Sensitivity Of ELISA Research Articles

CRYPTOSPORIDIOSIS PROVOKING DIARRHEA AMONG INFANTS, TODDLERS AND PRESCHOOL CHILDREN

Cryptosporidium intestinal coccidian is a preeminent account for infectious diarrhea with reference to children. Oocyst may escape ordinary microscopic analysis or immune detection; molecular techniques are of convenient use. This study intent to detect cryptosporidiosis infection among children, and debates risks for infection. A total of 189 children with age range from under one to five complaining of gastrointestinal manifestations were examined coproscopically, by acid fast stain, ELISA and nested PCR (nPCR) for Cryptosporidium from June to September. Infection was proved by nPCR among 13 (6.8%) samples. ELISA sensitivity merit stool staining (77%, 53.8%, respectively), however all positive samples by microscopy were settled by nPCR. Age groups (P = 0.02) were statistically associated with infection. Source ofwater (OR=0.069, 95% CI= 0.004/1.165, P = 0.01) and presence of undigested food particles on microscopy (OR= 1.058, 95% CI= 0.521/6.154, P = 0.02) were risks for infections. No other variables were predicated risks. Infection was potent among preschool children (46.2%), toddlers (38.5%) and then infants (15.4%). Cryptosporidiosis should be doubtful among children with diarrhea during preschool age. Source of water for ordinary drinking activities or for infantswith non-exclusive breast or complementary feeding should be considered as hazards aspects for acquiring Cryptosporidium.

Comparative diagnostic techniques in ruminant Fasciolosis: fecal sedimentation, indirect ELISA, liver inspection and serum enzyme activities

Different diagnostic tests have some disadvantages in diagnosing Fasciolosis. A cross-sectional study was conducted to estimate the prevalence and to compare the different diagnostic techniques in ruminant Fasciolosis: ELISA, biochemical analysis, liver and coprological examination. Out of the 324 cattle, 350 sheep and 385 goats slaughtered at Bishoftu, ELFORA Export Abattoir, 125 (38.5%), 4 (1.1%) and 2 (0.5%) and 108 (33%) 4 (1.1%) and 2 (0.5%) were found to be positive for lesions of fasciolosis and Fasciola eggs, respectively. The results of different diagnostic techniques in randomly selected 134 cattle, 68 sheep and 22 goats samples were compared. Out of the 134 examined cattle, 100 (75%) were found to be positive for antibodies against Fasciola specific f2 antigens followed by 54 (41%) for lesions of Fasciolosis, 40 (30%) for Fasciola eggs and 56 (42%) and 100 (75%) of the samples had an increased level of serum enzyme GGT and LDH above the normal values, respectively. The overall assessment indicates the test agreement of ELISA findings with the results of fecal examination is weak (Kappa=0.236), ELISA findings with the results of liver lesions is moderate (Kappa=0.373), liver lesions with GGT determination is moderate (Kappa=0.332) and liver lesions with level of LDH is absent (Kappa=-0.066). However, there was a strong relationship (Kappa=0.758) between fecal examination and liver lesions. In small ruminants, the prevalence of Fasciolosis was lower both in coproscopy and indirect ELISA tests: only one out of 68 sheep was positive coproscopically and 3 were positive serologically; and one out of 22 goats tested was positive both at coproscopy and serological examinations. The sensitivity of ELISA was higher compared to the others techniques in diagnosing ruminant fasciolosis. Coprological examination should be repeated and supported by other diagnostic methods.Keywords: Coproscopy; ELISA; Fasciola; Liver enzymes; Ruminants

An evaluation of serological methods to diagnose tick-borne encephalitis from serum and cerebrospinal fluid

