Development of an Indirect ELISA to Detect African Swine Fever Virus pp62 Protein-Specific Antibodies

African swine fever (ASF) is a highly detrimental viral disease caused by African swine fever virus (ASFV). The occurrence and prevalence of this disease have become a serious threat to the global swine industry and national economies. At present, the detection volume of African swine fever is huge, more sensitive and accurate detection techniques are needed for the market. pp62 protein, as a protein in the late stage of infection, has strong antigenicity and a high corresponding antibody titer in infected pigs. In this study, the CP530R gene was cloned into expression vector pET-28a to construct a prokaryotic expression plasmid, which was induced by IPTG to express soluble pp62 protein. Western blot analysis showed that it had great reactivity. Using the purified recombinant protein as an antigen, an indirect ELISA method for detecting ASFV antibody was established. The method was specific only to ASFV-positive serum, 1:1600 diluted positive serum could still be detected, and the coefficients of variation (CV) of the intra assay and inter assay were both <10%. It turns out that the assays had excellent specificity, sensitivity, and repeatability. This provides an accurate, rapid, and economical method for the detection of ASFV antibody in clinical pig serum samples.

Lateral Flow Immunoassay for Visible Detection of Human Brucellosis Based on Blue Silica Nanoparticles

Brucellosis is a highly contagious zoonosis chronic infectious disease with a strong latent capability to endanger human health and economic development via direct or indirect ways. However, the existing methods for brucellosis diagnosis are time-consuming and expensive as they require a tedious experimental procedure and a sophisticated experimental device and performance. To overcome these defects, it is truly necessary to establish a real-time, on-site, and rapid detection method for human brucellosis. Here, a lateral flow immunoassay (LFIA) with a rapid, sensitive, and alternative diagnostic procedure for human brucellosis with a high degree of accuracy was developed based on blue silica nanoparticles (SiNPs), Staphylococcal protein A (SPA), and surface Lipopolysaccharide of Brucella spp. (LPS), which can be applied for rapid and feasible detection of human brucellosis. To our knowledge, this is the first report that uses blue SiNPs as a signal probe of LFIA for the rapid diagnosis of human brucellosis. The precursor of blue SiNPs@SPA such as colorless SiNPs and blue SiNPs was synthesized at first and then coupled with SPA onto the surface of blue SiNPs by covalent bond to prepare blue SiNPs@SPA as a capture signal to catch the antibody in the brucellosis-positive serum. When SPA was combined with the antibodies in the brucellosis-positive serum, it was captured by LPS on the test line, forming an antigen–antibody sandwich structure, resulting in the T line turning blue. Finally, the results showed that it is acceptable to use blue SiNPs as visible labels of LFIA, and standard brucellosis serum (containing Brucella spp. antibody at 1,000 IU/ml) could be detected at a dilution of 10−5 and the detection limit of this method was 0.01 IU/ml. Moreover, it also demonstrated good specificity and accuracy for the detection of real human serum samples. Above all, the blue SiNPs-based LFIA that we developed provides a rapid, highly accurate, and inexpensive on-site diagnosis of human brucellosis, and shows great promise in clinical diagnostics for other diseases.

Autoimmune encephalitis with coexistent LGI1 and GABABR1 antibodies: case report

Background Autoimmune encephalitis (AE) with multiple auto-antibodies is of great clinical significance because its complex clinical manifestations and atypical imaging increase the difficulty of diagnosis, differential diagnosis and treatment, which may aggravate the disease, increase the recurrence rate and mortality. The coexistence of anti-Leucinie-rich Glioma Inactivated 1 (LGI1) and anti-γ-aminobutyric acid-beta-receptor 1 (GABABR1) has not been published before. Case presentation We herein present the case of a 60-year-old man with slow response, behavioral changes, psychosis and sleep disorders. Laboratory test included serum hyponatremia, positive serum LGI1 and GABABR1 antibodies using transfected cell-based assays. Electroencephalogram exhibited moderate diffusion abnormality. The patient responded well to steroid impulse treatment and sodium supplement therapy, and did not recur during the follow-up. Conclusions Here we report the first AE characterized by positive LGI1 and GABABR1 antibodies, as well as summarizing AE with multiple auto-antibodies reported so far, hopefully to provide experience for clinical practice.

IL28B-Gene Polymorphisms (rs12979860) in HCV Liver Parenchymal changes legitimize Screening for SNPs before DAAs Therapy

Background and objective: IL28B-gene polymorphisms show inconclusive relationships with CHCV DAAs-treatment outcomes on evaluation by serum-PCR. Solitary intra-PBMCs HCV-RNA antisense-strands are independently found in naïve and experienced cases regardless to viremia or hepatic-parenchymal changes. We correlated frequencies of IL28B-gene SNPs and alleles with HCV induced liver-changes during SVR evaluation by PBMCs-PCR after DAAs-therapy. Methods: Twelve weeks after completing DAAs-therapy, the impacts of IL28B-gene-SNPs were evaluated in three groups of patients: group-I (n=25) with negative serum and PBMCs HCV-PCR, group-II (n=52) had solitary intra-PBMCs HCV-RNA, and group-III (n=25) had positive serum HCV-RNA. All cases were subjected to IL28B-gene-SNP analyses and correlated with their ultrasonographic liver changes.Results: IL28B-genotyping in post-DAAs-treatment HCV-SVR and viral relapse revealed: a) dominant CC-genotype and C allele in normal hepatic parenchyma in group-I compared to group-II (P=0.0047, 0.0007) and group-III (P=0.0564, 0.000003) b) frequent CT-SNP and T-allele in bright hepatic parenchyma in group-II when compared with group-I (P=0.0077, 0.002 ) and group-III (P=0.0363, 0.0005) c) increased TT-SNP and T-allele frequencies in coarse liver in group-III compared to group-I (P=0.02256, 0.000130) and group-II (P=0.08647, 0.004308). Conclusions: Outcomes of HCV treatment with DAAs are dependent on host IL28B-gene polymorphisms and HCV induced liver changes. SVR is achieved when wild type-CC-genotype and C-allele are dominant in normal hepatic parenchyma; solitary intra-PBMCs-relapse occurs in increased frequency of CT-genotype when liver tissues are fibrotic; serologic-relapse is dominant when TT-genotype and T-allele are frequent in cirrhotic liver. IL28B-gene SNP analyses in relation to hepatic parenchymal changes are recommended before treating CHCV-infection with DAAs.

