decades. We recently conducted a molecular epidemiology study of dengue in the Jember regency, East Java province, Indonesia. Dengue is endemic in the region and outbreaks occur annually. We investigated the clinical characteristics and etiology of dengue-like febrile illness in this regency to understand the disease dynamics. A total of 191 patients with clinical symptoms similar to dengue were recruited during an 11-month study in 2019-2020. Children accounted for the majority of cases and dengue burden was estimated in 41.4% of the cases based on NS1 antigen, viral RNA, and IgG/IgM antibody detection with the majority (73.4%) being primary infections. Secondary infection was significantly associated with a higher risk of severe dengue manifestation. All four DENV serotypes were detected in Jember. Strikingly, we observed the predominance of DENV-4, followed by DENV-3, DENV-1, and DENV-2. Genotype determination using Envelope gene sequence revealed the classification into Genotype I, Cosmopolitan Genotype, Genotype I, and Genotype II for DENV-1, -2, -3, and -4, respectively. The predominance of DENV-4 in Jember may be associated with a new wave of DENV infections and spread in a non-immune population lacking a herd-immunity to this particular serotype.

Acute and subacute toxicity tests of goat bile in BALB/c mice.

AIM: The aim of this study was to investigate the toxicity of goat bile in BALB/c mice since some Indonesian people consume raw goat gallbladder to treat malaria and increase stamina. MATERIALS AND METHODS: Acute toxicity test was done in six groups of BALB/c mice using 100%, 50%, 25%, 12.5%, and 6.75% of goat bile and negative control. The death of mice was observed within 14 days. In the subacute toxicity test, the body weight and hematology parameters on day 0 and day 4 post-treatment were evaluated. The mice were closely observed for 28 days before plasma collection for the blood biochemistry evaluation. RESULTS: Mild diarrhea was observed in acute and subacute toxicity tests. No death of mice was observed in acute test. Goat bile did not inhibit the increase of the body weight of mice. A slight reduction in hemoglobin and hematocrit levels in mice treated with 25% and 50% goat bile, however, remained normal in mice treated with 100% goat bile. The red and white blood cell count were not affected. Liver and kidney functions were not affected by goat bile treatment as revealed by the plasma level of aspartate aminotransferase and alanine aminotransferase, blood urea nitrogen, and creatinine, which remained in the normal range. CONCLUSION: Goat bile treatment in BALB/c mice caused mild toxicity in mice. Hydrophobic bile acids may cause the toxicity of goat bile in mice; therefore, it is recommended that goat bile consumption not to be taken oftenly to avoid its harmful effect.

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.

Detection and Serotyping of Dengue Viruses in Aedes aegypti and Aedes albopictus (Diptera: Culicidae) Collected in Surabaya, Indonesia from 2008 to 2015.

Aedes aegypti and Aedes albopictus are the primary and secondary vectors, respectively, of dengue, the most important arboviral disease in the world. The aim of this study was to detect and serotype dengue viruses (DENV) in the vectors Ae. aegypti and Ae. albopictus in Surabaya, Indonesia. Between 2008 and 2015, 16,605 Aedes mosquitoes were collected in 15 sub-districts of Surabaya. Ae. aegypti was dominant (90.9%), whereas few Ae. albopictus were collected (9.1%). A total of 330 pools of adult Aedes mosquitoes were subjected to the serotyping of DENV by RT-PCR. DENV-1 (52.3%) was the most frequently detected serotype, followed by DENV-2 (40.3%), DENV-4 (4.6%), and DENV-3 (2.8%). The average minimum infection rate for Ae. aegypti in various sub-districts of Surabaya was 7.2 per 1,000 mosquitoes, while that for Ae. albopictus was 0.7 per 1,000 mosquitoes. The results showed that the predominantly circulating DENV serotype in mosquitoes continuously shifted from DENV-2 (2008) to DENV-1 (2009-2012), to DENV-2 again (2013-2014), and then back to DENV-1 (2015). The circulating DENV serotypes in mosquitoes were generally consistent with those in humans. Therefore, the surveillance of infected mosquitoes with DENV might provide an early warning sign for the risk of future dengue outbreaks.

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.

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.

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

BACKGROUND: Early 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. METHODS: The 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. RESULTS: Using 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. CONCLUSIONS: We 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.