Use of dengue NS1 antigen for early diagnosis of dengue virus infection

Accurate and timely diagnosis of dengue virus is important for early detection of dengue virus infection. In this study, the usefulness of the dengue NS1 antigen test was evaluated as a routine, rapid diagnostic test for dengue virus infection. A total of 208 sera from patients suspected of having dengue virus infection were collected and tested for dengue antibody, dengue genome and dengue NS1 antigen. Dengue antibody test, dengue PCR test and dengue antigen test were able to detect dengue virus infection from Days 1 to 8 in 72.8, 52.8 and 44.0% of samples, respectively. Of the 208 sera tested, 69.2% (144/208) of the acute sera were positive for dengue virus infection based on IgM antibody, IgG antibody, NS1 antigen and PCR tests. Thirty-two point two percent of the samples (67/208) were found positive for dengue NS1 antigen, 38.5% (80/208) were PCR positive, 40.9% (85/208) were IgM positive and 36.1% (75/208) were IgG positive for dengue virus. The results reveal the detection rate of dengue virus infection was similar for PCR and dengue antibody (65.9%) and for NS1 antigen and dengue antibody (62.0%) combinations. Therefore, the dengue NS1 antigen test can be used to complement the current antibody test used in peripheral laboratories. Thus, the combination of the NS1 antigen and antibody tests could increase the diagnostic efficiency for early diagnosis of dengue infection.     

NS1 antigen: A new beam of light in the early diagnosis of dengue infection

Objective: To evaluate NS1 antigen detection ELISA for the early laboratory diagnosis of dengue virus infection. Methods: The present study was conducted to evaluate the overall positivity of NS1 antigen detection ELISA and its comparison with viral RNA detection via real time PCR and Ig M antibodies detection by ELISA. Results: A total of 1 270 serum samples were tested 86%(1 097/1 270) were detected positive by one or more than one diagnostic test. Out of 1 270, 64%(807/1 270) were positive by NS1 ELISA and 52%(662/1 270), 51%(646/1 270) were positive by real-time RT-PCR and Ig M ELISA respectively.Conclusions: NS1 antigen detection ELISA is highly suitable diagnostic tools and it also has great value for use in outbreak and epidemic situation.     

Facilitation of cell adhesion by immobilized dengue viral nonstructural protein 1 (NS1): arginine-glycine-aspartic acid structural mimicry within the dengue viral NS1 antigen

Dengue virus infection causes life-threatening hemorrhagic fever. Increasing evidence implies that dengue viral nonstructural protein 1 (NS1) exhibits a tendency to elicit potentially hazardous autoantibodies, which show a wide spectrum of specificity against extracellular matrix and platelet antigens. How NS1 elicits autoantibodies remains unclear. To address the hypothesis that NS1 and matrix proteins may have structural and functional similarity, cell-matrix and cell-NS1 interactions were evaluated using a cell-adhesion assay. The present study showed that dengue NS1 immobilized on coverslips resulted in more cell adhesion than did the control proteins. This cell adhesion was inhibited by peptides containing arginine-glycine-aspartic acid (RGD), a motif important for integrin-mediated cell adhesion. In addition, anti-NS1 antibodies blocked RGD-mediated cell adhesion. Although there is no RGD motif in the NS1 protein sequence, these data indicate that RGD structural mimicry exists within the NS1 antigen.     

双单克隆抗体夹心酶联免疫吸附试验检测鼠疫F1抗原的实验观察

目的 观察双单克隆抗体(F1-McAb)夹心酶联免疫试验(DMcAbS-ELISA)快速检测鼠疫F1抗原的敏感性和特异性.方法 采用鼠疫细菌学检验、DMcAbS-ELISA和反向间接血球凝集试验(RIHA)对比检测鼠疫感染鼠和阴性对照鼠脏器标本.结果 共检测225份阴性对照鼠脏器标本,鼠疫细菌学检验、DMcAbS-ELISA和RIHA法检测F1抗原均为阴性.共检测308只鼠疫感染鼠脏器标本,鼠疫细菌学检验、DMcAbS-ELISA、RIHA法阳性率分别为92.21％(284/308)、90.91％(280/308)和89.61％(276/308),3种方法比较,差异无统计学意义(x2=5.65,P＞0.05).DMcAbS-ELISA法与鼠疫细菌学检验结果符合率为97.00％[(274+243)/533],Kappa值为0.940;与RIHA法符合率为99.25％[(276+253)/533],Kappa值为0.985.脏器标本F1抗原检测的真实性比较:DMcAbS-ELISA法敏感性为96.48％(274/284),特异性为97.59％(243/249),阳性预测值为97.86％(274/280),阴性预测值为96.05％(243/253),一致性为96.99％11/4×(274/280+274/284+ 243/253+243/249)|,Youden指数为0.9407;RIHA法的敏感性为96.13％(273/284),特异性为98.80％(246/249),阳性预测值为98.91％(273/276),阴性预测值为95.72％(246/257),一致性为97.39％[1/4×(273/276+273/284+246/257+246/249)],Youden指数为0.9492.DMcAbS-ELISA法对鼠疫菌检测灵敏度为2.7×104cfu/ml,RIHA法为2.2×105 cfu/ml;两种方法检测F1抗原灵敏度均为10 μg/L.结论 DMcAbS-ELISA法检测鼠疫F1抗原具有敏感、特异、简便、快速的特点,是有应用价值的鼠疫快速诊断技术.     

