Incidence of dengue in a rural hospital, Chinakakani, Andhra Pradesh, South India and comparison of two commercially available enzyme linked immunosorbent assays with immunochromatographic rapid test.

Background: Dengue is one of the most serious mosquito borne arboviral infections affecting tropical and subtropical countries in the world. Since there is no immune prophylactic or specific anti-viral therapy available, timely and rapid diagnosis plays a vital role in patients management and implementation of control measures. This work has been taken up 1. To study the incidence of dengue cases in our rural hospital, Chinakakani, South India. 2. To compare the performances of Pan Bio capture ELISA [PanBio], J Mitra Microlisa [J Mitra] and SD BIO dengue duo rapid test [SD RT]. Methods: A total of 1180 serum samples from clinically suspected dengue cases were collected over a period of seven months. All the samples were subjected to NS1 antigen and IgM antibody Pan Bio ELISA. The same were tested for J Mitra Microlisa and SDRT and were compared with Pan Bio. Measure of discrimination i.e. sensitivity and specificity was calculated for each observed test value and an receiver operating characteristic [ROC] curve was constructed to compare the area under curve‟s [AUC] of different test kits thereby identifying the test with the best discriminative value. Results: Out of 1180 samples tested, Pan Bio has shown an incidence rate of 284 [24.06%] (NS1 156+IgM 128), J Mitra 280 [23.72%] (NS1 156 + IgM 124) and SDRT 292 [24.74%] (NS1 156+IgM 136). As far as NS1 is concerned the same 156 samples were positive in all the three tests giving the sensitivity, specificity, positive and negative predictive values 100%. Remaining 1024 samples were negative for NS1. But this was not the case with IgM. AUC of IgM Pan Bio is 0.944 with sensitivity 90.32% and specificity 98.48%. AUC of IgM J Mitra is 0.932 with sensitivity 81.62% and specificity 98.75%. AUC of IgM SD RT is 0.992 with sensitivity 99.2% and specificity 99.1%. „Z‟ test revealed that there is statistically significant difference between AUC‟s of SD RT when compared to Pan Bio (p value: 0.05) and J Mitra (p value 0.0001) The p values explain that SD RT is superior to Pan Bio and J Mitra in classifying between diseased and non-diseased. Conclusion: High incidence rate was noticed in our region during monsoon and post-monsoon season which calls for timely preventive and control measures. SDRT is a valuable screening test in laboratories with minimal resources.

A comparative evaluation of dengue diagnostic tests based on single acute serum samples for laboratory confi rmation of acute dengue

A prospective study was carried out to evaluate the sensitivity of dengue NS1 antigen-capture ELISA in comparison with dengue virus isolation, conventional RT-PCR and real-time RT-PCR for laboratory confi rmation of acute dengue based on single-acute serum samples. Four primary healthcare centres were involved to recruit patients with clinical diagnosis of dengue illness. Patient’s demographic, epidemiological and clinical information were collected on a standardized data entry form and 5 ml of venous blood was collected upon consent. In the laboratory, six types of laboratory tests were performed on each of the collected acute serum sample. Of the 558 acute serum samples collected from 558 patients with clinical diagnosis of dengue from mid-August 2006 to March 2009, 174 serum samples were tested positive by the dengue NS1 antigen-capture ELISA, 77 by virus isolation, 92 by RT-PCR and 112 by real-time RT-PCR. A total of 190 serum samples were tested positive by either one or a combination of the four methods whereas, only 59 serum samples were tested positive by all four methods. Thus, based on singleacute serum samples, 190 of the 558 patients (34.1%) were laboratory-confi rmed acute dengue. The overall test sensitivity was 91.6%, 40.5%, 48.4% and 58.9% for dengue NS1 antigen-capture ELISA, virus isolation, conventional RT-PCR and real-time RT-PCR respectively. Statistically, dengue NS1 antigen-capture ELISA was the most sensitive and virus isolation was the least sensitive test for the laboratory confi rmation of acute dengue based on single-acute serum specimens. Real-time RT-PCR was signifi cantly more sensitive than the conventional RT-PCR.

