[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.

[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.

[Reactive epitope analysis of agB subunit antigens of Echinococcus granulosus by using synthetic peptides].

OBJECTIVE: To analyze reactive epitope of three subunit antigens AgB1, AgB2 and AgB4 of Echinococcus granulosus by using synthetic peptides. METHODS: Five synthetic peptides, KK36, RK30, B4-1, B4-2, and B4-3, derived from the sequences of AgB1, AgB2, and AgB4 subunit of E. granulosus, and the three recombinant subunits were used for the detection of serum antibodies by ELISA. A panel of 209 serum samples from patients with cystic echinococcosis (115), alveolar echinococcosis (54), cysticercosis (22), and healthy persons (18) was used in the study. The diagnostic efficiency of the recombinant subunits and peptides for serum detection was estimated and compared using receiver-operating characteristics (ROC) curve. RESULTS: The sensitivity and specificity of peptides KK36 and RK30 in patients with cystic echinococcosis were 89.2% and 62.5%, 85.0% and 59.4%, respectively. Their diagnostic efficiency (84.8% and 80.4%) was similar to AgB1 and AgB2 antigen (84.5% and 81.2%). The ROC curves of peptides KK36 and RK30 were well fitted by that of recombinant subunit AgB1 and AgB2. For the three peptides derived from AgB4 subunit, serum detection indicated that the diagnostic efficiency of B4-1, B4-2 and B4-3 were 49.4%, 57.9%, and 77.4%, respectively. Peptides B4-3 showed best reactivity and B4-2 also showed certain reactivity to the sera from patients with cystic echinococcosis. CONCLUSION: Peptides KK36 and RK30 contain the reactive epitope region of AgB1 and AgB2 subunits. B4-2 and B4-3 contain partial region of the reactive epitope of AgB4. The epitope region of AgB4 may be in the central and back part.

[Comparative analysis of nucleotide sequences of lactate dehydrogenase (LDH) gene and LDH epitopes of Plasmodium vivax and Plasmodium falciparum].

OBJECTIVE: To analyze the difference of nucleotide sequences of lactate dehydrogenase (LDH) gene and LDH epitopes of Plasmodium vivax and P. falciparum. METHODS: Specific primers were designed to amplify the full-length LDH gene sequence of P. vivax and P. falciparum (GenBank accession number: DQ198262 and DQ060151 respectively). The PCR products were sequenced and compared. The epitopes of objective LDH antigens were predicted by SYFPEITHI software. Pv-LDH and Pf-LDH genes were cloned into prokaryotic plasmid pET28a, then expressed in E. coli BL21(DE3) with isopropyl beta-D-1-thiogalactopyranoside (IPTG) induction. The immunogenicity of the recombinants Pv-LDH and Pf-LDH was analyzed by Western blotting and neutralization ELISA assays. RESULTS: Pf-LDH gene was same to reference sequences(DQ198262), while there is a single nucleotide difference at the position 666 between Pv-LDH gene and reference sequences (DQ060151). The coding region of the two genes contained 951 bp encoding a 316-amino-acid residue. Compared with Pf-LDH, Pv-LDH showed a nucleotide sequence identity of 75.1%, and an amino acid sequence identity of 90.2%. T cell epitope prediction indicated that there were 28 human leukocyte antigen (HLA) types which could recognize pLDH antigen epitopes. The common or similar epitopes accounted for about 75% of the predicted 180 epitopes. The number of specific epitopes of Pv-LDH and Pf-LDH proteins was 38 and 45, respectively. Western blotting analysis showed that the Pv-LDH recombinant antigen reacted with the sera of malaria patients, and the reactivity was much lower than that of sera of immunized rabbit. Neutralization ELISA showed that about 70.3% reactivity of Pv-LDH polyclonal antibodies could be suppressed by Pv-LDH, while only 30.5% by Pf-LDH. CONCLUSION: There are differences in DNA sequences of LDH gene and LDH epitopes between P. vivax and P. falciparum. The antibodies induced by the specific epitopes account for a small proportion in the antibody repertoire.

[Investigation on contamination of Cryptosporidium and Giardia in drinking water and environmental water in Shanghai].

OBJECTIVE: To understand the contamination status of Cryptosporidium sp. and Giardia lamblia in drinking water, source water and environmental water in Shanghai. METHODS: All water samples collected from drinking water, source water and environmental water were detected by a procedure of micromembrane filtration, immune magnetic separation (IMS), and immunofluorescent assay (IFA). RESULTS: Cryptosporidium oocysts and Giardia cysts were not found in 156 samples of the drinking water including finished water, tap water, or pipe water for directly drinking in communities. Among 70 samples either source water of water plants (15 samples), environmental water from Huangpu River(25), canal water around animal sheds(15), exit water from waste-water treatment plants(9), or waste water due to daily life(6), Cryptosporidium oocysts were detected in 1(6.7%), 2(8.0%), 7(46.7%), 1(11.1%), and 1(16.7%) samples, respectively; and Giardia cysts were detected in 1(6.7%), 3(12.0%), 6 (40.0%), 2(22.2%), and 2(33.3%), respectively. The positive rate of Cryptosporidium oocysts and Giardia cysts was 17.1% (12/70) and 20.0% (14/70), respectively. CONCLUSION: No Cryptosporidium oocysts and Giardia cysts have been detected in drinking water, but found in source water and environmental water samples in Shanghai.