Identification of a Theileria mutans-specific antigen for use in an antibody and antigen detection ELISA

Summary Purified piroplasms of Theileria mutans were used to immunize BALB/c mice to generate monoclonal antibodies (MoAbs). The MoAbs recognized an antigen of a relative molecular mass of 32 kDa in Western blots. This antigen was also recognized by sera from cattle which had recovered naturally from experimental tick-transmission or infections induced by the blood stages of T. mutans. The MoAbs did not react, in indirect immunofluorescence or enzyme-linked immunosorbent assays (ELISA), with the common haemoparasites of cattle, namely, T. parva, T. annulata, Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma congolense, T. vivax or T. brucei. An antigen capture ELISA was established with two of the MoAbs which recognized different epitopes on the 32 kDa molecule. Using this test it was possible to detect circulating antigens or immune complexes in sera collected from cattle during the acute or chronic phases of infection. When the purified 32 kDa protein was used as antigen in a micro-ELISA to detect circulating antibodies in both experimental and field cattle sera, it was found that the titres of antibodies ranged between 1:20 and 1:10 240. Results of this study indicate that the antigen and immune complex capture assays and the antibody detection ELISA can be complementary in the immunodiagnosis of acute and chronic T. mutans infections. Moreover, the tests are useful in the differential diagnosis of the disease and for epidemiological studies.     

Immunodiagnosis of infection with Schistosoma mansoni: enzyme-linked immunosorbent assay for detection of antibody to circulating antigen.

A circulating antigen, a negatively charged polysaccharide from the trematode Schistosoma mansoni, was noncovalently bound to the surface of poly(L-lysine)-coated wells in polystyrene trays, which were then used in a micro-enzyme-linked immunosorbent assay (ELISA) test. The method provides an immunodiagnostic test for schistosomiasis of exceptional sensitivity with a high degree of specificity. Comparison of Bell egg counts and ELISA titers revealed a good correlation (r congruent to 0.80) in young individuals with low to moderate worm burdens, but this relationship was less marked in older individuals or those with high egg counts.     

Identification of a Theileria mutans-specific antigen for use in an antibody and antigen detection ELISA.

Purified piroplasms of Theileria mutans were used to immunize BALB/c mice to generate monoclonal antibodies (MoAbs). The MoAbs recognized an antigen of a relative molecular mass of 32 kDa in Western blots. This antigen was also recognized by sera from cattle which had recovered naturally from experimental tick-transmission or infections induced by the blood stages of T. mutans. The MoAbs did not react, in indirect immunofluorescence or enzyme-linked immunosorbent assays (ELISA), with the common haemoparasites of cattle, namely, T. parva, T. annulata, Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma congolense, T. vivax or T. brucei. An antigen capture ELISA was established with two of the MoAbs which recognized different epitopes on the 32 kDa molecule. Using this test it was possible to detect circulating antigens or immune complexes in sera collected from cattle during the acute or chronic phases of infection. When the purified 32 kDa protein was used as antigen in a micro-ELISA to detect circulating antibodies in both experimental and field cattle sera, it was found that the titres of antibodies ranged between 1:20 and 1:10,240. Results of this study indicate that the antigen and immune complex capture assays and the antibody detection ELISA can be complementary in the immunodiagnosis of acute and chronic T. mutans infections. Moreover, the tests are useful in the differential diagnosis of the disease and for epidemiological studies.     

