Extremely Soft, Stretchable, and Self-Adhesive Silicone Conductive Elastomer Composites Enabled by a Molecular Lubricating Effect.

Softness, adhesion, stretchability, and fast recovery from large deformations are essential properties for conductive elastomers that play an important role in the development of high-performance soft electronics. However, it remains an ongoing challenge to obtain conductive elastomers that combine these properties. We have fabricated a super soft (Young's modulus 2.3-12 kPa), highly stretchable (up to 1500% strain), and underwater adhesive silicone conductive elastomer composite (SF-C-PDMS) by incorporating dimethyl silicone oil as a lubricating agent in a cross-linked molecular network. The resultant SF-C-PDMS not only exhibits superior softness but also can readily recover after a strain of 1000%. The initial resistance only decreases by 8% after 100000 cycles of tensile fatigue test (100% strain, 0.5 Hz, 15 mm/s). This multifunctional silicone conductive elastomer composite is obtained in a one-step preparation at room temperature using commercially available materials. Moreover, we illustrate the capabilities of this composite in motion sensing.

Proteomic and Immunological Identification of Diagnostic Antigens from Spirometra erinaceieuropaei Plerocercoid.

Human sparganosis is a food-borne parasitic disease caused by the plerocercoids of Spirometra species. Clinical diagnosis of sparganosis is crucial for effective treatment, thus it is important to identify sensitive and specific antigens of plerocercoids. The aim of the current study was to identify and characterize the immunogenic proteins of Spirometra erinaceieuropaei plerocercoids that were recognized by patient sera. Crude soluble extract of the plerocercoids were separated using 2-dimensional gel electrophoresis coupled with immunoblot and mass spectrometry analysis. Based on immunoblotting patterns and mass spectrometry results, 8 antigenic proteins were identified from the plerocercoid. Among the proteins, cysteine protease protein might be developed as an antigen for diagnosis of sparganosis.

Human Cases of Fascioliasis in Fujian Province, China.

Fascioliasis is a foodborne zoonotic parasitic disease. We report 4 cases occurring in the same family, in whom diagnosis of acute fascioliasis was established after series of tests. One case was hospitalized with fever, eosinophilia, and hepatic lesions. MRI showed hypodense changes in both liver lobes. The remaining 3 cases presented with the symptom of stomachache only. Stool analysis was positive for Fasciola eggs in 2 adult patients. The immunological test and molecular identification of eggs were confirmed at the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, China. The results of serological detection were positive in all the 4 patients. DNA sequencing of PCR products of the eggs demonstrated 100% homology with ITS and cox1 of Fasciola hepatica. The conditions of the patients were not improved by broad-spectrum anti-parasitic drugs until administration of triclabendazole.

Four Human Cases of Diphyllobothrium nihonkaiense (Eucestoda: Diphyllobothriidae) in China with a Brief Review of Chinese Cases.

We described 4 human infection cases of zoonotic fish-tapeworm, Diphyllobothrium nihonkaiense, identified with morphological and molecular characters and briefly reviewed Chinese cases in consideration of it as an emerging parasitic disease in China. The scolex and mature and gravid proglottids of some cases were seen, a rosette-shaped uterus was observed in the middle of the mature and gravid proglottids, and the diphyllobothriid eggs were yellowish-brown in color and displayed a small knob or abopercular protuberance on the opposite end of a lid-like opening. The average size of the eggs was recorded as 62-67×42-45 µm. The parasitic materials gathered from 4 human cases were morphologically identified as belonging to the genera Diphyllobothrium and Adenocephalus. The phylogenetic analysis based on the nucleotide sequences of cytochrome c oxidase subunit 1 gene of the etiologic agents confirmed that the 4 cases were D. nihonkaiense infection. The finding of 4 additional D. nihonkaiense cases suggests that D. nihonkaiense might be a major causative species of human diphyllobothriasis in China. A combined morphological and molecular analysis is the main method to confirm D. nihonkaiense infection.

[Digital Description and Identification of 11 Kinds of Principal Parasite Eggs].