BackgroundTick-borne encephalitis (TBE) is an infectious disease endemic to large parts of Europe and Asia. Diagnosing TBE often relies on the detection of TBEV-specific antibodies in serum and cerebrospinal fluid (CSF) as viral genome is mostly not detectable once neurological symptoms occur. ObjectivesWe evaluated the performance of TBEV IgM and IgG ELISAs in both serum and CSF of confirmed TBEV patients and discuss the role of (CSF) serology in TBEV diagnostics. Study designFor the assay evaluation we collected specimen from confirmed TBEV patients. Assay specificity was assessed using sera from patients with a related flavivirus infection or other acute infection. A selected ELISA assay was used to analyze TBEV-specific antibodies in CSF and to evaluate the use in confirming TBE diagnosis. ResultsIn this study the overall sensitivity of the IgM TBEV ELISAs was acceptable (94 -100 %). Four out of five IgM ELISA’s demonstrated an excellent overall specificity from 94 -100% whereas a low overall specificity was observed for the IgG TBEV ELISAs (30-71%). Intrathecal antibody production against TBEV was demonstrated in a subset of TBE patients. ConclusionsIn four out of five ELISAs, IgM testing in serum and CSF of TBE patients is specific and confirmative. The lack of IgG specificity in all ELISAs emphasizes the need of confirmatory testing by virus neutralisation, depending on the patient’s background and the geographic location of exposure to TBEV. A CSF-serum IgG antibody index can support the diagnosis specifically in chronic disease or once IgM has disappeared.

Application of Helicobacter pylori stool antigen test to survey the updated prevalence of Helicobacter pylori infection in Taiwan.

The reported prevalence of Helicobacter pylori infection in Taiwan was 54.4% in 1992. An updated prevalence of H.pylori infection in asymptomatic adults is lacking in Taiwan. We aimed to assess the updated age-standardized prevalence of H.pylori infection in asymptomatic subjects and in patients with dyspepsia and to assess the accuracy of H.pylori stool antigen (HpSA) test for screening of H.pylori in Chinese population. Asymptomatic adult subjects (N=189) were screened for H.pylori infection using HpSA, serology, and 13 C-urea breath test (13 C-UBT) in 2016-2017. Adult patients with dyspepsia (N=145) were screened for H.pylori using 13 C-UBT, HpSA, serology, rapid urease test, and histology during 2016-2018. Two types of HpSA, including the Diagnostec HpSA ELISA Kit (HpSA ELISA) and Rapid Test Kit (HpSA Rapid), were used in this study. Sensitivity, specificity, and accuracy of the HpSA tests were calculated using the 13 C-UBT as golden standard test. The unadjusted prevalence of H.pylori was 21.2% in asymptomatic adults and 37.9% in patients with dyspepsia (P<0.001). The age-standardized prevalence of H.pylori was 28.9% in asymptomatic adults in Taiwan. Of the 334 patients included for analysis, the area under the curve of HpSA ELISA test was 0.978, and the optimal cutoff value of optical density was 0.03. The sensitivity, specificity, and accuracy of the HpSA ELISA were 0.929, 0.983, and 0.967, respectively. The sensitivity, specificity, and accuracy of the HpSA Rapid were 0.929, 0.958, and 0.949, respectively. The prevalence of H.pylori infection has decreased in Taiwan. HpSA test is an accurate tool for screening of H.pylori in Chinese population.

Classical swine fever in Victorian domestic pigs: evidence of disease freedom.

Australia is currently regarded as free of classical swine fever (CSF), a highly contagious disease of pigs caused by a pestivirus. This study aimed to provide additional evidence that the Victorian domestic pig population is free of CSF. A structured representative sero-prevalence survey of Victorian domestic pigs at slaughter. Three-hundred and ninety-one pigs from 23 holdings were sampled at the time of slaughter between March 2016 and October 2017. All samples were negative for CSF virus Ab on ELISA. Because of uncertainty in the sensitivity of the CSF Ab ELISA, estimates of the true prevalence of CSF were calculated using Bayesian methods. The median and upper bound of the 95% credible intervals for the true prevalence of CSF was zero when the diagnostic sensitivity of the CSF Ab ELISA was assumed to range from 0.75 to 0.95. These results provide evidence that the population of domestic pigs in Victoria in 2016-2017 was free of CSF.