Cutaneous manifestations of chikungunya during a recent epidemic in Calicut, north Kerala, south India.

Background: There was a recent epidemic of chikungunya (CKG) in Calicut and other northern districts of Kerala, South India, affecting thousands of people. Aims: To study the cutaneous manifestations of CKG and to have a serological and histopathological correlation. Methods: A total of 162 patients (63 males and 99 females) with cutaneous manifestations of CKG were enrolled in the study and serological confirmation was done with capture IgM ELISA for CKG. Skin biopsy was done in all representative cases. Results: Cutaneous manifestations were found more in females. There were 23 children, the youngest being 39 days old. Generalized erythematous macular rash was the most common finding. Vesicles and bullae were also common especially in infants. Localized erythema of the nose and pinnae, erythema and swelling of the pre existing scars and striae and toxic epidermal necrolysis-like lesions sparing mucosae were the other interesting findings. Different types of pigmentation were observed with a striking nose pigmentation in a large number of patients, by looking at which even a retrospective diagnosis of CKG could be made. Hence we suggest this peculiar pigmentation may be called "chik sign". There was flare up of existing dermatoses like psoriasis, lichen planus and unmasking of Hansen's disease with type 1 reaction. Serological tests were positive in 97%. Some hitherto unreported histopathologic findings like melanophages in the erythematous rashes were observed. Conclusion: A spectrum of cutaneous manifestations of CKG with a wide variety of unusual presentations with confirmed serological and histopathological evidence was encountered.

High molecular mass fraction in clinical isolates of Paracoccidioides brasiliensis / Fração de alta massa molecular em isolados clínicos de Paracoccidioides brasiliensis

INTRODUCTION: Different serum levels of the IgG/IgE for Paracoccidioides brasiliensis high mass molecular (hMM) fraction (~366kDa) in the acute and chronic forms of the disease have been reported. Considering the nonexistence of hMM fraction investigation involving clinical isolates of P. brasiliensis, the present study aimed to investigate the presence of the hMM fraction (~366kDa) in cell free antigens (CFA) from P. brasiliensis clinical isolates. METHODS: CFA from 10 clinical isolates and a reference strain (Pb18) were submitted to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by gel image capturing and densitometer analysis. Additionally, CFA from 20 isolates and Pb18 were analyzed by capture ELISA (cELISA) using polyclonal (polAb) or monoclonal (mAb) antibodies to the hMM fraction. RESULTS: The presence of the hMM component was observed in CFA of all samples analyzed by SDS-PAGE/densitometry and by cELISA. In addition, Pearson's correlation test demonstrated stronger coefficients between hMM fraction levels using pAb and mAb (R = 0.853) in cELISA. CONCLUSIONS: The soluble hMM fraction was present in all the P. brasiliensis clinical isolates analyzed and the reference strain Pb18, which could be used as a source of this antigen. The work also introduces for first time, the cELISA method for P. brasiliensis hMM fraction detection. Analysis also suggests that detection is viable using polAb or mAb and this methodology may be useful for future investigation of the soluble hMM fraction (~366kDa) in sera from PCM patients.