Objective: To facilitate the identification of parasite eggs using computer technology, establish the automation-based applications, and propose an algorithm for egg classification. Methods: Eggs of 11 parasites, Clonorchis sinensis, Taenia solium, Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichiura, Spirometra mansoni, Diphyllobothrium latum, Ancylostoma duodenale, Schistosoma japonicum, Paragonimus westermani and Fasciolopsis buski, were selected and divided into two groups, the training group and the testing group, and were microphotographed. The eigenvalue was extracted using the VC++-based method. The eigenvalue database was constructed, and the training data set was tested with a variety of classification algorithms. The classifier was constructed using algorithm with the highest efficiency and an identification method was established by multi-feature fusion. Results: After removal of images with invalid values, the training group received 19 844 egg images, and the testing group, 3 721 images. Based on the 14 eigenvalues, there were significant differences in the size and color among the eggs of 11 parasite species. For example, the length, width, area and brightness of the smallest parasite egg of Clonorchis sinensis were 292.24 µm, 192.64 µm, 43 416.61 µm2 and 53.84, respectively, while those of the largest parasite egg of Fasciolopsis buski were 945.31 µm, 610.88 µm, 536 002.60 µm2 and 100.54, respectively. When using dynamic weights to construct the classifier, the discrimination rate on the training data set was 88.89%（17 641/19 844）, and that on the verification data set was 91.83%（3 004/3 271）, with an average modeling time of 0.01 s. Conclusion: The algorithm for egg classification has been established, which pravides a basis for further study on its feasibility.

[Sequencing and Phylogenetic Tree Construction of 18S Ribosomal DNA from Five Species of Plasmodium from Yunnan Border between China and Myanmar and Other Areas].

Objective: To analyze sequence variation and construct phylogenetic tree based on 18S ribosomal DNA among five species of Plasmodium in Yunnan border between China and Myanmar and other areas. Methods: Blood samples (or DNA samples)from malaria patients were collected from 2000 to 2015 in Yunnan border and Myanmar and other areas. DNA was extracted from blood samples, and the 18S rDNA fragment was amplified, sequenced and aligned with relevant sequences available in the GenBank. The phylogenetic tree was constructed by methods of neighbor joining (NJ), maximum likelihood (ML), and maximum parsimony (MP), respectively. Results: A total of 94 blood samples or DNA from malaria patients were collected. The 18S rDNA was successfully amplified from all the samples. Sequence alignment revealed variations of 0-0.2%, 0-0.1%, 0-0.1%, 0-0.1% and 0 for 18S rDNA sequence among Plasmodium falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi, respectively. The phylogenetic tree constructed with the three method showed consistency. Phylogenic analysis revealed that there were five big branches of Plasmodium spp. studied. The P. falciparum branch clustered with the isolates from Cameroon（KC428741, KC428742）, Brazil（KC906718）, and Malaysia（HQ283221） in GenBank. The P. vivax branch clustered with isolates from Cameroon（HF945443）, India （HM014361, JQ627158）, and Colombia （U83877）. However, the samples Pv11, Pv18 and Pv21 formed a small branch that showed closer phylogenetic relationship with P. cynomolgi（L07559）, an isolate from Macaca fascicularis. Moreover, P. malariae samples from Yunnan Province including Pm1, Pm3 and Pm4 clustered to form a small branch, and then clustered with samples from Hainan Province, showing geographical diversity. All the isolates of P. ovale clustered with isolates from Vietnam（EU935736 and AF387038）. All the isolates of P. knowlesi clustered into a branch, and showed close relationship with those from Myanmar (GU816250 and GU816246). Conclusion: There is no significant difference in 18S rDNA gene of the five species of Plasmodium from Yunnan border between China and Myanmar and other areas. The phylogenetic tree constructed with the NJ, MP and ML methods shows consistency.

[Analysis on parasitic infection of clinical samples from hospitals in Shanghai during 2011-2013].

