[Analysis on the application of three methods for malaria diagnosis].

Objective: To test the usage of microscopic examination, antigen detection(rapid dignostic test, RDT) and nucleic acid test(PCR) for detection of malaria cases. Methods: The blood test results for malaria and suspected malaria cases during 2012-2015 were retrospectively reviewed. Taking the confirmed cases as a gold standard, the three methods were compared in aspects of diagnosis indices, specificity of identification species, and cost effectiveness. Results: A total of 212 samples were included, each analyzed with the three methods. Based on the results of the three tests, 167(78.8%) were determined to be positive for malaria, and 45 negative (21.2%). Of the positive samples, 120(71.9%) were infected with Plasmodium falciparum,22(13.2%) with P. vivax,17(10.2%) with P. ovale, 6 (3.6%) with P. malariae, and 2(1.2%) with mixed infections. The method of PCR had the highest diagnostic efficiency (96.2%,204/212), followed by RDT (93.2%,192/206; P > 0.05 vs. PCR) and the microscopic method (88.2%,187/212; P < 0.05 vs. RDT and PCR). Similarly, the PCR method had the highest overall coincidence rate to the confirmed cases (95.3%,202/212), followed by RDT (93.2%,192/206) and microscopy (88.2%,187/212; P < 0.05 vs. PCR). As to the identification specificity among species, the PCR method(95.6%, 43/45) was superior to microscopy (91.1%, 41/45; P > 0.05 vs. PCR) and RDT (68.9%, 31/45; P < 0.05 vs. PCR). As to the identification of a particular species (P. falciparum), RDT performed best (100%,116/116), followed by PCR (93.3%,112/120) and microscopy (84.2%,101/120). Based on the comprehensive evaluation on 14 indicators including if it is a diagnostic criterion, equipment and technical requirement, diagnostic performance, time cost, and the need of technical training and promotion, we found that the RDT method had the highest score(37 of 42), while microscopy and PCR were scored 26 and 27, respectively. Conclusion: Under the falciparum malaria-dominated epidemiological situation, PCR and RDT show a higher detection efficiency, PCR and microscopy perform better in species identification, and RDT has the highest cost-effectiveness.

[Application of Semi-solid Culture and Rapid Screening Strategy in the Preparation of Monoclonal Antibody against Plasmodium falciparum Histidine-rich Protein].

OBJECTIVE: To prepare the monoclonal antibody of Plasmodium falciparum histidine-rich protein, and analyze the roles of semi-solid culture technique and screening strategies in the preparation of monoclonal antibodies. METHODS: BALB/c mice was immunized with the recombinant antigen of Plasmodium falciparum histidine-rich protein (rePf-HRP). The spleen cells of immunized mice were fused with SP20 cells. The fused cells were cultured in semi-solid medium containing 2% methylcellulose to promote colony growth. The single cell clone was transferred to a liquid medium. Testing methods for screening culture supernatant was established based on the immune antigen and other related antigens. The positive cell clones by coarse screening and specific screening were preserved in liquid nitrogen. The positive cell lines were used for ascite antibody preparation, identification of IgG subclass, recognition sites, antibody affinity and application analysis on sensitivity of detecting antigen. RESULTS: A total of 915 cell clones were obtained in semi-solid culture after cell fusion. The positive rate by coarse screening was 37.8% (346/915). The positive rate of specific screening accounted for only 2.6% (9/346) of the coarse screening-positive clones, 0.98% (9/915) of the total number of clones. Eight specific antibody-secreting cell clones were obtained after liquid nitrogen frozen recovery tests. After further detection, 2 specific cell clones could be used as a pair of antibody for rapid detecting circulating antigen in the blood of patients with falciparum malaria. CONCLUSION: Semi-solid culture method can provide enough fused cells for screening. Combined strategy of coarse and specific screening ensures the rapid selection of specific clones.

[Investigation on Entamoeba histolytica infection in diarrhea patients from general hospitals in Shanghai City].

OBJECTIVE: To understand the status of Entamoeba histolytica infection in diarrhea patients in general hospitals, so as to provide the evidences for the prevention and control of the disease. METHODS: The diarrhea patients in intestinal disease clinics of 3 general hospitals in Shanghai City were chosen as the investigation objectives, and their fecal and blood samples were collected, and then were detected by the normal saline direct smear method and iodine solution staining, immunochromatographic method and ELISA respectively to understand the infection status of E. histolytica, and the characteristics of the infected persons were analyzed. RESULTS Totally 1 015 fecal samples were detected, and among which 36 positive ones were detected by parasitological examinations, with a general positive rate of 3.55%. There were no statistically significant differences among the positive rates of patients from the three hospitals (P > 0.05), nor between or among those of the patients with different sexes, ages, occupations and education levels (all P > 0.05). The positive rate of E. histolytica in bloody purulent stools was higher than those in loose stools and watery stools (both P < 0.01). The peak period of infection was from July to September. Among the 36 infected people detected by parasitological examination, 88.90% of them complained about abdominal pain, and the red blood cells and leucocyte cells were found in the stool samples of 75.00% and 22.23% of the cases, respectively. The positive rates of E. histolytica were 8.18% (83/1 015) and 7.12% (48/675) respectively when detected by the immunochromatographic method and ELISA. CONCLUSIONS: Summer and autumn are the high risk seasons for E. histolytica infection, and the surveillance should be strengthened in this period. The positive rate of E. histolytica in samples of bloody purulent stools is high, and the combined application of several detection methods can increase the detection rate.