INTRODUÇÃO: Diferentes níveis sorológicos de IgG/IgE contra a fração de alta massa molecular (hMM) (~366kDa) de Paracoccidioides brasiliensis têm sido encontrados na PCM aguda e crônica. Considerando a inexistência de investigação sobre esta fração em isolados clínicos de P. brasiliensis, o objetivo deste estudo foi investigar a presença da fração hMM (~366kDa) no preparado livre de células (CFA) de P. brasiliensis obtidos de isolados clínicos. MÉTODOS: CFA de 10 isolados e de cepa de referência (Pb18) foram submetidas à eletroforese em gel de SDS-poliacrilamida (SDS-PAGE) seguida de captura de imagem e análise por densitometria. Adicionalmente, CFA de 20 isolados e de Pb18 foram analisados por ELISA captura (cELISA) utilizando anticorpos policlonal (polAb) ou monoclonal (mAb) para fração hMM. RESULTADOS: A presença do componente de hMM foi observada em todas as amostras analisadas por SDS-PAGE/densitometria e por cELISA. Adicionalmente, o teste de correlação de Pearson demonstrou forte relação entre os níveis de fração hMM usando pAb e mAb (R = 0.853) no cELISA. CONCLUSÕES: Conclui-se que a fração hMM está presente em todos os isolados clínicos de P. brasiliensis analisados e no isolado referencial, sugerindo a possibilidade dos mesmos serem utilizados como fonte desta fração antigênica. Este trabalho também introduz pela primeira vez o método de cELISA para detecção da fração hMM de P. brasiliensis, sugerindo que detecção utilizando anticorpos polAb ou mAb é viável e essa metodologia poderá ser útil para investigação futura desta fração solúvel (~366kDa) em soros de pacientes com PCM.

Development of a Polyclonal Antibody-based AC-ELISA and Its Comparison with PCR for Diagnosis of Canine Parvovirus Infection / 中国病毒学·英文版

A polyclonal antibody-based antigen-capture ELISA (AC-ELISA) has been developed for detection of Canine parvovirus (CPV) antigens in faecal samples of dogs. The assay uses rabbit anti-CPV polyclonal antibody as the capture antibody, guinea pig anti-CPV polyclonal antibody as tracing antibody and anti-guinea pig HRPO conjugate as the detection system. The optimum dilution of the capture antibody and the tracing antibody capable of detecting the CPV-2 antigens was found to be 1:1 600 and 1:400, respectively, in the check-board titration. In this study, a total of 152 samples (129 faecal samples and 23 cell culture supernatant) were tested both by AC-ELISA and by polymerase chain reaction (PCR). Of the samples tested, 69 and 78 samples were found positive by AC-ELISA and PCR, respectively. The AC-ELISA had relative sensitivity, relative specificity and accuracy of 88.4%, 100.0% and 91.4% respectively. The analytical sensitivity of AC-ELISA was estimated to be 102.8 TCID50/mL whereas PCR sensitivity was 100.8 TCID50/mL. The AC-ELISA is a simple, quick and reliable method for screening large numbers of faecal samples of dogs suspected of CPV infection.

Development of Capture ELISA Using a Biotinylated Monoclonal Antibody for Detection of Botulinum Neurotoxin Type A

A capture enzyme-linked immunosorbent assay (capture ELISA) was developed to detect Clostridium botulinum neurotoxin type A (BoNT/A) in assay buffer and human serum. The assay is based upon affinity-purified rabbit polyclonal and biotinylated monoclonal antibodies directed against the BoNT/A complex purified from C. botulinum ATCC19397. For the capture ELISA, the optimized amount (2 microgram/ml) of rabbit polyclonal antibody was immobilized on ELISA plates to detect BoNT/A (ranging from 0 to 500 ng/ml), which was recognized by 2 microgram/ml of the monoclonal antibody. From three independent repeated experiments, standard curves were linear over the range of 0~31.25 ng/ml BoNT/A and the coefficients (r(2)) ranged from 0.9951~0.9999 for all assays. The inter-variations were typically 0.50~6.93% and the specificity was confirmed by showing no cross-reactivity against BoNT/B and /E. The detection limit of capture ELISA was 0.488 ng/ml, which was close to mouse LD(50). In addition, application with BoNT/A-spiking human sera showed a possibility to detect BoNT/A with capture ELISA from the contaminated human sera. Taken together, the newly developed capture ELISA could serve as a rapid and sensitive screening tool for detecting BoNT/A simultaneously from massive specimens.