Using hapten cross-reactivity to screen heterologous competitive antigens for improving the sensitivity of ELISA

In this study, heterologous competitive antigens (HCAs) suitable for improving the sensitivity of ELISA were successfully screened based on their cross-reactivities (CRs) with 19 quinolone analogues; each containing the norfloxacin amino derivative (NOR0) coupled with bovine serum albumin (BSA) as a coating antigen. HCAs prepared with hapten analogues (CRs of 0.77%–49.92%) remarkably enhanced the sensitivity of the subsequent ELISA. ELISA sensitivity for NOR detection improved 26-fold when moxifloxacin–BSA was used as a heterologous coating antigen relative to when NOR0–BSA was used as a homologous coating antigen. This work, therefore, represents a detailed screening method to select suitable heterologous competitive antigens that improve ELISA sensitivity. Secondly, we present new theoretical tools to estimate hapten structures for use in the method, which may also be applied to improve the sensitivity of other immunoassays.

Development of a recombinant NP protein based indirect ELISA for the detection of antibodies against Newcastle disease virus and differentiation of infected or recombinant vaccine immunized chickens.

Newcastle disease (ND) is a highly contagious and economically important disease in the poultry industry caused by avian avulavirus-1, historically known as Newcastle disease virus (NDV). Control of ND primarily relies on prophylactic vaccination of flocks, and many vaccines are available on the market, both conventional and more recently introduced new generation recombinant types. To assess the protection level achieved by vaccination ELISA tests are typically used, they also are to track an infection with field strains in non-vaccinated flocks. Special modifications of ELISA can be used as a screening tool to detect infection in flocks vaccinated with new generation vaccines. In this study, we have developed an ELISA test for the detection of antibodies against the nucleoprotein (NP) of NDV and for differentiation of chickens vaccinated with commercial and prototype in-house recombinant vector vaccines from those infected with field NDV strains. The NP gene of LaSota NDV strain expressed in a baculovirus vector was used as a coating antigen in the ELISA. The developed test was optimized, validated and compared to other serological tests. The sensitivity, specificity and accuracy of recombinant NP protein-based ELISA were respectively 96.1%, 96.3%, and 96.2%. Inter-rater (kappa) agreement between the NP-ELISA and the gold standard HI test was calculated to be 0.995. In our comparisons, commercially available ELISA tests revealed different specificities ranging from 95.5-100% and sensitivities at variance, ranging from 90.1 to 99.0%. A high level of maternally derived antibodies was measured in the serum of 1-day-old broilers in the NP-ELISA assay. These antibodies had disappeared and were undetected at 3, 5 and 6 weeks post-vaccination but birds became positive again at 2 weeks after control infection with a velogenic NDV strain. In SPF chickens, antibodies against NP protein were detected only after a challenge. The recombinant NP protein-based ELISA test is sensitive, specific and accurate when compared to the gold standard HI test and commercially available kits. Moreover, the method could be also used for the differentiation between vaccinated and infected birds.

Echinococcus granulosus protoscolex antigen used in serodiagnosis of hydatidosis by nano-gold dot-ELISA

