Kesesuaian Uji Antigen Capture Enzyme Linked Immunosorbant Assay dan Nested Multiplex Polymerase Chain Reaction untuk Diagnosis Rutin Bovine Viral Diarrhea

Bovine Viral Diarrhea (BVD) is one of the main causes of impaired productivity and reproduction of cows. Antigen capture Elisa (ACE) is one of the serological technique that is sensitive, reliable and used regularly for detecting persistent BVD infection individually which simpler than multiplex nested PCR. The aim of this study was to determine the agreement between ACE and multiplex nested PCR as a routine laboratory diagnostic technique to detect the presence of BVD infection. A total of 128 cow serum samples consisting of 63 positive and 65 negative samples based on ACE were used in this study. The samples were collected from active and passive surveillance in dairy and beef cattle conducted by Balai Besar Veteriner (BBVet) Wates. The serum samples were then tested molecularly using multiplex nested PCR against BVD. The result showed 48 out of 63 BVDV-1 positive samples were found positive BVD antigen whereas 57 of 65 BVDV-1 negative samples were negative using multiplex nested PCR, . The agreement value between the two different assays based on statistic analysis using Kappa method was 0.64 and classified a good one. The result concluded that the ACE BVD assay was equally suitable as routine diagnosis to determine BVD infected cattle in the farm. Keywords: Antigen capture ELISA; Bovine viral diarrhea; Kappa; Multiplex nested PCR. Abstrak Bovine Viral Diarrhea (BVD) merupakan salah satu penyebab gangguan produktivitas dan reproduksi sapi. Antigen capture ELISA (ACE) merupakan salah satu teknik serologis yang sensitif, dapat diandalkan dan digunakan secara teratur untuk mendeteksi infeksi BVD persisten secara individual yang lebih sederhana daripada multiplex nested PCR. Tujuan penelitian ini adalah untuk mengetahui kesesuaian antara uji ACE dan multiplex nested PCR sebagai teknik diagnostik laboratorium rutin untuk mendeteksi adanya infeksi BVD. Sebanyak 128 sampel serum sapi yang terdiri dari 63 sampel positif dan 65 negatif berdasarkan ACE BVDV Antigen Test Kit/Serum Plus (Idexx®) digunakan dalam kajian ini. Sampel serum sapi merupakan koleksi dari surveilans aktif dan pasif pada sapi perah dan potong yang dilakukan Balai Besar Veteriner (BBVet) Wates. Sampel serum kemudian diuji secara molekuler menggunakan multiplex nested PCR terhadap BVD. Hasil penelitian menunjukkan bahwa dengan teknik multiplex nested PCR, 48 dari 63 sampel positif BVDV-1 ditemukan positif untuk antigen BVD sedangkan 57 dari 65 sampel negatif BVDV-1 negatif untuk antigen BVD. Analisis statitik berdasarkan perhitungan metoda Kappa menunjukkan nilai kesesuaian antara dua uji sebesar 0,64 dan tergolong bagus. Hasil penelitian menunjukkan kesimpulan bahwa uji ACE BVD sesuai sebagai diagnosis rutin untuk menentukan ternak yang terinfeksi BVD di peternakan. Kata kunci: Antigen capture ELISA; Bovine viral diarrhea; Kappa; Multiplex nested PCR.

Development of Monoclonal Antibody Based IgG and IgM ELISA for Diagnosis of Severe Fever with Thrombocytopenia Syndrome Virus Infection