Clinical application of anti-proteinase 3 capture ELISA in diagnosis of Wegener's granulomatosis / 临床检验杂志

Objective To investige the sensitivity and specificity of anti-proteinase 3 (PR3) capture ELISA in diagnosis of Wegener's granulomatosis (WG),and the correlation between the capture ELISA and indirect immunofluorescence assay (IIF).Methods Anti-PR3 antibody and anti-neutrophil cytoplasmic antibody (cANCA) in sera from 72 patients with WG,206 healthy blood donors and 24 patients with autoimmune diseases were detected by classic ELISA,capture ELISA and IIF.Results The sensitivities of classic ELISA and capture ELISA for detection of anti-PR3 in WG diagnosis were 73.6% and 87.5% respectively.The specificities of both the ELISAs were identical (100%).Detection of anti-PR3 by ELISA or IIF alone led to the serological hit rate of 87.5% and 84.7% for WG respectively,but the combination of capture ELISA and IIF increase the hit rate up to 91.6%.Conclusions The sensitivity of anti-PR3 capture ELISA as well as its correlation with IIF is prior to classic anti-PR3 ELISA.The combined detection of anti-PR3 capture ELISA and IIF may increase the diagnosis rate of clinically suspected WG.

Detection of Platelet Specific Antibodies by Modified Antigen Capture ELISA Test / 대한진단검사의학회지

BACKGROUND: Autoimmune thrombocytopenia (AITP) is characterized by autoantibody-induced platelet destruction. Although several studies have shown that pathogenic autoantibodies are mainly IgG directed platelet glycoproteins (GP), a platelet GP specific test is not available in clinical laboratories. The aim of this study was to evaluate the clinical usefulness of a Modified Antigen Capture Enzyme-linked immunosorbent assay (MACE) test in the diagnosis of AITP. METHODS: We investigated fifty-seven patients who showed a platelet count lower than 100 x 10(9)/L and underwent a bone marrow examination. They were classified into primary AITP (P-AITP) (n=21), secondary AITP (S-AITP) (n=15), and non-immune thrombocytopenia (NITP) (n=21) by bone marrow findings and clinical diagnosis. Platelet GP (IIb/IIIa, Ia/IIa, Ib/IX, IV)-specific antibodies and anti-HLA class I antibody were detected by MACE test. RESULTS: Among 57 samples, platelet GP specific antibodies were detected in 8 (22.2%) of 36 patients with AITP and 1 (4.8%) of 21 patients with NITP. The specificities were as follows: GP IIb/IIIa (n=4), GP Ia/IIa (n=5), GP Ib/IX (n=3) and GPIV (n=2). Of the nine patients with platelet GP specific antibodies, four (44.4%) had more than two platelet GP specific antibodies. The sensitivity, specificity, positive predictive value and negative predictive values of the MACE test for AITP were 22.2%, 95.2%, 88.9%, 41.7%, respectively. A previous transfusion history was associated with a higher detection rate of anti-HLA class I antibodies (P<0.05). CONCLUSIONS: The MACE test is a convenient method to detect platelet GP specific antibody and is very specific to diagnose AITP. In clinical practice, even though it is not sensitive, the MACE test would be useful in differentiating AITP from NITP.

Development of a novel antigen capture-ELISA using IgY against porcine interleukin-6 and its application

