Molecular detection and species identification of Plasmodium spp. infection among imported cases in Eastern China, 2012-2018.

Due to the increasing number of returnees from malaria endemic areas, imported malaria has become a public health challenge in China. To better understand the characteristics of imported Plasmodium species and adjust appropriate strategies for malaria prevention and control in Eastern China, we conducted molecular detection and species identification on 1282 imported malaria cases in Shandong Province between 2012 and 2018. The findings showed that P. falciparum was predominant, particularly in cases imported from Africa. P. vivax was the dominant species imported from Asian countries. Additionally, imported P. ovale and P. malariae emerged in the province. Further surveillance and control of imported malaria among returnees from Africa and Southeast Asia is needed to be strengthened in Eastern China.

High genetic diversity in Plasmodium falciparum isolates among Chinese migrant workers returnee from Africa.

Malaria is one of the most important parasitic diseases that causes a serious public health problem. The genetic diversity of malaria parasites may affect malaria transmission and malaria control strategies. In China, imported malaria was significantly increased in recent years, among which numerous migrant workers were infected with Plasmodium falciparum from Africa. However, little was known about genetic diversity of these populations in China. In this study, we evaluated genetic polymorphism and allele frequencies of msp1, msp2, and glurp genes in P. falciparum among Chinese migrant workers returnee from Africa between 2013 and 2017. Of the 381 P. falciparum isolates, 89.0% for msp1 gene, 71.7% for msp2 gene, and 78.0% for glurp gene were successfully genotyped. In msp1, 29 different alleles were observed, among which the K1 allelic family (71.7%) was predominant. In msp2, 21 different alleles were detected, of which the 3D7 allelic family (91.2%) was more frequent than FC27 allelic family (72.5%). For glurp, 12 individual alleles were detected in the samples. Taken together, the findings showed a high genetic diversity of these isolates, which provided the baseline data for African P. falciparum population imported to China.

Comparative Proteomics Analysis for Elucidating the Interaction Between Host Cells and Toxoplasma gondii.

Toxoplasma gondii, a representative model organism belonging to the phylum Apicomplexa, can infect almost all warm-blooded organisms, including humans. The invasion of host cells via host-parasite interaction is the key step for T. gondii to complete its life cycle. Herein we performed tandem mass tag analysis to investigate global proteomic changes in host cells (human foreskin fibroblasts, HFFs) [HFFs infected with T. gondii (HT) vs. HFFs (H)] and T. gondii [HT vs. T. gondii (T)] during intracellular infection. Overall, 3477 and 1434 proteins were quantified, of which 375 and 1099 proteins were differentially expressed (adjusted p-value < 0.05 and >1.5 or <0.67-fold change) in host cells and T. gondii, respectively. T. gondii invasion relies on the secretion of numerous secretory proteins, which originate from three secretory organelles: micronemes, rhoptries, and dense granules. In the HT vs. T group, few secretory proteins were upregulated, such as microneme proteins (MICs: MIC6, MIC10), rhoptry bulb proteins (ROPs: ROP5, ROP17), and dense granule proteins (GRAs: GRA4, GRA5, GRA12). In contrast, dozens of known secretory proteins were significantly downregulated in T. gondii-infected HFFs. In HFFs, gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed a large number of differentially expressed proteins (DEPs) enriched in metabolic processes and immune-associated signaling pathways, such as NF-κB, cAMP, and Rap1 signaling pathways. Further, in case of T. gondii, DEPs were involved in ribosome biogenesis, citrate cycle, and galactose metabolism, indicating that cell biosynthesis and metabolism of T. gondii were altered after host cell invasion. These findings reveal novel modifications in the proteome of host cells as well as T. gondii, helping us better understand the mechanisms underlying host-parasite interaction.

Protective immunity induced by a DNA vaccine cocktail expressing TgSAG1, TgROP2, and the genetic adjuvant HBsAg against Toxoplasma gondii infection.