OBJECTIVE: To analyze the results of parasitic pathogen detection on clinical samples from Shanghai hospitals during 2011-2013. METHODS: Samples of serum, stool, sputum, body fluid and biopsy were collected from hospitals. The etiological, serological and molecular biology methods were used to detect parasitic infection cases. RESULTS: During 2011-2013, a total of 16,151 clinical samples were collected. 855 parasitic infection were found from 5939 samples by pathogen detection, belonging to 32 species, with a detection rate of 14.4%. The positive rate of Blastocystis hominis and Entamoeba histolytica was 8.3% (494/5939) and 3.1% (186/5939), respectively. The rate of intestinal protozoa infection in under 20-year-old age group was higher than other age groups (P<0.05). No significant difference was found between males and females (P>0.05). Totally 10,212 serum samples were examined, the total antibody-positive rate was 7.1% (730/10,212). In the 730 positive samples, 173 (23.7%), 143 (19.6%), 139 (19.0%), 132 (18.1%), and 128 (17.5%) showed positive for the antibodies against Cysticercus cellulosae, Schistosoma japonicum, Paragonimus westermani, Toxoplasma gondii and Sparganum mansoni, respectively. The main source regions of protozoal infection were Shanghai (269 cases), Jiangsu (142 cases), Anhui (106 cases) and Zhejiang (82 cases). 89 cases were worm infection, the main source were Zhejiang (24 cases), Shanghai (18 cases), Jiangxi (11 cases). CONCLUSION: Among the samples from hospitals, the major intestinal protozoans are Blastocystis hominis and Entamoeba histolytica, and the sero-positive cases are mainly Cysticercus cellulosae and Schistosoma japonicum infection.

The protective effects of BMSA1 and BMSA5-1-1 proteins against Babesia microti infection.

The intracellular parasite Babesia microti is among the most significant species causing human babesiosis and is an emerging threat to human health worldwide. Unravelling the pathogenic molecular mechanisms of babesiosis is crucial in developing new diagnostic and preventive methods. This study assessed how priming with B. microti surface antigen 1 (BHSA 1) and seroreactive antigen 5-1-1 (BHSA 5-1-1) mediate protection against B. microti infection. The results showed that 500 µg/ml rBMSA1 and rBMSA5-1-1 partially inhibited the invasion of B. microti in vitro by 42.0 ± 3.0%, and 48.0 ± 2.1%, respectively. Blood smears revealed that peak infection at 7 days post-infection (dpi) was 19.6%, 24.7%, and 46.7% in the rBMSA1, rBmSA5-1-1, compared to the control groups (healthy mice infected with B. microti only), respectively. Routine blood tests showed higher white blood cell, red blood cell counts, and haemoglobin levels in the 2 groups (BMSA1 and BMSA5 5-1-1) than in the infection control group at 0-28 dpi. Moreover, the 2 groups had higher serum interferon-γ, tumor necrosis factor-α and Interleukin-17A levels, and lower IL-10 levels than the infection control group throughout the study. These 2 potential vaccine candidate proteins partially inhibit in vitro and in vivo B. microti infection and enhance host immunological response against B. microti infection.

[Establishment of the experimental animal model of Babesia microti].

OBJECTIVE: To establish the experimental animal model for the study of Babesia microti. METHODS: BALB/c mice, immunosuppressive BALB/c mice, SCID mice and NOD-SCID mice were inoculated with B. microti-infected red blood cells (RBC) by intraperitoneal injection respectively. After inoculation, thin blood smears were prepared every day, stained with Giemsa staining and examined for the presence of parasitemia. Three mice were dissected to examine the infectivity in bone marrow, brain, spleen, heart, lung, kidney and liver tissues. The infection rate of erythrocytes in different tissues was recorded, and the relationship between the infectivity of tissues and infection rate in peripheral blood was analyzed. Blood samples infected with B. microti were preserved in liquid nitrogen with dimethyl sulfoxide (DMSO) for 2 months. The thawed parasitized blood was injected into the BALB/c mice by same route and the parasitemia was monitored. RESULTS: The four kinds of mice were all infected by B. microti with parasitemia. The percentage of parasitized red blood cells from peripheral blood were 82.4% (BALB/c mice, d7), 73.2% (immunosuppressive BALB/c mice, d5), 86.4% (SCID mice, d8) and 72.5% (NOD-SCID mice, d8) at the maximum, respectively. Parasitemia decreased rapidly in BALB/c mice, whereas decreased slowly in immunosuppressive BALB/c mice. Only the parasitemia in SCID mice and NOD-SCID mice decreased significantly and tended to picking up again. The parasites were observed in RBCs from bone marrow, brain, spleen, heart, lung, kidney and liver tissues. The infection rate of erythrocytes in tissues increased with an increase of infection in peripheral blood. After cryopreservation, the parasites proliferated in BALB/c mice. Parasitemia appeared after inoculation with frozen infected blood two days later than that of fresh infected blood. The infection rate reached its peak after inoculation with frozen infected blood one day later than that of fresh infected blood. CONCLUSION: The experimental animal model of B. microti has been established. The infection rate of erythrocytes is related to the immune status of the host mice.