Improvement of Bovine Pestiviral diagnosis by the development of a cost-effective method for detecting viral RNA in fresh specimens and samples spotted in filter papers.

Bovine pestiviruses are the causative agents of Bovine viral diarrhea, a disease that generates severe economic losses in cattle. The aim of this study was to improve its diagnosis by means of developing a RT-qPCR to detect bovine pestiviruses A, B and H; and to set a protocol for collecting, shipping and conserving bovine pestiviral RNA in filter papers. The developed RT-qPCR showed high sensitivity in detecting these viruses in different matrixes: viral stocks, semen and serum samples. Regarding the possibility of using the technique to test serum pools, it was possible to identify a positive serum sample within a pool containing 30 sera. In addition to evaluating the qPCR from fresh samples, the use of filter papers to sow bovine samples was analyzed. The sampling method in two different filter papers using bovine blood drops was a useful alternative for diagnosis purposes and allowed the preservation of pestiviral RNA up to 12 months under refrigeration.

Development of a Dual ELISA for the Detection of CD2v-Unexpressed Lower-Virulence Mutational ASFV

African swine fever virus (ASFV) is an important viral pathogen infecting pigs worldwide throughout the pig industry. CD2v (an outer-membrane glycosylated protein of ASFV)-unexpressed lower-virulence mutants have appeared in China and other countries in recent years. Using OIE-recommended quantitative PCR and ELISA methods, people can accurately judge whether pigs are infected with wild-type ASFV. However, the strategy has failed to distinguish ΔCD2v lower-virulence mutants and wild-type ASFV infection. Here, we expressed and purified the CD2v and p30 proteins via CHO cells and successfully established a dual enzyme-linked immunosorbent assay (ELISA), which can be used to differentiate pigs infected with wild-type ASFV or with CD2v-unexpressed lower-virulence mutants. The dual ELISA showed excellent specificity without cross-reactions with antibodies of PRRSV, CSFV, JEV, PRV, or PPV. The dual ELISA could detect ASFV-infected positive serum samples up to dilutions of 5120 times, possessing high sensitivity. Therefore, the application of this dual ELISA approach can play an important role in ASFV epidemiology study and fill the gaps in differential diagnosis.

Anti-NMDAR encephalitis presenting after immature teratoma resection

This is a case of a young woman who developed neurological and psychiatric symptoms 3 days after resection of an immature teratoma. She was diagnosed with anti-NMDA receptor encephalitis via positive serum antibody titres, which was later confirmed with cerebrospinal fluid antibody titres. Given her cancer diagnosis, she underwent treatment with bleomycin, etoposide and cisplatin chemotherapy in addition to 5 days of high-dose steroids (1 g of intravenous methylprednisolone) for the encephalitis. This treatment regimen led to significant clinical improvement 3 weeks after completion of one cycle of chemotherapy.

An uncommon cause of vertigo: Neuromyelitis optica spectrum disorder

Neuromyelitis optica spectrum disorder (NMOSD) is an uncommon antibody-mediated disease of the central nervous system. Its classic presentation includes long segments of spinal cord inflammation, optic neuritis with or without intractable vomiting, and hiccups. Here, we described a case of a 39-year-old woman with an atypical presentation of vertigo, which was finally diagnosed as NMOSD by a positive serum aquaporin-4 antibody.

SYMPTOMS ASSOCIATED WITH DIFFERENT DEGREES OF MEGAESOPHAGUS IN CHAGAS DISEASE

ABSTRACT BACKGROUND: Dysphagia is the most frequent digestive symptom in Chagas disease, although other symptoms are reported. These symptoms can be associated with the degree of radiological impairment of the esophagus and the duration of dysphagia. OBJECTIVE: This investigation aimed to assess the symptoms and the time of dysphagia related to the different degrees of megaesophagus in patients with Chagas disease. METHODS: A total of 29 patients aged 48 to 73 years participated in this investigation. All of them had dysphagia and a positive serum result for Chagas disease. They were submitted to the assessment of symptoms and radiological examination of the esophagus to assess the degree of megaesophagus, which ranged from I (mild change) to IV (intense change). Dysphagia was quantified with the Eating Assessment Tool (EAT-10). RESULTS: Twelve (41%) patients had megaesophagus degree I, 9 (31%) had degree II, and 8 (28%) had degrees III (6) and IV (2). The intensity of dysphagia was not related to the result of the radiological examination, with EAT-10 median of 5.5 for the degree I, 9.0 for degree II, and 5.5 for degrees III and IV (P>0.25). Choking (14%), regurgitation (21%), voice complaint (21%), weight loss (17%), and odynophagia (17%) were not related to the degree of megaesophagus. Voice changes and odynophagia were related to the patients’ time of dysphagia. Likewise, the frequency of symptoms and EAT-10 values were related to the duration of dysphagia. CONCLUSION: The longer the patient had dysphagia, the more frequent were the symptoms reported by the patients. There was no relationship between the degrees of megaesophagus and the symptoms and intensity of dysphagia.