Evaluation of partially purified soluble egg antigens in colloidal gold immunochromatography assay card for rapid detection of anti-Schistosoma japonicum antibodies.

Shistosomiasis is one of the important parasitic diseases in developing countries and especially remains a threat to public health in China. Many immunodiagnostic kits have shown cross-reactions with other parasitic diseases and need large volumes of serum for the tests. In this study, we evaluated partially purified soluble egg antigen (SEA) in a colloidal gold immunochromatography assay (GICA) card kit for rapid detection of anti-Schistosoma japonicum antibodies using 5 microl of serum. Partially purified SEA from S. japonica was purified by Sephacryl S-300 chromatography. The optional reaction system and detection level of GICA using partially purified SEA were established by improving conjugated concentration and formulation of sample buffer and labeled solution. GICA showed 93.7% sensitivity in detecting schistosomiasis patients, 97.6% specificity in healthy population and patients with other parasitic diseases and a Youden's index value of 0.91. Cross-reaction with other parasitic diseases, such as paragonimiasis (1 case) and toxoplasmosis (1 case) is significantly lower compared to using crude SEA. Partially purified SEA in GICA is practical for detection of schistosomiasis in the field as it requires a small volume of serum, has high sensitivity, and has low cross-reaction rate.

[Development and evaluation of colloidal gold immunochromatography assay (GICA) for detection of antibody against Schistosoma japonica in smaller serum].

OBJECTIVE: To develop a rapid colloidal gold immunochromatography assay (GICA) for detection of the antibody to schistosome in 5 microl sera. METHODS: A soluble egg antigen (SEA) from Schistosomajaponica was separated with sephacryl S-300 column to retain the active antigen fractions to the antibody of schistosome. The optional reaction system and detection kit with 5 microl sera were established by improving conjugated concentration and formulas of sample buffer and labeled solution. RESULTS: The sensitivity of detecting schistosomiasis patients whose stool examinations for schistosome eggs were positive was 93.7%, the specificity to health population 97.1%, the cross reaction rate to patients with paragonimiasis 5.6%. The Youden' s index value was 0.91. There was 96.1% crude coincidence of GICA and ELISA in detecting 507 cases of floating population and the Kappa value was 0.81. CONCLUSION: GICA kit is practical for detection of schistosomiasis in the field because of its advantages such as smaller serum needed, the high sensitivity, lower cross reaction rate and spread application for human and animals.

[Investigation on food contamination with parasites in Shanghai market].

OBJECTIVE: To understand the contamination status of food with parasites in Shanghai market, so as to provide the evidence for formulating the surveillance strategy in parasitic diseases and provide the technical support in the food safety. METHODS: The freshwater fish products, marine products, vegetables, snails and frogs were sampled with the cluster random method in the farmer-trades and supermarkets of the 18 districts in Shanghai City during 2005-2010 period, and all the specimen were screened by the digestion method, or crushing method or dissection method or saline floatation method to check the parasite metacercaria or larvae or eggs. RESULTS: A total of 5 185 specimen in 23 species of fishes were screened in fresh-water products, with parasite infection rate of 1.93%. About 4 033 specimens of 20 species of fishes and shrimps were screened and 1.76% of samples were infected with Clonorchis sinensis. Among all kind of fishes, the highest infection rate was 7.83% (48/613) in Pseudorasbora parve. No any infected specimen was found among 1 152 specimen of fresh water crustacean screened. Anisakis spp. were found in 12.7% of 433 specimens of 23 species of seawater products, among them, the higher infection rate of Anisakis spp. was found in Pneumatophorus japonicas and Trichiurus haumela with their infection rates of 50.00% (13/26) and 23.46% (42/179), respectively, which much higher than those found in other seawater products (P < 0.01). In 37 kinds of vegetables, the parasite eggs were found in one of 428 specimens with its infection rate of 0.47%, while no any parasite egg was found in 103 specimens of 10 kinds of fruits. No any Angiostrongylus cantonensis larvae were found in 330 snails, 31.37% of 102 frogs were found infected with Spirometra mansoni spargana. No any contamination with parasites was found in 116 meat specimens of pigs and cattle. In the same time, the intestinal parasite infection rate of residents was 0.42% (131/31 239). CONCLUSIONS: It is found that some of foods in Shanghai markets are contaminated with parasites. Therefore, it is necessary to enforce the activities in health education as well as to take integrated prevention measures in order to ensure the food safety.