Enhanced phosphatidylserine exposure and erythropoiesis in Babesia microti-infected mice.

Introduction: Babesia microti (B. microti) is the dominant species responsible for human babesiosis, which is associated with severe hemolytic anemia and splenomegaly because it infects mammalian erythrocytes. The actual prevalence of B. microti is thought to have been substantially underestimated. Methods: In this study, Bagg's albino/c (BALB/c) mice were intraperitoneally injected with B. microti-infected erythrocytes, and parasitemia was subsequently measured by calculating the proportion of infected erythrocytes. The ultrastructure of infected erythrocytes was observed using scanning and transmission electron microscopes. Quantifying phosphatidylserine (PS) exposure, oxidative stress, intracellular Ca2+, and erythropoiesis of erythrocytes were done using flow cytometry. The physiological indicators were analyzed using a Mindray BC-5000 Vet automatic hematology analyzer. Results: Of note, 40.7 ± 5.9% of erythrocytes changed their structure and shrunk in the B. microti-infected group. The percentage of annexin V-positive erythrocytes and the levels of reactive oxygen species (ROS) in the erythrocytes were higher in the B. microti-infected group than in the control group at 10 dpi. Significant splenomegaly and severe anemia were also observed following B. microti infection. The parasitemia level in the B. microti-infected splenectomized group was higher than that of the B. microti-infected sham group. The population of early erythroblasts increased, and the late erythroblasts decreased in both the bone marrow and spleen tissues of the B. microti-infected group at 10 dpi. Discussion: PS exposure and elevated ROS activities were hallmarks of eryptosis in the B. microti-infected group. This study revealed for the first time that B. microti could also induce eryptosis. At the higher parasitemia phase, the occurrence of severe anemia and significant changes in the abundance of erythroblasts in B. microti-infected mice group were established. The spleen plays a critical protective role in controlling B. microti infection and preventing anemia. B. microti infection could cause a massive loss of late erythroblasts and induce erythropoiesis.

Angiostrongylus cantonensis: identification and characterization of microRNAs in male and female adults.

Angiostrongylus cantonensis causes eosinophilic meningitis and eosinophilic pleocytosis in humans and is of significant socio-economic importance globally. microRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial roles in gene expression regulation, cellular function and defense, homeostasis and pathogenesis. They have been identified in a diverse range of organisms. The objective of this study was to determine and characterize miRNAs of female and male adults of A. cantonensis by Solexa deep sequencing. A total of 8,861,260 and 10,957,957 high quality reads with 20 and 23 conserved miRNAs were obtained in females and males, respectively. No new miRNA sequence was found. Nucleotide bias analysis showed that uracil was the prominent nucleotide, particularly at positions of 1, 10, 14, 17 and 22, approximately at the beginning, middle and the end of the conserved miRNAs. To our knowledge, this is the first report of miRNA profiles in A. cantonensis, which may represent a new platform for studying regulation of genes and their networks in A. cantonensis.

Molecular Characterization and Immunological Evaluation of Truncated Babesia microti Rhoptry Neck Protein 2 as a Vaccine Candidate.

