Establishment and Preliminary Application of a Rapid Fluorescent Focus Inhibition Test (RFFIT) for Rabies Virus

The World Health Organization (WHO) standard assay for determining levels of the rabies virus neutralization antibody (RVNA) is the rapid fluorescent focus inhibition test (RFFIT), which is used to evaluate the immunity effect after vaccination against rabies. For RFFIT, CVS-11 was used as the challenge virus, BSR cells as the adapted cells, and WHO rabies immunoglobulin (WHO STD) as the reference serum in this study. With reference to WHO and Pasteur RFFIT procedures, a micro-RFFIT procedure adapted to our laboratory was produced, and its specificity and reproducibility were tested. We tested levels of RVNA in human serum samples after immunization with different human rabies vaccines (domestic purified Vero cell rabies vaccine (PVRV) and imported purified chick embryo cell vaccine (PCECV)) using different regimens (Zagreb regimen and Essen regimen). We analyzed the levels of RVNA, and compared the immune efficacy of domestic PVRV and imported PCECV using different immunization regimens. The results showed that the immune efficacy of domestic PVRV using the Zagreb regimen was as good as that of the imported PCECV, but virus antibodies were generated more rapidly with the Zagreb regimen than with the Essen regimen. The RFFIT procedure established in our laboratory will enhance the comprehensive detection ability of institutions involved in rabies surveillance in China.

Microdilution inhibition test of Chinese herbs to assess their effect against clinical strains of Ureaplasma urealyticum in vitro

Objective：To explore the antibacterial effect of Chinese crude drugs against clinical strains of Ureaplasma urealyticum（UU）, including eight pure herbs and three compound herbs, and determine their minimal inhibitory concentrations（MICs）. Methods：Isolates were collected from clinical patients with UU infection, and cultured in UU broth. In order to test the different effects on clinical strains of UU, the assays were performed by microdilution inhibition tests, and MICs of the herbs against the clinical strains of UU were calculated. Results：The MICs of eight pure herbs against clinical strains of UU were as follows： Galla Chinensis：0.313-1.25 g/L; Fructus Forsythiae： 1.25-5.00 g/L; Cortex Phellodendri： 1.25-5.00 g/L; Radix Paeoniae Rubra： 1.25-2.50 g/L; Semen Plantaginis：2.50-10.00 g/L; Herba Lysimachiae： 5.00-20.00 g/L; Rhizoma Coptidis： 5.00-20.00g/L, Herba Houttuyniae： 10.00-20.00 g/L. The MICs of compound herbs were： Liuheji： 0.625-2.50 g/L; Bazhengsan： 5.00-20.00 g/L; Wulinsan：2.50-20.0 g/L.Conclusion：Galla Chinensis, Fructus Forsythiae, Cortex Phellodendrim, Radix Paeoniae Rubra, and Semen Plantaginis, exerted the stronger antibacterial effect against clinical strains of UU, whereas Herba Lysimachiae, Rhizoma Coptidis and Herba Houttuyniae, had relatively weaker activity against UU. Compound herbs, Bazhengsan and Wulinsan, and particularly Liuheji, also had antibacterial effects against UU. Further studies of the effects and mechanisms of action of Chinese crude drugs against UU infections are worthwhile.

EFFECTS OF SDS,PBS SOLUTIONS UPON FLUORESCENCE VALUES OF DERMATOPHAGOIDES PTERRONYSSINUS RADIOALLERGOSORBENT TEST INHIBITION

Objective To explore the effects of SDS, PBS re-dissolvent solutions on fluorescence values of radioallergosorbent test (RAST) inhibition. Methods Dermatophagoides pterronyssinus allergen immunoCAP and UniCAP 100 System were used. The Sera Pool consisted of 20 Dermatophagoides pterronyssinus allergic patients sera, their specific IgE fluorescence values were between 12 505 and 24 776. Results Fluorescence value percentages decreased: 62.9%, 54.1%, 43.5%, 6.7%, 3.7%, 2.6%, 2.2%, and 1.4% respectively, when SDS concentrations were at 2%, 1%, 0.5%, 0.25%, 0.1%, 0.05%, 0.025%, and 0.01%. Fluorescence values decreased more than 5% with SDS concentrations equal to 0.25% or higher. PBS in 0.1 and 0.01 mol/L concentrations decreased fluorescence values 2.9% and 0.9% respectively. Conclusions SDS is a commonly used surfactants in allergen extract and re-dissolvent prepared allergen precipitation for RAST inhibition. Thus effects of surfactants (e.g. SDS) upon the RAST inhibition tests must be considered when they were used as re-dissolvent agents to improve protein resolution in RAST inhibition.