Background: Echinococcus granulosus protoscolex antigen (PSAg) is a protein with significant immunological properties having higher sensitivity and specificity in ELISA. It lowers cross-reaction with antibodies of other parasites and thus its application is recommended in serological diagnosis. Labelling of ELISA with nano-gold particles improved the diagnostic abilities of the laboratory technique in hydatidosis detection. Objective: To evaluate the use of nano-gold dot-ELISA for isolation of E. granulosus PSAg and its application in serodiagnosis of hydatidosis in humans and animals in comparison with dot-ELISA. Material and Methods: Hydatid cyst PSAg was isolated and used for immunization of rabbits to raise IgG polyclonal antibodies (pAb) in antisera. These sera were labeled with horseradish peroxidase (HRP) and used for detection of circulating PSAg in sera of human cases and camels and sheep by dot-ELISA and nano-gold dot-ELISA. Results: Conjugation of the anti-protoscolex pAb with gold nano-particles increased the sensitivity of antigen detection by nano-gold dot-ELISA to 94.4% and specificity to 90%, with positive and negative predictive values of 94.4% and 90%, and an accuracy of detection of 92.9% in both human and animal sera. Conclusion: Nano-gold dot-ELISA technique is more sensitive than dot-ELISA for detection of hydatidosis antigen both in human and animal samples

Sensitivity and specificity of diagnostic tests for Lassa fever: a systematic review

BackgroundLassa fever virus has been enlisted as a priority pathogen of epidemic potential by the World Health organization Research and Development (WHO R & D) Blueprint. Diagnostics play a crucial role in epidemic preparedness. This systematic review was conducted to determine the sensitivity and specificity of Lassa fever diagnostic tests for humans.MethodsWe searched OVID Medline, OVID Embase, Scopus and Web of Science for laboratory based and field studies that reported the performance of diagnostic tests for Lassa fever in humans from 1 January 1990 to 25 January 2019. Two reviewers independently screened all the studies and included only studies that involved the evaluation of a Lassa fever diagnostic test and provided data on the sensitivity and specificity. The quality of the studies was assessed using the QUADAS-2 criteria. Data on the study location, study design, type of sample, index test, reference tests and diagnostic performance were extracted from the studies.ResultsOut of a total of 1947 records identified, 1245 non-duplicate citations were obtained. Twenty-six (26) full-text articles examined which identified 08 studies meeting pre-defined criteria. Only one study was a field evaluation study. The sensitivity and specificity of the point of care (RDT) against the Nikisins qPCR were 91.2%(95% CI:75.2–97.7) and 86%(95% CI: 71.4–94.2) at temperatures 18–30 °C, while the sensitivity and specificity of the single IgM ELISA assay against standard RT-PCR were 31.1%(95%CI: 25.6–37) and 95.7%(95%CI:92.8–97.7). The sensitivity of the combined ELISA Antigen/IgM assay(against virus isolation), the recombinant IgM/IgG ELISA(against standard RT-PCR), and the IgM/IgG immunoblot(against IFA) were 88%(95%CI:77–95), 25.9%(95%CI:20.8–31.6), and 90.7%(95%CI:84.13–97.27) respectively. The specificity of the combined ELISA Antigen/IgM assay(against virus isolation), the recombinant IgM/IgG ELISA(against standard RT-PCR), and the IgM/IgG immunoblot(against IFA) were 90%(95%CI:88–91), 100%(95%CI:98.2–100), and 96.3%(95%CI:92.2–100) respectively.ConclusionLassa fever has assays for antigenaemia, IgM, IgG and PCR detection. The RDT reportedly performed well but more data are needed from other countries and at temperatures above 30 °C. Most combined immunoassays perform better than the single IgM. Multiplex and pan-Lassa assays are needed. More well conducted field studies are needed.Trial registrationProspero registration number: CRD42018091585.

Usefulness of dark field microscopy (DFM), IGM ELISA and microscopic agglutination test (MAT) for the early diagnosis of acute leptospirosis

Usefulness of dark field microscopy (DFM), IGM ELISA and microscopic agglutination test (MAT) for the early diagnosis of acute leptospirosis - IJMR- Print ISSN No: - 2394-546X Online ISSN No:- 2394-5478 Article DOI No:- 10.18231/j.ijmr.2019.036, Indian Journal of Microbiology Research-Indian J Microbiol Res

Association of common polymorphisms in IL-6 and IL6ST genes with levels of inflammatory markers and coronary stenosis