Toxoplasma gondii is an intracellular obligate parasitic protozoon that can infect all warm-blooded animals, causing zoonotic toxoplasmosis. So far, there is no commercial toxoplasmosis vaccine for human use. In the present study, we constructed a DNA vaccine cocktail which includes the surface protein (SAG1) and the rhoptry protein ROP2 denoted as pEGFP-N1-SAG1-ROP2. In order to improve the efficacy, HBsAg was used as a genetic adjuvant to construct pEGFP-N1-HBsAg-SAG1-ROP2. Two eukaryotic plasmids were transiently transfected into HEK293T cells and the expression was examined using fluorescence microscopy and western blotting. We then immunized Kunming mice intramuscularly with the DNA vaccine. After three immunizations, the immune response was evaluated by measuring antibody levels, cytokine production, percentages of CD4+ and CD8+ T lymphocytes, and the survival times of the T. gondii RH strain challenged mice. The results showed that the two DNA vaccines stimulated Th1 responses, and had a higher antibody titer, IL-2, IL-12, and IFN-γ levels, and percentage of CD4+ and CD8+ T lymphocytes than the control group. In addition, mice immunized with the pEGFP-N1-HBsAg-SAG1-ROP2 vaccine showed increased survival times compared with pEGFP-N1-SAG1-ROP2.

Prediction of Toxoplasma gondii virulence factor ROP18 competitive inhibitors by virtual screening.

BACKGROUND: Rhoptry protein 18 (ROP18) is a key virulence factor of Toxoplasma gondii. The host's immune responses mediated by immune-related GTPases (IRGs) could be blocked by ROP18's kinase activity. ROP18 also interacts with various substrates, such as activating transcription factor 6 beta (ATF6ß) and affects multiple physiological functions within host cells, thereby inducing intense virulence. In this study, competitive inhibitors targeted to ROP18 were subjected to virtual screening based on the principle of structure-based drug design (SBDD). METHODS: The preparation of the ROP18 structure was conducted using the "Structure Prepare" function of Molecular Operating Environment (MOE) software. The ATP-binding pocket was selected as the starting point for virtual screening. Construction of the pharmacophore model used Extended Hückel Theory (EHT) half-quantitative measurement and construction, as well as the characteristics of Type I kinase inhibitors. The pharmacophore model of ROP18 was imported into the Specs database for small molecule similarity screening using EHT pharmacophore measurement. Hit compounds were selected using the functions of London dG and generalized-born volume integral/weighted surface area (GBVI/WSA) scoring. The top 100 hits were analyzed by molecular docking and structure activity relationships (SAR) analysis. RESULTS: The final pharmacophore comprised three typical characteristics: three hydrogen bond acceptors/donors, two ring aromatic features occupying the hydrophobic core, and one cation group feature targeted to the terminus of ATP. A total of 1314 hit compounds analogous to ROP18 pharmacophore were passed through the Specs. After two rounds of docking, 25 out of 100 hits were identified as belonging to two main scaffold types: phthalimide ring structure, thiazole ring and styrene structure. Additionally, the screen also identified 13 inhibitors with distinct scaffold types. The docking models and SAR analysis demonstrated that these hits could engage in multiple hydrogen bonds, salt bridges halogen bonds, and hydrophobic interactions with ROP18, and para-position halo substituents on the benzene ring may enhance their affinity scoring. CONCLUSIONS: A pharmacophore against the ROP18 ATP-binding pocket was successfully constructed, and 25 representative inhibitors from 15 scaffold clusters were screened using the Specs database. Our results provide useful scaffold types for the chemical inhibition of ROP18 or alternative conformations to develop new anti-toxoplasmosis drug leads.

Mutation Profile of pfdhfr and pfdhps in Plasmodium falciparum among Returned Chinese Migrant Workers from Africa.

We evaluated markers of sulfadoxine-pyrimethamine (SP) resistance in Plasmodium falciparum among 254 returned migrant workers in China from Africa from 2013 to 2016. High prevalences of pfdhfr (97.2%) and pfdhps (96.5%) mutations were observed. The partially resistant genotype was homogeneously distributed in Africa with a modestly high prevalence (48%), whereas the super resistant genotype was only found in West Africa with a very low frequency (1.2%). The findings provided baseline data about the molecular markers of SP resistance.

Increasing number of imported Plasmodium ovale wallikeri malaria in Shandong Province, China, 2015-2017.