[Epitope structure prediction of antigen B of Echinococcus granulosus and construction of multi-epitope recombinant antigen].

OBJECTIVE: To improve the reactivity of Echinococcus granulosus antigen B (EgAgB) multi-epitope antigens using the gene fragment from 3 subunits, EgAgB1, EgAgB2, and EgAgB4. METHODS: Discovery Studio Visualizer software and I-TASSER on-line server were used to predict protein structure gene sequence of the 3 subunits and their combinations in different way. The epitope or subunit combination higher prediction scores was selected for gene recombination. The target sequence was amplified with primers and by using overlap extension PCR technology. The target sequence was then cloned to pET32a constructing expression plasmid and expressing recombinant proteins. The expressed products were served recombinant antigens after purification. The immuno-response of the recombinant multi-epitope antigens were Western blotting analysis. RESULTS: Structure prediction showed that all the three subunits EgAgB1, EgAgB4 are in a "Z" word structure. The epitope region is located in the central part of the sequence. For from the three subunits and four reactive epitopes (KK36, RK30, B4-2, and B4-3), 57 different combinations tried for structure prediction. Six of them were selected for recombination and expression. Western blotting six multi-epitope antigens (MEA-8, MEA-20, MEA-26, MEA-36, MEA-49, and MEA-52) suggested reactivity of multi-epitope antigen was much stronger than AgB subunit antigens when the positive echinococcosis were used. CONCLUSION: By using protein tertiary structural prediction and screening the higher prediction score of combinations, six multi-epitope recombinant antigens were constructed. Western blotting shows that the hand reactivity of multi-epitope antigen is much stronger than that of AgB subunit antigens.

[Detection of Cryptospordium spp. in environmental water samples by FTA-PCR].

OBJECTIVE: To establish a FTA-polymeras chain reaction (FTA-PCR) method in detection of Cryptospordium spp. in different sources of water. METHODS: The semi automated immunomagnetic separation (IMS) of Cryptospordium oocysts in environmental water samples was performed firstly, and then genomic DNA of Cryptospordium oocysts was extracted by FTA filters disk. Oligonucleotide primers were designed based on the DNA fragment of the 18 S rRNA gene from C. parvum. Plate DNA was amplified with primers in PCR. The control DNA samples from Toxoplasma gondii,Sarcocystis suihominis, Echinococcus granulosus, and Clonorchis sinensis were amplified simultaneously. All PCR products were detected by agar electrophoresis dyed with ethidium bromide. RESULTS: The 446 bp fragment of DNA was detected in all samples of C. parvum, C. andersoni, and C. baileyi, while it was not detected in control groups in laboratory. No positive samples were found from 10 samples collected from tape water in 5 districts of Shanghai City by FTA-PCR. Nine positive samples were detected totally from 70 different environmental water samples, there were 0 out of 15 samples from the source of tape water, 2 out of 25 from the Huangpu River, 5 out of 15 from rivers around the animal farmers, 1 out of 9 from output water of contaminating water treatment factory, 1 out of 6 from the out gate of living contaminating water. The 446 bp fragment was detected from all the amplified positive water samples. CONCLUSIONS: FTA-PCR is an efficient method for gene detection of Cryptospordium oocysts, which could be used in detection of environmental water samples. The contamination degree of Cryptospordium oocysts in the river water around animal farms is high.

[Impairment of learning and memory ability in mice with latent infection of Toxoplasma gondii].