Background: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly emerged virus that possesses a great threat to human health because of the high fatality rate. Method: To develop sensitive and specific sero-diagnosis systems for SFTSV infections, monoclonal antibodies (MAbs) against recombinant SFTSV nucleocapsid (rSFTSV-N) protein were developed by immunizing BALB/C mice with rSFTSV-N protein and fusing the spleen cells with SP2/0 myeloma cells. Three hybridoma cell lines secreting MAbs against rSFTSV-N were obtained. MAb based IgG sandwich enzyme linked immunosorbent assay (ELISA) and IgM capture ELISA systems were established by using the newly developed MAbs. One hundred fifteen clinical suspected SFTS patient serum samples were used to evaluate the newly established systems by comparing with the total antibody detecting sandwich ELISA system and indirect ELISA systems. Results: The MAb based sandwich IgG ELISA was perfectly matched with that of the total antibody sandwich ELISA and the indirect IgG ELISA with a sensitivity and specificity of 100%. IgM capture ELISA results perfectly matched with that of the total antibody sandwich ELISA while was more sensitive comparing with the indirect IgM ELISA. Conclusions: The MAbs against rSFTSV-N protein offer new tools for SFTSV studies and our newly developed MAb-based IgG and IgM capture ELISA systems would offer safe and useful tools for diagnosis of SFTS virus infections and epidemiological investigations.

Capacity of a Multiplex IgM Antibody Capture ELISA to Differentiate Zika and Dengue Virus Infections in Areas of Concurrent Endemic Transmission

Serological cross-reactivity has proved to be a challenge to diagnose Zika virus (ZIKV) infections in dengue virus (DENV) endemic countries. Confirmatory testing of ZIKV IgM positive results by plaque reduction neutralization tests (PRNTs) provides clarification in only a minority of cases because most individuals infected with ZIKV were previously exposed to DENV. The goal of this study was to evaluate the performance of a ZIKV/DENV DUO IgM antibody capture ELISA (MAC-ELISA) for discriminating between DENV and ZIKV infections in endemic regions. Our performance evaluation included acute and convalescent specimens from patients with real-time reverse transcription polymerase chain reaction (RT-PCR)-confirmed DENV or ZIKV from the Sentinel Enhanced Dengue Surveillance System in Ponce, Puerto Rico. The ZIKV/DENV DUO MAC-ELISA specificity was 100% for DENV (N = 127) and 98.4% for ZIKV (N = 275) when specimens were tested during the optimal testing window (days post-onset of illness [DPO] 6–120). The ZIKV/DENV DUO MAC-ELISA sensitivity of RT-PCR confirmed specimens reached 100% for DENV by DPO 6 and for ZIKV by DPO 9. Our new ZIKV/DENV DUO MAC-ELISA was also able to distinguish ZIKV and DENV regardless of previous DENV exposure. We conclude this novel serologic diagnostic assay can accurately discriminate ZIKV and DENV infections. This can potentially be useful considering that the more labor-intensive and expensive PRNT assay may not be an option for confirmatory diagnosis in areas that lack PRNT capacity, but experience circulation of both DENV and ZIKV.

Immobilization of Proteinase K for urine pretreatment to improve diagnostic accuracy of active tuberculosis

The World Health Organization (WHO) calls for the development of a rapid, biomarker-based, non-sputum test capable of detecting all forms of tuberculosis (TB) at the point-of-care to enable immediate treatment initiation. Lipoarabinomannan (LAM) is the only WHO-endorsed TB biomarker that can be detected in urine, an easily collected sample matrix. For obtaining optimal sensitivity, we and others have shown that some form of sample pretreatment is necessary to remove background from patient urine samples. A number of systems are paper-based often destined for resource limited settings. Our current work presents incorporation of one such sample pretreatment, proteinase K (ProK) immobilized on paper (IPK) and test its performance in comparison to standard proteinase K (SPK) treatment that involves addition and deactivation at high temperature prior to performing a capture ELISA. Herein, a simple and economical method was developed for using ProK immobilized strips to pretreat urine samples. Simplification and cost reduction of the proposed pretreatment strip were achieved by using Whatman no.1 paper and by minimizing the concentration of ProK (an expensive but necessary reagent) used to pretreat the clinical samples prior to ELISA. To test the applicability of IPK, capture ELISA was carried out on either LAM-spiked urine or the clinical samples after pretreatment with ProK at 400 μg/mL for 30 minutes at room temperature. The optimal conditions and stability of the IPK were tested and validation was performed on a set of 25 previously analyzed archived clinical urine samples with known TB and HIV status. The results of IPK and SPK treated samples were in agreement showing that the urine LAM test currently under development has the potential to reach adult and pediatric patients regardless of HIV status or site of infection, and to facilitate global TB control to improve assay performance and ultimately treatment outcomes.

Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

Infection with Zika virus (ZIKV), a member of the Flavivirus genus of the Flaviviridae family, typically results in mild self-limited illness, but severe neurological disease occurs in a limited subset of patients. In contrast, serious outcomes commonly occur in pregnancy that affect the developing fetus, including microcephaly and other major birth defects. The genetic similarity of ZIKV to other widespread flaviviruses, such as dengue virus (DENV), presents a challenge to the development of specific ZIKV diagnostic assays. Nonstructural protein 1 (NS1) is established for use in immunodiagnostic assays for flaviviruses. To address the cross-reactivity of ZIKV NS1 with proteins from other flaviviruses we used site-directed mutagenesis to modify putative epitopes. Goat polyclonal antibodies to variant ZIKV NS1 were affinity-purified to remove antibodies binding to the closely related NS1 protein of DENV. An antigen-capture ELISA configured with the affinity-purified polyclonal antibody showed a linear dynamic range between approximately 500 and 30 ng/mL, with a limit of detection of between 1.95 and 7.8 ng/mL. NS1 proteins from DENV, yellow fever virus, St. Louis encephalitis virus and West Nile virus showed significantly reduced reactivity in the ZIKV antigen-capture ELISA. Refinement of approaches similar to those employed here could lead to development of ZIKV-specific immunoassays suitable for use in areas where infections with related flaviviruses are common.

Seroprevalence of Dengue Infection Using IgG Capture ELISA in India, 2017–2018

We conducted a nationally representative population-based survey in 60 districts from 15 Indian states covering all five geographic regions during 2017–2018 to estimate the age specific seroprevalence of dengue. Of the 12,300 sera collected, 4,955 were positive for IgG antibodies against dengue virus using IgG Indirect ELISA indicating past dengue infection. We tested 4,948 sera (seven had inadequate volume) positive for IgG antibodies on indirect ELISA using anti-dengue IgG capture ELISA to estimate the proportion of dengue infections with high antibody titers, suggestive of acute or recent secondary infection. Of the 4,948 sera tested, 529 (10.7%; 95% CI: 9.4–12.1) were seropositive on IgG capture ELISA. The proportions of dengue infections with high titers were 1.1% in the northeastern, 1.5% in the eastern, 6.2% in the western, 12.2% in the southern, and 16.7% in the northern region. The distribution of dengue infections varied across geographic regions, with a higher proportion of infections with high antibody titer in the northern and southern regions of India. The study findings could be useful for planning facilities for clinical management of dengue infections.

Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

Infection with Zika virus (ZIKV), a member of the Flavivirus genus of the Flaviviridae family, typically results in mild self-limited illness, but severe neurological disease occurs in a limited subset of patients. In contrast, serious outcomes commonly occur in pregnancy that affect the developing fetus, including microcephaly and other major birth defects. The genetic similarity of ZIKV to other widespread flaviviruses, such as dengue virus (DENV), presents a challenge to the development of specific ZIKV diagnostic assays. Nonstructural protein 1 (NS1) is established for use in immunodiagnostic assays for flaviviruses. To address the cross-reactivity of ZIKV NS1 with proteins from other flaviviruses we used site-directed mutagenesis to modified putative epitopes. Goat polyclonal antibodies to variant ZIKV NS1 were affinity-purified to remove antibodies binding to the closely related NS1 protein of DENV. An antigen-capture ELISA configured with the affinity-purified polyclonal antibody showed a linear dynamic range between approximately 500 to 30 ng/mL, with a limit of detection of between 1.95 and 7.8 ng/mL. NS1 proteins from DENV, yellow fever virus, St. Louis encephalitis virus and West Nile virus showed significantly reduced reactivity in the ZIKV antigen-capture ELISA. Refinement of approaches similar to those employed here could lead to development of ZIKV-specific immunoassays suitable for use in areas where infections with related flaviviruses are common.