Since 2012, no indigenous malaria case have been reported in Shandong Province, China, whereas the number of imported cases and the genetic diversity of Plasmodium spp. have increased. Beginning in 2015, the number of Plasmodium ovale cases were increased and the P. ovale wallikeri malaria case began to arise. From 2015 to 2017, a total of 676 imported malaria cases were detected and 76 P. ovale spp. isolates were identified, of which 48 were P. ovale curtisi and 28 P. ovale wallikeri. The number of P. ovale wallikeri malaria cases increased yearly, six were identified in 2015, eight were identified in 2016, and 14 were identified in 2017. All cases with an attached travel history from Africa, with represented source countries of Equatorial Guinea (n = 9), Republic of the Congo (n = 4), and Democratic Republic of the Congo (n = 3). P. ovale wallikeri is increasing among travelers returning to Shandong Province from Africa. Although the P. ovale spp. infection rarely progressed to severity, this species is suspected to generate hypnozoites which have the potential to relapse. The ability to cause relapse is the threat to public health and should be concerned.

Surveillance of Antimalarial Resistance Pfcrt, Pfmdr1, and Pfkelch13 Polymorphisms in African Plasmodium falciparum imported to Shandong Province, China.

Antimalarial drug resistance is a major public health problem in China. From 2012 to 2015, more than 75% of malaria cases in Shandong Province were P. falciparum returned from Africa. However, molecular marker polymorphisms of drug resistance in imported P. falciparum cases have not been evaluated. In this study, we analyzed polymorphisms of the Pfcrt, Pfmdr1, and Pfkelch13 genes in 282 P. falciparum cases returned from Africa to Shandong between 2012 and 2015. Among the isolates, polymorphisms were detected in codons 74-76 of Pfcrt and 86, 184, 1246 of Pfmdr1, among which K76T (36.6%) and Y184F (60.7%) were the most prevalent, respectively. Six Pfcrt haplotypes and 11 Pfmdr1 haplotypes were identified and a comparison was made on the prevalence of haplotypes among East Africa, West Africa, Central Africa and South Africa. One synonymous and 9 nonsynonymous mutations in Pfkelch13 were detected in the isolates (4.6%), among which a candidate artemisinin (ART) resistance mutation P553L was observed. The study establishes fundamental data for detection of chloroquine resistance (CQR) and ART resistance with molecular markers of the imported P. falciparum in China, and it also enriches the genetic data of antimalarial resistance for the malaria endemic countries in Africa.

Protective immune response against Toxoplasma gondii elicited by a novel yeast-based vaccine with microneme protein 16.

Toxoplasma gondii is an obligate intracellular protozoan that can invade all eukaryotic cells and infect all warm-blood animals, causing the important zoonosis toxoplasmosis. Invasion of host cells is the key step necessary for T. gondii to complete its life cycle and microneme proteins play an important role in attachment and invasion of host cells. Microneme protein 16 (TgMIC16) is a new protective protein in T. gondii and belongs to transmembrane microneme proteins (TM-MIC). The TM-MICs are released onto the parasite's surface as complexes capable of interacting with host cell receptors. In the present study, we expressed the TgMIC16 protein on the surface of Saccharomyce cerevisiae (pCTCON2-TgMIC16/EBY100) and evaluated it as a potential vaccine for BALB/c mice against challenge infection with the RH strain of T. gondii. We immunized BALB/c mice both orally and intraperitoneally. After three immunizations, the immune response was evaluated by measuring antibody levels, lymphocyte proliferative responses, percentages of CD4+ and CD8+ T lymphocytes, cytokine production, and the survival times of challenged mice. The results showed that the pCTCON2-TgMIC16/EBY100 vaccine stimulated humoral and cellular immune responses. In addition, mice immunized with the pCTCON2-TgMIC16/EBY100 vaccine showed increased survival times compared with non-immunized controls. In summary, TgMIC16 displayed on the cell surface of S. cerevisiae could be used as potential vaccine against toxoplasmosis.

Cysticercosis in Shandong Province, Eastern China.

We analyzed demographic and clinical data and estimated the incidence of cysticercosis in Shandong Province, China, during 1975-2014. Our analyses showed that a cysticercosis-endemic area is present in Shandong Province, especially in its western regions. Improved surveillance and control are needed to address the elevated risk for cysticercosis in this region.

Expression of codon-optimized TgMIC16 in three Escherichia coli strains.