OBJECTIVE: To detect the learning and memory ability in mice model of latent Toxoplasma gondii infection with object recognition test and Morris water maze test. METHODS: Thirty-six Kunming mice were divided into control group, infection group with 6 cysts each mouse (low infection group), and infection group with 12 cysts each mouse (high infection group) averaged. Mice in the two infection groups were orally infected with T. gondii Prugniaud (PRU) low virulence strain. Object recognition test was conducted at the 63rd day after infection. After the first day of adaptation and the second day of familiarization in the test, the time expended on exploring new and familiar objects was recorded on the third day and the discrimination index (DI) was calculated. Morris water maze test was conducted at the 66th day. The ability of spatial learning, spatial memory retention and working memory capacity was evaluated by place navigation test, spatial probe test, and working memory test, respectively. The mice were sacrificed at the 74th day after infection. The left cerebral hemisphere of mice was fixed, sliced, and stained with eosin-hematoxylin for pathological examination. The right hemisphere was used to detect the activity of superoxide dismutase (SOD) and malondialdehyde (MDA) content. RESULTS: The results of object recognition test showed that the discrimination index of high infection group and low infection group was (14.3 +/- 5.2)% and (17.5 +/- 5.6)%, respectively, significantly lower than the control [(28.9 +/- 7.1)%] (P < 0.01). In the place navigation test, the latency to find the platform in the two infection groups was longer than the control, with significant difference on the second and third day (P<0.05). In the spatial probe test, the percentage of the distance across the platform quadrant in the total swimming distance of high infection group and low infection group were (19.9 +/- 5.0)% and (23.9 +/- 6.8)%, respectively, significantly lower than the control [(27.4 +/- 3.6)%] (P < 0.05). In the working memory test, at the fourth day of test the latency of high infection group and low infection group [(365 +/- 14.2) s and (35.3 +/- 13.7) s] was significantly longer than the control [(30.4 +/- 12.5) s] (P<0.05). In all the tests, there was no statistical significance between low infection group and high infection group (P > 0.05). The brain sections of two infection groups showed cysts of T. gondii, proliferation of glial cells, widened gap around small blood vessels, and a phenomenon of "vascular cuff". The activity of SOD in the mice brains of two infection groups was significantly lower than the control, while MDA level was significantly higher (P < 0.05). SOD and MDA showed no significant difference between two infection groups (P > 0.05). CONCLUSION: Latent infection of T. gondii may lead to learning and memory impairment in mice.

[Effect of IgG3 antibody purified from sera of Microtus fortis against Schistosoma japonicum].

IgG3 antibody reaction to soluble antigens prepared from schistosomula (SSA), adult worms (SAWA) and eggs (SEA) in laboratory-bred Microtus fortis (Mf), BALB/c mice and Kunming (Km) mice challenged by cercariae of Schistosoma japonicum was detected by indirect ELISA. The effect of purified IgG3 antibody on in vitro killing schistosomula and protecting mice from infection of S. japonicum was evaluated. The IgG3 antibody level in Mf against SSA and SAWA increased significantly by 79.6 percent and 49.6 percent after the fourth week of challenge infection, but no significant increase in BALB/c mice. Purified IgG3 antibody from laboratory-bred Mf and wild Mf effectively killed schistosomula, and that of the wild Mf induced higher worm-reduction rate. The death rate of schistosomula due to IgG3 antibody purified from sera of laboratory-bred Mf and wild Mf was 2.35 and 5.88 times as high as that of Km mice respectively. The results suggest that IgG3 antibody from Microtus fortis may play an important role in immunity against S. japonicum.

[Development of immunoblot kit for the detection of anti-Toxoplasma antibodies].

OBJECTIVE: To develop a diagnostic kit for the detection of anti-Toxoplasma antibodies in human sera by immunoblot technique. METHODS: The cytoplasm proteins of Toxoplasma gondii were extracted from collected ascites in Kunming strain mice infected by tachyzoites of Toxoplasma gondii (RH strain). Toxoplasma gondii soluble antigens were separated by SDS-PAGE electrophoresis and transferred to pyroxylin membrane. Using nonpoisonous tetramethylbenzidine (TMB) as horseradish peroxidase substrate for immunoblot, the optimal experimental conditions were selected through comparison of antigen preparation, reagents for blocking or washing, dilution concentration, reaction time, and frequencies of reacting band. Sensitivity, specificity, Youden index and stability were evaluated as the standard for the kit. RESULTS: In 30 sera with anti-Toxoplasma [gM antibodies and 28 with IgG antibodies, the sensitivity for IgM and IgG antibody detection was 90.0% (27/30) and 85.7% (24/28) respectively, the specificity was all 100% in examining 40 healthy control sera, and the Youden index was 0.9 and 0.86 respectively. The kit was stable at 49 degrees C for 6 months. CONCLUSION: The immunoblot kit shows an easy operation, fast reaction and reliable result, and may be practical.

[A rapid procedure to purify serum IgG from Microtus fotis].

OBJECTIVE: To evaluate the procedure to purify IgG antibodies from Microtus fotis serum. METHODS: IgG antibodies from sera of three groups of Microtus fotis were purified by protein G or protein A affinity chromatography, their purity and binding capacity were compared. RESULTS: The protein G affinity chromatography was more efficient than protein A affinity chromatography. The antibodies isolated from protein G affinity chromatography showed a higher purity and better activity than that from protein A affinity chromatography monitored by SDS-PAGE and ELISA. The ability of the purified IgG to bind the second antibodies were 8.5 times and 3.1 times that of non-IgG proteins and unpurified sera, respectively. CONCLUSION: The protein G affinity chromatography is a rapid, convenient and reliable procedure for Microtus fotis serum IgG purification.