Inflammation is an important factor in the onset and development of pathological lesions of coronary arteries. Various cytokines, including interleukin-6 (IL-6), and pattern recognition receptors, such as C-reactive protein (CRP), are the key inflammatory mediators. We aimed to identify associations of single nucleotide polymorphisms (SNPs) rs1800795 of the IL-6 gene and rs3729960 of the IL6ST gene with levels of inflammatory markers and coronary lesions.The study included 316 men and 131 women (mean age 61.2 ± 9.4 years) subjected to coronary angiography. IL-6 levels were assayed by ELISA and high sensitivity CRP (hsCRP) levels were measured by turbidimetry. The rs1800795 and rs3729960 genotypes of the IL-6 and IL6ST genes, respectively, were determined by real time PCR. Data were analyzed by the SPSS software. In women, the CC genotype (N = 31) of rs1800795 of the IL-6 gene is associated with lower concentrations of hsCRP in the dominant model (GG + CG; N = 38 and N = 62, respectively) and the corresponding hsCRP levels were significantly different (P = .015) at 1.70 mg/l (25% 0.75; 75% 3.55) and 3.30 mg/l (25% 1.20; 75% 6.18). In men, these genotypes were not associated with hsCRP levels. Distribution of allele frequency of rs1800795 of the IL-6 gene in groups with and without coronary lesions indicate that the G allele has significantly higher frequency in men but not in women with coronary atherosclerosis (OR 2.45; 95%CI 1.31–4.72; P = .003). Thus, in women, rs1800795 of the IL-6 gene is associated with hsCRP levels and in men, rs1800795 is associated with presence of coronary lesions.

Canine Brugia malayi microfilarial excretory/secretory protein-based antibody assay for the diagnosis of brugian filariasis in dogs.

Brugian filariasis is reported in dogs in Kerala, India. Antibody detection kits are not available worldwide, for detection of Brugian filariasis in dogs. A study was carried out to develop Indirect plate ELISA using excretory secretory antigen isolated from canine brugian microfilariae and compare the sensitivity and specificity with that of blood smear examination. Identification of microfilariae was done by acid phosphatase staining using Naphthol AS-TR method and Polymerase Chain Reaction for Hha 1 repeat sequence. The microfilariae were identified as Brugia malayi. Isolation of brugian microfilariae from canine blood was done by gradient centrifugation method. The isolated microfilariae were maintained in RPMI-1640 media. The pooled media was then concentrated to obtain excretory secretory protein (ESP). This ESP was used to develop Indirect ELISA. The sensitivity and specificity of the plate ELISA developed was 84 and 100 per cent respectively when compared with blood smear examination. This is the first report of successful isolation of ESP from Brugia malayi microfilariae from dogs and standardization of plate ELISA using the antigen.

Simply Mixing Poly Protein G with Detection Antibodies Enhances the Detection Limit and Sensitivity of Immunoassays.

An insufficient amount of detection antibodies bound to their antigens usually limits the sensitivity of immunoassays. Here, we describe a simple method to improve the detection limit and sensitivity of various immunoassays by mixing detection antibodies with a soluble poly protein G (named 8pG). 8pG was developed by fusing eight repeated fragment crystallizable (Fc) binding domains of streptococcal protein G to a linear polymer. Simply mixing detection antibodies with 8pG to form an antibody/8pG complex largely increased the accumulation of detection antibody to target molecules, which dramatically enhanced the sensitivity in direct ELISA, sandwich ELISA, Western blot, and flow cytometry systems, separately. The detection limit of Western blot for low-abundance PEGylated interferon (Pegasys) and recombinant human CTLA4 (rhCTLA4) improved by at least 13-fold and 31-fold, respectively, upon mixing detection antibodies with 8pG. Moreover, the nanoscale size of the antibody/8pG complex did not influence the granularity and dimension of target cells in the flow cytometry system. Collectively, we provide a quick and easy-to-operate method to make various immunoassays to sensitively detect low-abundance target molecules by just mixing their detection antibodies with 8pG.