In a previous study, we found that rabbit anti-Toxoplasma gondii serum was capable of recognizing truncated T. gondii microneme protein 16 (TgMIC16), indicating that TgMIC16 is an essential antigenic T. gondii protein. However, the broad application of this recombinant protein is limited by its low expression level. In this study, we performed codon optimization of TgMIC16 by changing the codon-adaptation index from 0.22 to 1.0 without altering the amino acid sequence and expressed the optimized gene in three different Escherichia coli strains, followed by comparison of soluble recombinant-protein expression and yield. Our results showed that the recombinant protein rTgMIC16 was expressed as inclusion bodies in all three strains following optimization of induction parameters, and western blot analysis revealed the presence of a ~72-kD recombinant protein as a specific band following purification. A shuffle-expression strain was selected to amplify incubation products and induce expression, resulting in an overall rTgMIC16 yield of ~20 mg/L. These findings provide a basis for further investigation of TgMIC16 to elucidate its functions and interaction partners.

Characteristics of Imported Malaria and Species of Plasmodium Involved in Shandong Province, China (2012-2014).

Malaria remains a serious public health problem in Shandong Province, China; therefore, it is important to explore the characteristics of the current malaria prevalence situation in the province. In this study, data of malaria cases reported in Shandong during 2012-2014 were analyzed, and Plasmodium species were confirmed by smear microscopy and nested-PCR. A total of 374 malaria cases were reported, 80.8% of which were reported from 6 prefectures. Of all cases, P. falciparum was dominant (81.3%), followed by P. vivax (11.8%); P. ovale and P. malariae together accounted for 6.4% of cases. Notably, for the first time since 2012, no indigenous case had been reported in Shandong Province, a situation that continued through 2014. Total 95.2% of cases were imported from Africa. The ratio of male/female was 92.5:1, and 96.8% of cases occurred in people 20-54 years of age. Farmers or laborers represented 77.5% of cases. No significant trends of monthly pattern were found in the reported cases. All patients were in good condition after treatment, except for 3 who died. These results indicate that imported malaria has increased significantly since 2012 in Shandong Province, especially for P. falciparum, and there is an emergence of species diversity.

Simvastatin downregulated C35 expression and inhibited the proliferation of colon cancer cells Lovo and HT29 in vitro.

The aim of this study was to investigate the antitumor effect of simvastatin in human colon cancer and the possible underlying mechanism. We found that simvastatin dose-dependently inhibited the proliferation of human colon cancer cells Lovo and HT29 using a MTT assay. Real-time PCR and Western blotting assays showed that simvastatin significantly suppressed C35 expression at both mRNA and protein levels. Since C35 is known to have a significant oncogenic role in cancer development via promoting cell proliferation and migration, results obtained in the current study imply that downregulation of C35 expression might be involved in the antitumor effect of simvastatin on colon cancer.

Epidemiological Investigation of Asymptomatic Dogs with Leishmania Infection in Southwestern China Where Visceral Leishmaniasis is Intractable.

Heishui county, located in northwest Sichuan province, southwestern China, is an endemic area of zoonotic visceral leishmaniasis (VL) and is the most intractable area. VL is never destroyed in it. Asymptomatic dogs (Leishmania parasites have been diagnosed but clinically healthy) are considered to be a potential reservoir host in zoonotic VL area, and most can lead to infection of individuals, that is a new challenge for controlling VL in humans. The present study aimed to assess the Leishmania infection rate of asymptomatic dogs in Heishui county. Total 105 asymptomatic domestic dogs were gathered from 4 districts in Heishui county to investigate the infection rate with serological and molecular methods based on ELISA and kinetoplast minicircle DNA(kDNA) PCR, respectively. Out of 105 dogs, 44 (41.9%) were positive by more than 1 method; 21 (20.0%) were positive by ELISA, and 30 (28.6%) were positive by kDNA-PCR. Our study showed that Leishmania infection of domestic dogs which is clinically healthy is prevalent in the studied district, and the asymptomatic dogs infected by Leishmania may be the primary reason for the prevalence of visceral leishmaniasis in the area.

[Prokaryotic Expression, Purification and Immunological Characterization of Micronemal Protein 16 of Toxoplama gondii].