Interleukin-6 (IL-6) is introduced as a marker of disease. At present, a variety of method may be used to quantify expression of this protein. Antigen capture-ELISA is a sensitive and accurate quantification method previously used with ovine, rat, and human IL-6 proteins. However, it has never been reported to quantify porcine IL-6 protein using capture ELISA. In this study, we generated and characterized a set of IgY and mono-specific polyclonal antibodies to recombinant porcine IL-6 (rpIL-6), and combining these with a sensitive and specific capture-ELISA for a diagnostic purpose. cDNA encoding the mature protein coding region of porcine IL-6 was cloned and expressed with pQE-30UA expression vector. rpIL-6 was then expressed and purified by using Ni-NTA resin. Protein mass of 24 kDa was found with SDS-PAGE and the identity of the protein was confirmed by Western-blot. Production of polyclonal antibodies against rpIL-6 was performed using the purified rpIL-6 in mice and hens. An antigen capture-ELISA was developed with the antibodies after their extraction. To compare the IL-6 level in the different sanitary state of farms, pig sera were randomly collected and concentration of IL-6 in the sera was measured with the antigen capture-ELISA. The capture-ELISA with the optimal concentration of antibodies, in this study, was able to detect about 10 ng/ml of rpIL-6. IL-6 levels determined with the capture-ELISA in pig sera showed positive correlation with the sanitary states of the farms. These results suggested that the developed antigen capture-ELISA could be a good tool for the screening of microbial infection in pig farms.

Clinical Significance and Detection of Antibodies Against Platelet GP Ib/I X and Gp II b/IIIa in Childhood Chronic Idiopathic Thrombocytopenic Purpura / 대한소아혈액종양학회지

BACKGROUND: Chronic idiopathic thrombocytopenic purpura is an autoimmune disorder caused by sequestration of antibody-sensitized platelets in the reticuloendothelial system. However, uncertainty as to the specificity, frequency and clinical significance of such antibodies still remains. So, we tried to further clarify the above uncertainty in childhood chronic idiopathic thrombocytopenic purpura. METHODS: We analyzed sera from 29 patients. Twenty six patients were chronic ITP who were admitted or followed up to the Department of Pediatrics, Severance Hospital, Yonsei University Medical College from August 1996 to March 1997 by employing a modified antigen-capture ELISA(MACE), flow cytometry and electrophoresis(SDS-PAGE) and immuno-blotting(IB) assays. Three patients with ITP less than 6 months after onset of ITP were included to know the possibility to differrentiate between acute ITP and chronic ITP in this study. RESULTS: 1) Glycoprotein(GP)-specific antibodies were found in 28% (8/29) of patients, with 2 patients having antibodies directed solely to Gp II b/III a, no patients holding antibodies specific only for GPI b/I X and 6 possessing antibodies against both anti-GP I b/I X and Gp II b/III a antigen. 2) The detection rate of GP-specific antibodies of flow cytometry was about 10%. The positivity of anti-GPI b/I X antibodies by MACE and immunoblotting was 14% (4/29), respectively, the positivity of anti-Gp II b/III a antibodies by MACE and immunoblotting was, 21 % (6/29) respectively. The concordance rate between two assays(MACE and IB) was 79% (23/29). None of the three methods was good enough to stand alone. 3) Serum antibodies were not more frequently detected in active(p=1.0) or non-splenectomized(p=.54) chronic ITP patients. 4) No association was found between antibody specificity(anti-GPI b/I X, anti-Gp II b/ III a) and platelet counts(p : .87). CONCLUSION: We conclude that in korean childhood chronic ITP, antibodies against both anti-GPI b/I X and Gp II b/III a antigen were predominant antibody. But, the longterm follow-up in more cases is needed to further clarify the clinical significance of antral-platelet antibody in chronic ITP should be assessed.

SEQUENTIAL DETERMINATION OF SPECIFIC IGM ANTIBODIES IN SERUM SAMPLES FROM PATIENTS WITH EPIDEMIC HEMORRHAGIC FEVER / 西安交通大学学报(医学版)

The specific IgM (SIgM) antibodies in serum samples from 32 patients with epidemic hemorrhagic fever (EHF) were sequentially determined by IgM antibody capture ELISA (MacELISA) and indirect immunofluorescence technique (IFA) sitnultaneously. Of the samples detected by the two methods, 99.46% had the same positive or negative results. The response curves of SIgM titres separately determined by MacELISA or IFA were parellel, whereas, the SIgM titres detected by MacELISA were comparatively higher than those detected by IFA. During the course of the disease, no significant defference was found between the SIgM titres of defferent illness types at the same illness day.