Babesia microti is a protozoan that infects red blood cells. Babesiosis is becoming a new global threat impacting human health. Rhoptry neck proteins (RONs) are proteins located at the neck of the rhoptry and studies indicate that these proteins play an important role in the process of red blood cell invasion. In the present study, we report on the bioinformatic analysis, cloning, and recombinant gene expression of two truncated rhoptry neck proteins 2 (BmRON2), as well as their potential for incorporation in a candidate vaccine for babesiosis. Western blot and immunofluorescence antibody (IFA) assays were performed to detect the presence of specific antibodies against BmRON2 in infected mice and the localization of N-BmRON2 in B. microti parasites. In vitro experiments were carried out to investigate the role of BmRON2 proteins during the B. microti invasion process and in vivo experiments to investigate immunoprotection. Homologous sequence alignment and molecular phylogenetic analysis indicated that BmRON2 showed similarities with RON2 proteins of other Babesia species. We expressed the truncated N-terminal (33-336 aa, designated rN-BmRON2) and C-terminal (915-1171 aa, designated rC-BmRON2) fragments of the BmRON2 protein, with molecular weights of 70 and 29 kDa, respectively. Western blot assays showed that the native BmRON2 protein is approximately 170 kDa, and that rN-BmRON2 was recognized by serum of mice experimentally infected with B. microti. Immunofluorescence analysis indicated that the BmRON2 protein was located at the apical end of merozoites, at the opposite end of the nucleus. In vitro red blood cell invasion inhibition studies with B. microti rBmRON2 proteins showed that relative invasion rate of rN-BmRON2 and rC-BmRON2 group is 45 and 56%, respectively. Analysis of the host immune response after immunization and B. microti infection showed that both rN-BmRON2 and rC-BmRON2 enhanced the immune response, but that rN-BmRON2 conferred better protection than rC-BmRON2. In conclusion, our results indicate that truncated rhoptry neck protein 2, especially its N-terminal fragment (rN-BmRON2), plays an important role in the invasion of host red blood cells, confers immune protection, and shows good potential as a candidate vaccine against babesiosis.

[Dynamic observation of chicken egg yolk antibodies against soluble egg antigen of Schistosoma japonicum obtained by two immunization routes].

OBJECTIVE: To observe the dynamic changes of the specific chicken egg yolk antibodies (IgY) against soluble egg antigens (SEA) of Schistosoma japonicum by two immunization routes. METHODS: Seven New Zealand rabbits were infected with S. japonicum cercariae (1500 per rabbit). After 42 days the rabbits were sacrificed to collect eggs and prepare SEA. Two groups each with 3 healthy hens were intravenously and subcutaneously immunized with 50 microg SEA, respectively. All hens received five immunizations by the same dose of antigen, with 2-week interval for the first two doses, and 4-week interval for the rest doses. Hen eggs were collected at pre-immunization and every two weeks after the first immunization. Crude IgY was extracted from egg yolk by water dilution method, and were analyzed by SEA-based ELISA, then purified by using EGG stract IgY Purification System from the 8th to 18th week after the first immunization. IgY concentration was determined by A260/A280 ratio. The expression of IgY was detected by agarose double diffusion method and SEA-based ELISA. The characteristics of IgY was analyzed by SDS-PAGE and Western blotting. RESULTS: The titer of IgY reached a peak at the 8th week in the intravenous group (A492 = 1.28) and at the 12th week in the subcutaneous group (A492 = 0.78), respectively, and maintained at a high level in the intravenous group until the 18th week after the first immunization. The concentration of purified IgY was about 6.5-9.0 mg/nml. Agarose double diffusion method and SEA-based ELISA demonstrated that the peak titer of IgY in the intravenous group was 1:16 and 1:51200, respectively. SDS-PAGE demonstrated that IgY contained two major protein bands (Mr 25,000 and 68,000). IgY purified from immunized egg yolk specifically reacted with SEA. CONCLUSION: The intravenous method is superior than the subcutaneous injection method in obtaining a high level of egg yolk antibodies against SEA of Schistosoma japonicum, and the purified IgY shows better specificity.

Establishment and application of the National Parasitic Resource Center (NPRC) in China.