Objective: To prokaryotically express three gene fragments of micronemal protein 16 （TgMIC16） of Toxoplasma gondii, and analyze the immunoreactivity of the three recombinant protein products. Methods: Primers were designed for three fragments of TgMIC16 gene which encode proteins within the functional domain. Reverse-transcription PCR was used to generate cDNA from RNA, and the three fragments were amplified on the cDNA by PCR using the designed primers. The PCR products were double-digested, inserted into the pET-32a（+） plasmid, and transformed into Escherichia coli TOP10 cells. Plasmids extracted from positive clones were confirmed by BamHⅠ/HindⅢ double digestion and sequencing, and further transformed into E. coli Rosetta cells. Protein expression was induced by IPTG, and confirmed by SDS-PAGE. The expressed recombinant proteins were purified with Ni-NTA affinity chromatography and their immunoreactivity analyzed with Western blotting. Results: The amplified three fragments were 1 806, 1 290 and 855 bp in size. Double digestion and sequencing results confirmed the successful construction of the three recombinant plasmids. SDS-PAGE analysis showed successful expression of the three recombinant proteins (M(r) 88 000, 68 000 and 52 000, respectively）, in the form of inclusion bodies. Western blotting showed that the three purified recombinant proteins reacted with His monoclonal antibody and rabbit anti-T. gondii antibody. Conclusion: The three fragments within the functional domain of TgMIC16 are successfully expressed in prokaryotic expression system and show immunoreactivity.

[Detection of Genetic Mutations Associated with Drug Resistance in Imported Plasmodium falciparum].

Objective: To investigate the mutation of genes associated with drug resistance (Pfcrt, Pfmdr1, Pfdhfr and K13） in imported Plasmodium falciparum in Shandong Province. Methods: Blood was collected from 94 falciparum malaria cases who returned from Africa in 2014. Genomic DNA for P. falciparum was extracted from the blood samples and nested PCR was performed using primers specifically designed for Pfcrt, Pfmdr1, Pfdhfr and K13. The PCR products were sequenced. Gene mutations were analyzed by sequence alignment. Results: The 94 imported cases were from 18 African countries. Nested PCR was successful on DNA from all the blood samples except for Pfcrt amplification in one sample. Sequence analysis revealed three types of mutations Pfcrt K76T （36.6%, 34/93）, Pfmdr1 N86Y （21.3%, 20/94）, and Pfdhfr S108N （98.9%, 93/94） （χ2=127.5， P<0.05）. K13 C580Y mutation was not found. Co-occurrence of K76T, N86Y, and S108N was found in 6 blood samples （6.5%）, which were imported from Liberia（2）, Angola（1）, Equatorial guinea（1）, Congo（1）, and Guinea（1）. Co-occurrence of K76T and S108N mutations was found in 28 samples（30.1%）, and that of N86Y and S108N in 14 samples （15.1%）. Forty-four samples（47.3%） harbored S108N mutation only, and one sample was null for any of the mutations. Conclusion: There are mutations in Pfcrt, Pfmdr1, and Pfdhfr in imported Plasmodium falciparum in Shandong Province. No mutation was found for the K13 gene.

[Cloning and Bioinformatics Analysis of Toxoplasma gondii ROP21 Gene].

The full-length gene sequence of Toxoplasma gondii ROP21 (TgROP21) gene was amplified with PCR. The signaling peptide and transmembrane domain of TgROP21 protein were predicted by SignaIP and TMHMM online predictive sites, and the hydrophilicity and antigenic index of this protein were ananlyzed with DNAStar software. Meanwhile, the functional domains and tertiary structure were modeled by combined use of ExPASY and PRODATA online sites. As expected, the PCR results revealed one band at 2,022 bp. The signaling peptide, transmembrane domain, hydrophilicity, antigen index, functional domain and 3D structure of TgROP21 were successfully predicted. This work may provide a theoretical foundation for further verification of TgROP21 function.

Establishment of lymphatic filarial specific IgG4 indirect ELISA detection method.

OBJECTIVE: To establish the lymphatic filarial specific IgG4 indirect ELISA detection method and develop the kits. METHODS: ELISA and the developed specific IgG4 reagent was used to explore the best way for detecting filarial specific IgG4. Combined with the production process of commercialized enzyme immunoassay kit to develop economical lymphatic filarial specific IgG4 test kit, and to explore the value of the kit in the laboratory. RESULTS: We determined the most optimal detective antigen was Malay adult filarial antigen and the optimal concentration of coating antigen was 1.0 µg/ml. The appropriate serum dilution was 1:20 to 40 and the work titers of specific IgG4 agents was 1:800. We determined the optimal reaction time for substrates and developed a reproducible and stable detection kit with sensitive and specificity, which was easy to operate. CONCLUSION: We successfully established the lymphatic filarial specific IgG4 indirect ELISA detection method and developed the kits with good reproducibility and stable result, which should be widely applied.