Evaluation of a Commercial SD Dengue Virus NS1 Antigen Capture Enzyme- Linked Immunosorbent Assay Kit for Early Diagnosis of. Dengue Virus Infection. ABSTRACTEarly definitive diagnosis of dengue virus infection may help in the timely management of dengue virus infection.
We evaluated. the Standard Diagnostics (SD, South Korea) dengue virus nonstructural protein NS1 antigen enzyme- linked immunosorbent assay. SD dengue NS1 Ag ELISA) for the detection of dengue virus NS1 antigen in patients' sera, using a total of 3. RT)- PCR, an in- house Ig. M capture (MAC)- ELISA, and a hemagglutination. HI) assay. Of the 3. NS1 antigen compared to 3. MAC- ELISA or RT- PCR, 1.
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RT- PCR, and 2. 26 (7. MAC- ELISA only. The assay was able to detect NS1 antigen. The NS1 detection rate is inversely proportional while the Ig.
The complement system is a part of the immune system that enhances (complements) the ability of antibodies and phagocytic cells to clear microbes and damaged cells from an organism, promotes inflammation, and attacks membrane. Antigen targeting 1, 2, 3, 4, 5 and adjuvancy schemes 6, 7 that respectively facilitate delivery of antigen to dendritic cells and elicit their activation have been explored in vaccine development. Evaluation of a Commercial SD Dengue Virus NS1 Antigen Capture Enzyme-Linked Immunosorbent Assay Kit for Early Diagnosis of Dengue Virus Infection.
M detection. rate is directly proportional to the presence of Ig. G antibodies. The overall sensitivity and specificity of the SD dengue.
NS1 Ag ELISA in the detection of “confirmed dengue virus” sera are 7. This suggests that the. SD kit is highly specific and sensitive for the detection of NS1 antigen. However, caution is needed when the kit is used. Combining this assay with an Ig. M. and/or Ig. G assay will increase the sensitivity of detection, especially in areas with a higher prevalence of secondary dengue. The global prevalence of dengue has grown dramatically in recent decades.
The disease is now endemic. Africa, the Americas, the Eastern Mediterranean, the Western Pacific, and particularly, in Southeast. Asia. The World Health Organization (WHO) estimates that more than 2. Among these infections, approximately 5. DHF), with 2. 4,0. It comprises four closely related but antigenically distinct serotypes, namely, DENV- 1, DENV- 2, DENV- 3, and DENV- 4.
All. these four serotypes of dengue virus can be distinguished by both serological and molecular methods. The dengue virus genome. RNA with an approximate size of 1.
NS1 does not form part of the virion but is released from the dengue virus- infected cells. Preliminary studies have shown. RNA replication, and it has been found in acute- phase blood samples.
This has suggested a possible major involvement of NS1 in dengue virus pathogenesis and its possible use as a suitable. Several approaches have been applied for laboratory diagnosis of dengue. These methods include detection of the virus (by cell culture or immunofluorescence), detection of virus.
Ag) (by enzyme- linked immunosorbent assay . Recently, commercially available kits for the detection of dengue virus NS1 antigen. NS1 antigen could be useful for the detection of early stages. In this study, we describe the evaluation of an ELISA kit manufactured by Standard Diagnostics, Inc. These blood samples were collected from patients admitted to the University. Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia, for acute viral infection.
These sera consisted of (i) 3. RT- PCR positive, (iii) 5. NS1 antigen was detected by both the Platelia dengue NS1 antigen kit (Bio- Rad Laboratories, Marnnes- la- Coquette. France) and pan- E dengue early ELISA kit (Panbio, Queensland, Australia), (iv) 5. Ig. M negative but seroconverted. Ig. M to dengue virus was detected, (viii) 2.
All samples were subjected to virus isolation, in- house Ig. M capture ELISA (MAC- ELISA). RT- PCR, and the hemagglutination inhibition (HI) assay. Sera that tested positive for the. RNA or serologically positive for recent acute dengue virus infection were categorized as confirmed.
Written informed. Ethical clearance was obtained from the Scientific and Ethical Committee at the UMMC. Virus was then detected by immunofluorescence with dengue virus- specific monoclonal antibodies obtained. Centers for Disease Control and Prevention (Fort Collins, CO). Twenty- five microliters of antigen was added to the first 1.
BABS was added to the last. Freshly prepared goose red blood.
The results. were then interpreted according to WHO guidelines (1. After washing thrice with phosphate- buffered saline (PBS)- Tween 2.
The plates were then washed three times with PBS- Tween. The plates were washed again with PBS- Tween 2. After three additional. The reaction was. The positive- versus- negative ratio was then calculated. The positive control/sample.
OD was divided by the mean of the negative OD to obtain a positive/negative ratio (P/N). A P/N ratio equal to or greater than. A result with a P/N ratio of less than 2. The thermal cycling conditions consisted of a 3. Following amplification, the melting curves were analyzed. This is to verify the specificity.
PCR product by looking at its melting temperature (Tm). Melting curve analysis consisted of a denaturation step at 9.
The Tm of each specific PCR product was analyzed using i. Cycler i. Q optical system software version 3.