Combined use of an antigen and antibody detection enzyme-linked immunosorbent assay for cysticercosis as tools in an epidemiological study of epilepsy in Burundi

OBJECTIVE: To evaluate the benefits of the detection of both circulating antibodies (Ab) and antigens (Ag) for the diagnosis of cysticercosis in people with epilepsy. Neurocysticercosis is a cause of neurological diseases world-wide, especially epilepsy. The clinical symptoms of neurocysticercosis are non-specific and diagnosis is often difficult. METHODS: Serum samples were collected from subjects in a matched case-control study for epilepsy in the Kiremba area, Burundi, between March and April 2001 (epileptic cases=303; controls without epilepsy=606). The enzyme-linked immunosorbent assay (ELISA) was used for the detection of antibodies (Ab-ELISA) and circulating Ag (Ag-ELISA). RESULTS: The Ab-ELISA revealed 58.7% positivity in epilepsy cases and 31.4% in healthy controls; and Ag-ELISA revealed 38.3% positivity in epilepsy cases and 20.0% in controls. The matched odds ratios were 3.6 (95% CI: 2.5-4.9) for Ab-ELISA, and 2.9 (95% CI: 2.1-4.3) for Ag-ELISA. CONCLUSION: Both Ag- and Ab-ELISA detected a significantly higher number of seropositives among people with epilepsy than among controls. The risk of epilepsy was high in cases with a positive Ag-ELISA, although less important than in cases with positivity for Ab-ELISA. Dead or degenerating cysticerci appear to be more frequently associated with epilepsy than living cysts. The high number of people with circulating Ag of Taenia solium suggests that the study area is a focus of active transmission of the parasite.     

酶斑点杂交法与双抗体夹心法检测汉坦病毒感染的比较

目的将酶标抗汉坦病毒NP抗原单链抗体应用于酶斑点杂交法和双抗体夹心法,检测汉坦病毒感染,探索价格低廉早期HFRS诊断试剂。方法以戊二醛偶联辣根过氧化物酶和抗汉坦病毒NP抗原单链抗体,制备酶标单链抗体,并应用于酶斑点杂交法和双抗体夹心法中,检测56份早期HFRS患者血清及66份阴性对照血清。结果56份HFRS患者血清中,双抗体夹心法检测出29例阳性,阳性率为51.79%,酶斑点杂交法检测出28例阳性,阳性率为50.00%,两方法检测66例阴性对照血清,结果均为阴性。统计学处理,两方法差异不显著。结论以酶标抗汉坦病毒NP抗原单链抗体制备的酶斑点杂交和双抗体夹心诊断试剂,均为稳定性好、假阳性率低、造价低廉、诊断可靠的诊断试剂。     

Evaluation of new fourth-generation human immunodeficiency virus antigen and antibody detection assay with enzyme-linked fluorescent immunoassay

We evaluated the fourth-generation HIV screening assay VIDAS HIV DUOII (DUOII) based on ELFA for simultaneous detection of anti-HIV-1 and anti-HIV-2 antibodies and HIV-1 p24 antigen through comparison with other HIV antigen-antibody detection assays. Materials were 1228 HIV-negative specimens, 95 HIV-antibody-positive specimens, and HIV commercial panels. The specificity of DUOII was 99.8% and sensitivity 100%, detecting all of HIV-1 group M subtype A, B, B', C, D, A/E, F, G, B/D, HIV-1 group O, and HIV-2. The sensitivity test to HIV-1 p24 antigen was 5pg/ mL, higher than other assays. DUOII was equivalent to or superior in detecting results earlier than other assays in an evaluation using 10 commercial HIV-1 seroconversion panels of primary infection. DUOII detects anti-HIV IgM antibody, so no negative sample was found in the second window between p24 antigen disappearance and raised anti-HIV IgG antibody. DUOII has sufficient specificity and sensitivity for HIV screening, and detects primary infection sooner than other assays. These results indicate that DUOII is useful and reliable in HIV screening.     

High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever

Infection with any 1 of 4 dengue viruses produces a spectrum of clinical illness ranging from a mild undifferentiated febrile illness to dengue fever (DF) to dengue hemorrhagic fever (DHF), a potentially life-threatening disease. The morbidity and mortality of DHF can be reduced by early hospitalization and careful supportive care. To determine its usefulness as a predictor of DHF, plasma levels of the secreted dengue virus nonstructural protein NS1 (sNS1) were measured daily in 32 children with dengue-2 virus infections participating in a prospective, hospital-based study. Free sNS1 levels in plasma correlated with viremia levels and were higher in patients with DHF than in those with DF. An elevated free sNS1 level (> or =600 ng/mL) within 72 h of illness onset identified patients at risk for developing DHF.     