Serological and molecular screening of umbilical cord blood for Toxoplasma gondii infection.

Human umbilical cord blood has proven to be a successful alternate source of hematopoietic stem cells for pediatric patients with major hematologic disorders. Toxoplasma gondii is a global opportunistic protozoan which cause fatal complications in immunocompromised individuals. Our goal is to study the prevalence of toxoplasmosis in umbilical cord blood (UCB) and to assess the sensitivity of ELISA and PCR for Toxoplasma infection screening. One hundred cord blood samples were collected immediately after delivery. Anti-Toxoplasma IgG and IgM antibodies were determined using ELISA method; Toxoplasma DNA was detected using nested PCR technique. Total nucleated cells (TNC) and HB were also determined. Demographic data and risk factors data related to the transmission of toxoplasmosis, were collected from mothers. Among 100 cord blood samples, 36 (36%) were positive for anti-Toxoplasma IgG antibodies and 6 (6%) were positive for anti-Toxoplasma IgM antibodies. The nested PCR showed 11 (11%) samples containing Toxoplasma DNA from which, 6 (55%) samples were IgM positive. There was no significant association between the risk of Toxoplasma transmission and cord blood positivity for toxoplasmosis. Owing to the prevalence of toxoplasmosis, its rapid progression and its fatal outcome in immunocompromised patients, cord blood screening for toxoplasmosis with nested PCR should be incorporated into cord blood bank screening protocols.

Using molecular descriptors for assisted screening of heterologous competitive antigens to improve the sensitivity of ELISA for detection of enrofloxacin in raw milk

The use of the heterologous competitive strategy has become a vital method to improve the sensitivity of ELISA. In this work, we prepared an anti-enrofloxacin (ENR) mAb with ENR-bovine serum albumin (BSA) as immunogen. The molecular descriptors of quinolones were then used to screen heterologous coating antigens for the detection of ENR based on an ensemble learning method to improve the sensitivity of the ELISA. Results indicated that 6 of the 7 selected heterologous competitive antigens could enhance the sensitivity of ELISA. The ELISA sensitivity for the detection of ENR with sarafloxacin-BSA as heterologous coating antigen was improved 10-fold (in PBS) and 6-fold (in milk) compared with that with ENR-BSA as homologous antigen. The strategy can effectively screen suitable heterologous competitive antigens to improve the sensitivity of ELISA, followed by preparation of mAb with no additional modification to the corresponding immunogen.

Lentiviral-mediated delivery of classical swine fever virus Erns gene into porcine kidney-15 cells for production of recombinant ELISA diagnostic antigen.

Classical swine fever virus (CSFV), a member of the Pestivirus genus within the Flaviviridae family causes contagious fatal disease in swine. Antibodies against E2, Erns and NS3 proteins of virus can be detected in infected animals. Development of an ELISA coating antigen to improve the sensitivity of detecting Erns-specific antibodies in pig sera is always desirable for diagnosis as well as for differentiation of infected from vaccinated animals. In present study, a lentivirus-based gene delivery system was used to develop a stable PK-15 cell line expressing Erns (PK-Erns) for production of diagnostic antigen. The Lenti-Erns virus was purified from the supernatant of co-transfected 293LTV cells and used to transduce PK-15 cells. The homogenous PK-Erns cell line was produced by single cell cloning by monitoring eGFP expression. The Erns gene in the genomic DNA and RNA transcripts in total RNA isolated from PK-Erns cells were detected by PCR and RT-PCR, respectively. Expression of 45kDa Erns glycoprotein was detected in western blot using CSFV-specific hyperimmune sera. The use of PK-Erns cell lysate as antigen in serial dilution and single dilution ELISAs with known positive and negative pig sera was investigated. The PK-Erns ELISA revealed sensitivity equivalent to commercial HerdChek ELISA kit. The sensitivity, specificity and accuracy of the PK-Erns ELISA was 95%, 100% and 96.66%, respectively compared to ELISA using purified CSFV as coating antigen. When field pig sera (n = 69) were tested in PK-Erns ELISA, a significant correlation between the titers from serial dilution and single dilution ELISA was observed. This indicated that PK-Erns cell line can serve as continuous source of ELISA diagnostic antigen for detection of CSFV-specific antibodies in pig sera.

