An epidemiological analysis of imported malaria in Shanghai during a COVID-19 outbreak.

BACKGROUND: The goal of this study was to understand the epidemiological characteristics of imported malaria in Shanghai specifically during the epidemic period of novel corona-virus pneumonia (COVID-19), to provide a reference for preventing the transmission of imported malaria after this disease had been previously eliminated. METHODS: The data of malaria cases reported in Shanghai from 2020 to 2021 were obtained from the China Information System for Disease Control and Prevention (CISDCP) and the Information System for Parasitic Disease Control and Prevention (ISPDCP). The characteristics of demographic and epidemiological distribution, travel-related information, diagnosis information, regions of infection acquisition and disposal information of epidemic situation were analysed with descriptive statistics. RESULTS: A total of 112 cases of malaria were reported in Shanghai from January 2020 to December 2021. There were 18 cases and 94 cases in 2020 and 2021, respectively, reaching the lowest and highest levels in the past 10 years. The incidence of malaria associated with seasons had an increasing trend (χ2 = 81.143, P < 0.05). These cases included Plasmodium falciparum (97, 86.61%), Plasmodium vivax (4, 3.57%), Plasmodium ovale (8, 7.14%) and Plasmodium malariae (3, 2.68%). The median age of patients with malaria was 38.0 years, the majority of these individuals were males (109, 97.32%), and most of them were labour personnel (93, 83.04%). Of the reported cases, 8 of these individuals (7.14%) reported experiencing malaria symptoms before their arrival in China after their stay overseas; 97 of these individuals (86.61%) reported experiencing symptoms within 14 days after their initial arrival from overseas; 15 of these individuals (13.39%) were diagnosed with 'severe malaria'; and 4 of these individuals (3.57%) were also diagnosed with COVID-19. All cases were imported from Africa, and there were no indigenous cases and deaths. CONCLUSION: Due to the impact of COVID-19, the number of imported malaria cases in Shanghai had greatly increased; however, prevention and control measures for imported malaria could be implemented to prevent re-transmission of this condition. Considering that the number of individuals returning from overseas labour is likely to increase in the next few years, it is necessary to strengthen the surveillance of imported malaria and to review the protocol for potential epidemic situations. Together, these measures could support the maintation of free-malaria status in Shanghai.

Development and Preliminary Application of a Triplex Real-Time Quantitative PCR Assay for the Simultaneous Detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum.

Background: The protozoan parasites including Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum can infect the human intestinal tract and cause serious diseases. In this study, we aimed to develop a triplex real-time quantitative PCR (qPCR) for the simultaneous differential detection of these three intestinal protozoa. Methods: Specific primers and TaqMan probes were designed for the 16S-like SSU rRNA sequence of E. histolytica, the gdh sequence of G. lamblia, and the 18srRNA sequence of C. parvum. A triplex qPCR assay was developed based on single-duplicate experiments to evaluate its limit of detection (LOD), specificity, stability, and reproducibility. Additionally, 163 fecal samples from patients with diarrhea who tested positive for copro-antigen were tested to verify the practicality of the assay. Results: The triplex qPCR assay could specifically detect E. histolytica, G. lamblia, and C. parvum without cross-reactivity amongst the target-specific TaqMan probes of these three intestinal protozoan parasites and did not produce amplification curves for any other non-target species, and had good specificity. Amplification of serial dilutions showed that the triplex qPCR detected as little as 500 copies/µL of standard plasmid DNA. The standard curve displayed good linearity between 5 × 102 and 5 × 108 copies/µL; qPCR assays were performed with an efficiency of more than 95% and R 2 values were greater than 0.99. The triplex qPCR assay had good repeatability with intra- and inter-assay coefficients of variation less than 1.92%. Among the 163 fecal samples, four samples were confirmed to be positive for C. parvum using the triplex qPCR assay. Conclusion: The triplex qPCR established in this study not only provides a rapid, sensitive, specific tool for the simultaneous detection of E. histolytica, G. lamblia, and C. parvum, but also has good practical application value.

GATA2/FGF21 Axis Regulates the Effects of High Glucose on the Apoptosis, Autophagy and Oxidative Stress of Human Umbilical Vein Endothelial Cell via PI3K/AKT/mTOR Pathway.

OBJECTIVE: We investigated the role and mechanism of GATA-binding factor 2/Fibroblast growth factor 21 (GATA2/FGF21) axis in high glucose (HG)-induced injury in human umbilical vein endothelial cells (HUVEC). METHODS: After HG treatment and transfection, the viability and apoptosis of HUVECs were determined via Cell Counting Kit-8 and Hoechst 33258 staining methods, and the content of lactate dehydrogenase (LDH) and reactive oxygen species (ROS) was measured via colorimetric assay and DCFH-DA staining. The potential transcription factor of FGF21 was predicted with bioinformatic analysis and confirmed via dual-luciferase reporter assay and chromatin immunoprecipitation. The expressions of GATA2/FGF21, apoptosis-, autophagy- and phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway-related factors were quantified with quantitative real-time polymerase chain reaction or Western blot. RESULTS: Overexpressed FGF21 abolished the effects of HG on repressing the expressions of FGF21 and cell viability, and promoting apoptosis, the levels of LDH and ROS and autophagy in HUVECs, with increased Bcl-2 and p62 expression yet decreased Bax, Cleaved PARP, Cleaved caspase-3, LC3 II/LC3 I ratio and Beclin 1. GATA2 was the transcription factor of FGF21 and was downregulated after HG treatment, and the effects of overexpressed FGF21 in HG-treated HUVECs were all reversed after the silence of GATA2. Besides, overexpressed FGF21 promoted the activation of PI3K/AKT/mTOR pathway, with increased phosphorylation levels of PI3K, AKT and mTOR, whereas silencing GATA2 abolished the trend. CONCLUSION: GATA2/FGF21 axis has a protective function against HG in HUVEC via regulating PI3K/AKT/mTOR pathway.