Bio- Rad Laboratories, Marnes- la- Coquette. France). The Tm for each sample was used to identify the dengue virus serotype, and the samples sharing the same Tm were interpreted as belonging to the same serotype. Detection for the presence of NS1 antigen was performed. The microplate was mixed using a vibrating mixer and incubated at 3. The plate was then washed five. After 1 h of incubation. The plate was further incubated at room temperature for 1.
The results were then interpreted. Statistical. analysis was performed with Statistica version 1.
Stat. Soft, Inc., United States). The sensitivity, specificity, efficiency. PPV), and negative predictive value (NPV) for the assays were calculated based on confirmed dengue.
PCR positive, seronegative acute- phase sera, acute primary, or acute secondary) using the following. The assay was evaluated against a panel of samples, including “confirmed positive sera” (validated by virus isolation. PCR), Ig. M- positive sera, NS1 antigen- positive sera, and sera from healthy donors and other disease agents. The. results are summarized in Table 1.
Of the 3. 20 dengue sera, 2. NS1 antigen (with the SD dengue NS1 Ag ELISA kit), compared to. RT- PCR, 2. 26 (7.
MAC- ELISA, and 3. RT- PCR or MAC- ELISA. Among. the 3. 0 samples that were positive by virus isolation, the SD dengue NS1 Ag ELISA detected 2. One sample. from the category that was clinically dengue but negative by laboratory tests, as well as another sample from the negative- control. SD dengue NS1 Ag ELISA. The detection rate by the SD dengue NS1 Ag ELISA in the absence of Ig.
M was 8. 2. 4. 2% (7. Ig. M. As the levels of Ig. M antibodies increased, the NS1 detection rate decreased. Further analysis.
NS1 detection rate is inversely proportional to the presence of Ig. G antibodies as depicted.
HI value (Fig. At lower levels of antibodies (HI . This suggests that the SD dengue NS1 Ag ELISA is more sensitive for diagnosis in the absence. HI value versus positive detection rate for MAC- ELISA, real- time RT- PCR, and NS1 antigen. The frequency of detection of NS1 through day 7 after the onset of fever was compared (Fig. For the first seven days of fever, 6.
NS1 antigen, compared to 5. RT- PCR and 6. 7.
MAC- ELISA, whereas for fever that lasted from day 8 through day 1. NS1 antigen, compared to 1. RT- PCR and 1. 00% (4. MAC- ELISA. 2. Comparison of RT- PCR, NS1 antigen detection, and Ig. M for detection after onset of fever. We further analyzed the data with regard to day of onset of disease symptoms.
NS1 antigen could be detected up to day 1. RT- PCR was only able to detect viral RNA up to. The kit was also used to detect dengue virus- positive samples comprising all four serotypes of dengue. Table 2. No significant difference was observed between the 4 serotypes with a P value of > 0. The overall sensitivity and specificity of the kit are 7. Isolation of viruses can take from 7 to 1.
Ig. M antibody or a rise in Ig. G antibody titer in paired acute- and convalescent- phase sera. Serological tests are generally. Ig. M capture ELISA formats. More than 9. 0%. of patients are Ig. M positive by the 4th day of illness, but the Ig.
M antibody may be due to infection up to 3 months earlier. These kits. do not require specialized training but their sensitivity and specificity is very variable. The choice of a test, therefore. Hence, diagnosis of dengue disease during the acute phase should be a priority for patients and for public. In- house Ig. M capture ELISAs that have been the mainstay of dengue diagnosis in many laboratories throughout. First, Ig. M is detected only after day 3 of the onset of symptoms, making early diagnosis. Second, because flaviviruses share common.
E) protein, a high degree of cross- reactivity is frequently observed. After. the onset of illness, the virus is found in serum, plasma, and circulating blood cells, as well as other tissues, for 4 to.
As such, during the early stages of the disease, virus isolation or nucleic acid or antigen detection can be used. At the end of the acute phase of infection, serology is the method of choice for diagnosis. Both the Pan. Bio dengue NS1 antigen capture ELISA and the Platelia dengue NS1 antigen assay were shown to be able to detect. NS1 antigen in acute- phase sera from both primary and secondary infections (1, 4, 1. Their sensitivity also decreased with increasing concentrations of antibody. In this study, we evaluated the use of the. SD dengue NS1 Ag ELISA for the diagnosis of dengue virus infections.
The analysis of all serum samples in the groups revealed. NS1 antigen was lowest in the RT- PCR- positive group (6.
Table 1). The sensitivity was highest in the primary acute dengue serum group (1. In both groups, the detection of NS1 in the convalescent phase. Although the analysis was being done on 1. NS1 antigen in acute patient sera, with the assay being more efficient at the acute phase of primary. This concurs with the findings of Dussart et al. Ig. G and Ig. M antibodies affected the detection of NS1.
However, NS1 was detectable up to day 1. The specificity for NS1 antigen was shown to be 9. NS1 antigen). Louis encephalitis virus, Rocio virus, and related viruses in this group, need to. Obtaining such sera that are dengue virus antibody negative.