Low population Japanese encephalitis virus (JEV) seroprevalence in Udayapur district, Nepal, three years after a JE vaccination programme: A case for further catch up campaigns?

The live attenuated Japanese encephalitis (JE) vaccine SA14-14-2 has been used in Nepal for catch-up campaigns and is now included in the routine immunisation schedule. Previous studies have shown good vaccine efficacy after one dose in districts with a high incidence of JE. The first well-documented dengue outbreak occurred in Nepal in 2006 with ongoing cases now thought to be secondary to migration from India. Previous infection with dengue virus (DENV) partially protects against JE and might also influence serum neutralising antibody titres against JEV. This study aimed to determine whether serum anti-JEV neutralisation titres are: 1. maintained over time since vaccination, 2. vary with historic local JE incidence, and 3. are associated with DENV neutralising antibody levels. We conducted a cross-sectional study in three districts of Nepal: Banke, Rupandehi and Udayapur. Udayapur district had been vaccinated against JE most recently (2009), but had been the focus of only one campaign, compared with two in Banke and three in Rupandehi. Participants answered a short questionnaire and serum was assayed for anti-JEV and anti-DENV IgM and IgG (by ELISA) and 50% plaque reduction neutralisation titres (PRNT50) against JEV and DENV serotypes 1–4. A titre of ≥1:10 was considered seropositive to the respective virus. JEV neutralising antibody seroprevalence (PRNT50 ≥ 1:10) was 81% in Banke and Rupandehi, but only 41% in Udayapur, despite this district being vaccinated more recently. Sensitivity of ELISA for both anti-JEV and anti-DENV antibodies was low compared with PRNT50. DENV neutralising antibody correlated with the JEV PRNT50 ≥1:10, though the effect was modest. IgM (indicating recent infection) against both viruses was detected in a small number of participants. We also show that DENV IgM is present in Nepali subjects who have not travelled to India, suggesting that DENV may have become established in Nepal. We therefore propose that further JE vaccine campaigns should be considered in Udayapur district, and similar areas that have had fewer vaccination campaigns.

Humoral immune response to repeated lumpy skin disease virus vaccination and performance of serological tests

BackgroundIn the presented study we investigated the development of the humoral immune response against LSDV during the process of re-vaccination of cattle over a time span of 5 months. In addition, the performance of different serological techniques for antibody detection against LSDV was compared. For sample collection, an area without previous LSD outbreak reports in Serbia was selected. Seventy-nine cattle from twenty farms vaccinated in 2016 and re-vaccinated in 2017 were included in the study. Two farms from the same area with good calving management were selected for investigation of passive LSDV antibody transfer from vaccinated mothers to new-borne calves.ResultsAll investigated cattle were healthy on the day of vaccination and during the whole study. Swelling at the injection site or other side effects of vaccination did not occur after re-vaccination in the study.Detection of LSD-specific antibodies was performed with the standard serological methods VNT and IFAT as well as a commercially available Capripox double antigen multi-species-ELISA. Capripoxvirus-specific antibodies were detected 46 to 47 weeks after vaccination in 2016, with VNT in 35.06% and with IFAT and ELISA in 33.77%. A secondary response was observed in all three tests 1 month after re-vaccination with a significant increase in seropositive animals compared to the results before re-vaccination. With all applied serological methods, the number of animals testing positive was significantly higher at 1 and 5 months post re-vaccination than before re-vaccination. No significant statistical difference (p > 0.05) was observed between the results of all three tests used. The sensitivity and specificity of ELISA was estimated to be SeELISA 91% and SpELISA 87% calculated by the results of VNT and SeELISA 88% and SpELISA 76% calculated by the results of IFAT. Passive antibody transfer from vaccinated mothers to new-born calves was investigated at 14 days after birth. Discrepancies for the detection of LSDV specific antibodies between cows and newborn calves at the age of 14 days were observed in VNT and IFAT, but not in ELISA.ConclusionOf all tests used the commercially available ELISA shows to be the most useful for high throughput analysis compared to VNT or IFAT.