From malaria elimination to post-elimination: a 10-year surveillance data study in Shanghai.

BACKGROUND: The aim of this study was to investigate and analyse the characteristics of malaria in Shanghai from 2010 to 2019 and to provide suggestions for areas with a similar elimination process in China in order to prompt development of strategies and interventions in the post-elimination stage. METHODS: This was a cross-sectional study exploring the malaria characteristics during 2010-2019 in Shanghai, China. Malaria data from the Infectious Diseases Information Reporting Management System (IDIRMS) between 2010 and 2012 and data from the Parasitic Diseases Information Reporting Management System (PDIRMS) between 2013 and 2019 were combined for analysis in this study. RESULTS: From 2010 to 2019, a total of 436 malaria cases were reported in Shanghai. Among them, 415 (95.18%) were imported from abroad, 19 (4.36%) were domestically acquired from other provinces, 1 (0.23%) case was caused by blood transfusion, and 1 (0.23%) had a long incubation. Only Plasmodium vivax was found in domestically indigenous cases; Plasmodium falciparum accounted for the largest proportion of imported cases. Domestically acquired cases were only reported in 2010-2011 and 88% occurred in June to September; no significant seasonal difference was observed for imported cases over the 10 years. No local transmission has occurred in Shanghai since 2012. The median interval from fever onset to diagnosis was 3 days. Between 2010 and 2019, among 308 foci, 33 were classified as potential transmission and dispersed in suburb areas (Minhang, Baoshan, Jiading, Pudong, Jinshan, Songjiang, Qingpu, Fengxian, and Chongming). Only Anopheles sinensis was present and the proportion of Anopheles sinensis in different species of mosquitoes under surveillance in Shanghai decreased from 2011 to 2019. CONCLUSIONS: Shanghai faces the challenge of malaria re-establishment caused by imported malaria in the post-elimination stage. Therefore, risk investigation and assessment should be carried out, and receptivity and susceptibility should be assessed for every point of focus. Training should be continued to strengthen facility staff capability, and multisectoral coordination and cooperation need to be conducted efficiently to maintain malaria elimination in Shanghai.

Laboratory and Field Evaluation of the Phytotoxic Activity of Sapindus mukorossi Gaertn Pulp Extract and Identification of a Phytotoxic Substance.

Interest in finding plant-based herbicides to supplement synthesized herbicides is increasing. Although the extract of Sapindus mukorossi Gaertn has been reported to have herbicidal activity, little is known about phytotoxic substances and their efficacy of weed control in the field. To identify phytotoxic substances, the bioassay-guided fractionation by column chromatography and high-speed counter-current chromatography (HSCCC) was carried out. The phytotoxic activity assay, performed by the agar medium method, showed that the 70% ethanol fraction exhibited strong root growth inhibition against Trifolium pratense with an 50% inhibitory concentration (IC50) value of 35.13 mg/L. An active compound was isolated from the 70% ethanol fraction and identified as hederagenin 3-o-ß-D-xylopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside (Compound A). Compound A had an IC50 value of 16.64 mg/L. Finally, a new formulation was prepared based on the 70% ethanol fraction, which exhibited good efficacy against broadleaf weeds in a carrot field. The fresh weight control efficacy was 78.7% by 45 days after treatment at the dose of 1500 g a. i./ha. Hence, the extract of S. mukorossi pulp could be a promising supplement to the synthesized herbicides. Furthermore, compound A from S. mukorossi may be responsible for its phytotoxic activity.

Simultaneous determination of five azadirachtins in the seed and leaf extracts of Azadirachta indica by automated online solid-phase extraction coupled with LC-Q-TOF-MS.

Neem (Azadirachta indica) extract is well-known as a natural pesticide for the control of agricultural pests. Azadirachtin A and its structural analogues are considered as active compounds. However, the amounts of azadirachtins varies in neem extracts, providing a variety of insecticidal activities. In this study, a novel method of automated online solid-phase extraction coupled with liquid chromatography/quadrupole-time-of-flight mass spectrometry (SPE-LC-Q-TOF-MS) was developed and validated for simultaneous quantification of five azadirachtins (azadirachtins A, B, D, H and I) in seed and leaf extracts of A. indica. Different experimental parameters (such as SPE cartridge, injection volume and washing step) were optimized. The optimized SPE-LC-Q-TOF-MS method showed good recovery (82.0-102.8%), linearity (r2 ≥ 0.9991) and precision (0.83-4.83%). The limit of detections (LODs) for the five analytes ranged from 0.34 to 0.76 ng mL-1. The validated method was successfully applied for determination of the analytes in the neem leaves and seeds from different locations and a neem formulation. The online SPE-LC-Q-TOF-MS method was found to be a simple, precise and accurate and can be used as a powerful tool for quality control of neem extracts or its formulations.

[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.