Comparison of RFFIT Tests with Different Standard Sera and Testing Procedures

The World Health Organization （WHO） standard assay for determining antibody level is the rapid fluorescent focus inhibition test （RFFIT） and is used to determine the degree of immunity after vaccination against rabies. To compare the difference in RFFIT results between the laboratories of The National Institute of Infectious Disease in Japan （NIID） and the Chinese Centre for Disease Control （CCDC） as well the influence of the choice of standard serum （STD） for the detection, the two laboratories detection methods were simultaneously manipulated by RFFIT. The reference serums used in NIID and the WHO standard serum used in CCDC were compared in the same RFFIT detection to determine the titer of four sera samples C1, Sl, S2 and S4 in parallel, and the titers of the detected sera samples were calculated using the standard formula for neutralizing antibody titer. No significant difference was found in RFFIT methods from the two laboratories and the RFFIT testing procedures of the two laboratories have good consistency. However, different titers were obtained with the tentative internal standard serum （TI-STD） produced by adjusting to 2.0 IU of WHO standard serum in NIID and the WHO STD. The titer determined with the TI-STD was higher than that determined with WHO STD, This difference appears to be significant and requires further investigation

Comparative Study of Serologic Results Obtained by HI Test of Avian Influenza and Newcastle Disease Performed in Four Mexican Laboratories

In the poultry industry, it is common to evaluate the humoral immune response associated to the vaccination calendar used for the different zootechnical purposes. When the vaccine against avian influenza and Newcastle disease currently used in a farm is going to be changed, an important parameter observed to choose a product over the others is based on the antibody titers reached by the application of the new vaccine. This study aimed to compare the serologic results obtained by hemagglutination inhibition （HI） test of avian influenza and Newcastle disease reported in four different national laboratories. One-day-old Ross broiler chickens were kept in Horsfall-Bauer isolation units and were vaccinated subcutaneously for the prevention of avian influenza and Newcastle disease. Then, serum of all the birds was extracted at three, six and seven weeks old and sent to four different national diagnostic laboratories, where HI test was performed for avian influenza and Newcastle disease. The treatments were designed in 4 × 3 factorial. Data showed significant statistical differences between laboratory results （up to six logarithms for influenza at six and seven weeks）. This study confirms that the results of the HI test can vary from one laboratory to another, thus it is important to consider this, when the vaccines against avian influenza and Newcastle disease are evaluated at field.

Detection of specific IgE antibodies to major and minor antigenic determinants in sera of penicillin allergic patients

Objective To investigate the mechanism (s) of penicillins allergic reaction. Methods The radioallergosorbent test (RAST) was used to detect 9 specific IgE antibodies, including major antigenic determinants: benzylpenicilloyl (BPO), ampicilloyl (APO), amoxicilloyl (AXO), phenoxomethylpenicilloyl (PVO) and flucloxacilloyl (FLUO), and minor antigenic determinants: benzylpenicillanyl (BPA), amoxicillanyl (AXA), 6-aminopenicillanic (APA) and phenoxomethylpenicillany (PVA), in the sera of 32 penicillin allergic patients. The relationship between specific IgE antibodies and penicillins chemical structures was studied by radioallergosorbent inhibition test. Results Nineteen of 32 patients (59.4%) were RAST positive, among whom, five cases were positive only to one or two antigenic minor determinants, and three cases were positive only to one or three major antigenic determinants. The remaining 11 patients were positive not only to major antigenic determinants but also minor antigenic determinants. In 9 specific IgE antibodies, the positive rate of PVA-IgE was the highest (34.38%), followed by BPO-IgE (31.25%). The positive rate of FLUO-IgE was the lowest (15.63%). Of the total patient group, 53.13% were positive to one or more minor antigenic determinants, while 37.5% (12/32) were positive to one or more major antigenic determinants. The percentage of patients with urticarial reactions who were positive to minor antigenic determinants (63.16%) was significantly higher than observed in the anaphylactic shock group (38.5%, P<0.05). Conclusions The minor antigenic determinant was important in allergic reaction. The combining sites of the specific IgE antibodies were likely to be the side-chain of drug or the overwhelming drug molecule.

The fourth case of rabies caused by organ transplantation in China

In January 2019,the fourth rabies case caused by organ transplantation was noticed in China,with the conditions of one per year for the recent four years.Different from the previous cases,there were no definite epidemiological histories of exposure or rabies-related symptoms from this patient.This case strongly supports the call for the legislation of establishing a national-level management that will incorporate the screening programs on donors prior to the practice of organ transplantation to reduce the risks on rabies caused by organ transplantation.