Evaluation of Enzyme-Linked Immunosorbent Assay Using Recombinant 56-kDa Type-Specific Antigens Derived from Multiple Orientia tsutsugamushi Strains for Detection of Scrub Typhus Infection.

Scrub typhus is caused by the intracellular bacterium Orientia tsutsugamushi. The 56-kDa type-specific antigen (TSA) displays a significant antigenic variation across different O. tsutsugamushi strains. To minimize the influence of the antigenic diversity of TSA on assay sensitivity, we developed a mixed-TSA enzyme-linked immunosorbent assay (mixed-TSA ELISA) using a mixture of recombinant TSAs of prototype (Karp, Gilliam, and Kato) and local (TW-1, TW-10, TW-19, and TW-22) O. tsutsugamushi strains as antigens to detect immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against O. tsutsugamushi. These four local strains covered a major part of the total genetic diversity of TSA gene of O. tsutsugamushi in Taiwan. A total of 109 acute-phase serum samples from O. tsutsugamushi polymerase chain reaction-positive, scrub typhus patients, and 82 negative control serum samples from non-scrub typhus cases were used for evaluation of the recombinant TSA-based ELISA. We compared the performance of the mixed-TSA ELISA with immunofluorescence assay (IFA), which is considered the gold standard method for the serological diagnosis of scrub typhus. The results indicated that the sensitivity of IgM mixed-TSA ELISA (80.7%) was significantly higher than that of IgM IFA (68.8%). We demonstrated that the mixed-TSA ELISA had a high sensitivity and specificity and can be used for screening of scrub typhus patient in the early phase of the disease.

Added value of IgA antibodies against Zika virus non-structural protein 1 in the diagnosis of acute Zika virus infections

Zika virus (ZIKV) is a mosquito-borne flavivirus posing a public health threat due to its association with neurological complications in newborns and adults. In flavivirus-endemic areas, coming mosquito seasons will require the differentiation of primary versus secondary and acute versus past ZIKV/flavivirus infections. This is complicated by two major difficulties: [i] secondary infections often present with low or undetectable titres of specific IgM and with early-positive IgG, [ii] previous flavivirus infection(s) or vaccinations cause elevated cross-reactivities. Here, we analysed the anti-ZIKV IgA, IgG, and IgM responses at different stages of infection in an endemic setting, scrutinising the diagnostic relevance of specific IgA. Anti-ZIKV antibodies were measured by ELISA based on ZIKV non-structural protein 1 (NS1) in paired sera from 31 patients with suspected primary or (flavivirus-primed) secondary ZIKV infection. The control panel comprised samples from 136 DENV-infected patients. Among ZIKV samples collected 8–16 days after symptom onset, ELISA sensitivities for detecting anti-ZIKV NS1 IgA, IgG, and IgM were 93.5%, 100%, and 48.4%, respectively. The proportion of cases with negative IgM but positive IgA was higher in suspected secondary (61.9%) than in primary (30.0%) ZIKV infections. Combined IgA/IgM detection yielded a sensitivity of 100% at a specificity of 97.1%. In conclusion, at time points after PCR can detect the virus, the determination of anti-ZIKV NS1 IgA may improve the accuracy in diagnosing acute ZIKV infection in flavivirus-endemic regions in the context of both primary and secondary infection, especially when IgM is undetectable.