胶体金免疫层析法检测鼠疫F1抗原的研究

Objective To study the sensitivity and specificity of gold-immunochromatography assay (GICA) for detection of Yersiniapestis(Y. pestis ) F1 antigen. Methods Viscera organ(liver and spleen) specimens of 308 mice with virulent Y. pestis infection and 225 control specimens of rats(217 Spermophilus dauricus, 5 mice,3 guinea pigs) were detected by GICA dipstick with monoclonal antibody against plague F1 antigen (F1MAb).Meanwhile, micro-method of reverse indirect hemagglutination assay(RIHA) and bacteria culture were carried out for parallel testing. Results Bacteriological examination of 225 control specimens, and F1 antigen detected with GICA and RIHA were all negative. No cross-reaction with related Yersinia pseudotuberculosis at 1 x 108 cfu/ml level was found in GICA and RIHA. Detection sensitivity of Y. pestis by GICA and RIHA were 2.5 × 105 cfu/ml and 2.0 × 105 cfu/ml, respectively, and of F1 antigen were 1μg/L and 10 μg/L, respectively. Coincidence was 97.94% (522/533) between GICA and bacteriological test, Kappa = 0.959, and the difference was statistically insignificant(x2 = 0.36, P ＞ 0.05); and 97.94%(522/533) between GICA and RIHA, Kappa = 0.959, with statistically significant difference in the positive detection rates(x2 = 9.09, P ＜ 0.05). Out of the 308 infected mice, 284 were positive of plague bacterial cultured, In 284 samples with positive bacterial culture, there were 280 of positive detected by GICA for F1 antigen, positive rate of F1 antigen was 98.59%, higher than that by RIHA[the positive rate of 96.13%(522/533)], with statistically significant difference(x2 = 5.14, P ＜ 0.05). Sensitivity of GICA was 98.59% (280/284), specificity was 97.19% (242/249), positive predictive value (PPV) was 97.56% (280/287),negative predictive value ( NPV ) was 98.37% (242/246), and Youden index was 0.9578. Conclusions GICA is sensitive and specific, fast and simple in detection of F1 antigen of the plague. It's a valuable detection technique for early and rapid diagnosis of plague.     

胶体金免疫层析法检测鼠疫F1抗原的研究

目的 研究胶体金免疫层析试纸条检测鼠疫F1抗原的敏感性和特异性.方法通过鼠疫F1抗原单克隆抗体(F1MAb)捕捉F1抗原的胶体金免疫层析(GICA)试纸条,检测308只强毒鼠疫耶尔森菌(鼠疫菌)感染鼠脏器标本和225份对照鼠(达乌尔黄鼠217只,小白鼠5只,豚鼠3只)标本,同时用鼠疫反向间接血球凝集试验(RIHA)微量法和细菌培养做平行检测.结果 225只对照鼠鼠疫细菌学检验及GICA和RIHA的F1抗原检测均为阴性,GICA和RIHA在1×108 cfu/ml水平上未发现与近缘假结核耶尔森菌有交叉反应.GICA、RIHA对鼠疫菌检测灵敏性为2.5×105、2.0×105cfu/ml;检测F1抗原灵敏性为1、10 μg/L.GICA与细菌学检验符合率为97.94%(522/533),Kappa值=0.959,阳性检出率组间比较,差异无统计学意义(x2=0.36,P＞0.05);与RIHA符合率为97.94%(522/533),Kappa值=0.959,阳性检出率组间比较差异有统计学意义(x2=9.09,P＜0.05).308只实验鼠样本细菌培养阳性284只,284份细菌培养阳性标本中,GICA有280份检测阳性,阳性率为98.59%,高于RIHA[阳性率为96.13%(522/533)],两组比较差异有统计学意义(x2=5.14;P＜0.05).GICA的敏感度为98.59%(280/284),特异度为97.19%(242/249),阳性预测值为97.56%(280/287),阴性预测值为98.37%(242/246),Youden指数为0.9578.结论 GICA检测鼠疫F1抗原敏感特异,快速简便,是鼠疫早期快速诊断中有应用价值的检测技术.     

Evaluation of the NS1 rapid test and the WHO dengue classification schemes for use as bedside diagnosis of acute dengue fever in adults

Because healthcare facilities in many dengue endemic countries lack laboratory support, early dengue diagnosis must rely on either clinical recognition or a bedside diagnostic test. We evaluated the sensitivity and specificity of the 1997 and 2009 World Health Organization (WHO) dengue classification schemes and the NS1 strip test in acute sera from 154 virologically confirmed dengue patients and 200 patients with other febrile illnesses. Both WHO classification schemes had high sensitivity but lacked specificity. The NS1 strip test had high specificity, but its sensitivity was significantly lower in secondary compared with primary dengue infections. Differences in viral serotypes did not affect the performance of any of the three diagnostic approaches. Taken collectively, our findings indicate that the 1997 WHO dengue case definition can be used to exclude dengue, and the NS1 strip test can be used to confirm dengue infection, although the latter should be interpreted with caution in regions where secondary dengue infection is prevalent.