The National Parasitic Resource Center (NPRC) was created in 2004. It is a first-level platform under the Basic Condition Platform Center of the Ministry of Science and Technology of China. The resource centre involves 21 depository institutions in 15 regions of the country, including human parasite and vector depository, animal parasite depository, plant nematode characteristic specimen library, medical insect characteristic specimen library, trematode model specimen library, parasite-vector/snail model specimen library, etc. After nearly 15 years of operation, the resource centre has been built into a physical library with a database of 11 phyla, 23 classes, 1115 species and 117,814 pieces of parasitic germplasm resources, and three live collection bases of parasitic germplasm resources. A variety of new parasite-related immunological and molecular biological detection and identification technologies produced by the resource centre are widely used in the fields of public health responses, risk assessments on food safety, and animal or plant quarantine. The NPRC is the largest and top level resource centre on parasitology in China, and it is a leading technology platform for collecting and identifying parasitic resources.

[Chicken egg yolk immunoglobulin and its application in medicine].

Egg yolk immunoglobulin (IgY) is a major immunoglobulin from birds, amphibians and reptiles. IgY does not activate mammalian complement system, and does not bind to mammalian rheumatoid factors, protein A or G. In addition, it has a high yield, and is easy to extract and purify. With these advantages, IgY has been widely used in veterinary science, functional food and bio-products, and proved potential application for disease control and treatment. This paper reviews biological characteristics of IgY, its extraction, purification, and application in immunodiagnosis and treatment.

Prevalence and risk factors of Fascioliasis in China.

Fascioliasis has emerged as a significant public health problem among ruminants and humans. Human fascioliasis is a neglected food-borne parasitic disease, which has emerged or reemerged in more than 60 countries worldwide. In China, the first case of human fascioliasis was reported in 1921 in Fujian Province. The first major outbreak of this parasitic disease in 29 patients occurred in 2012 in Yunnan Province. Nonetheless, the prevalence of fascioliasis in China is probably underestimated due to the poor sensitivity of diagnostic tests, limited epidemiological data, and a poor understanding of the impact of subclinical illness. This study aimed to review the prevalence and risk factors of fascioliasis in China so as to improve the prevention and control of this disease.

Molecular characterization of Babesia microti seroreactive antigen 5-1-1 and development of rapid detection methods for anti-B. microti antibodies in serum.

Babesiosis has become a new global threat impacting human health, and most human babesiosis cases are caused by Babesia microti. Until now few antigens of B. microti have been described which can be used for the diagnosis of human babesiosis. In the present study, we report on the bioinformatic analysis, cloning and expression of the sequence encoding the B. microti seroreactive antigen 5-1-1 to investigate its potential incorporation in serologic diagnostic tools for babesiosis. Bioinformatic analysis and recombinant gene expression were performed to molecularly characterize seroreactive antigen 5-1-1. Enhanced chemiluminescence (ECL)-Western blot methods were used to detect specific antibodies in infected mice. Immunofluorescence antibody assays (IFA) were performed to detect the localization of BmSA5-1-1 in B. microti parasites. ELISA and immunochromatographic (ICT) tests were developed using recombinant BmSA5-1-1 to evaluate its potential use in rapid detection methods for B. microti antibodies and for the diagnosis of babesiosis. A recombinant expression plasmid was constructed by inserting the target gene fragment in the pET28a vector after double digestion with BamHI and XhoI restriction enzymes. The recombinant BmSA5-1-1 protein was expressed in Escherichia coli (rBmSA5-1-1) and purified by means of Ni-nitrilotriacetic acid (NTA) agarose columns. Polyclonal antibodies were generated against rBmSA5-1-1. Based on indirect immunofluorescence assay results, BmSA5-1-1 appeared to localize on the surface of B. microti. ELISA tests using the rBmSA5-1-1 antigen detected specific antibodies from infected mice as early as 4 days post-infection. Our results indicate that the two methods we developed can detect specific antibodies in mice at different stages of infection with sensitivities of 100% (rBmSA5-1-1 ELISA) and 90% (ICT). The specificity of the two methods was 100%. Sera of patients suffering from other closely related parasitic diseases, such as malaria and toxoplasmosis, produced negative results. In conclusion, seroreactive antigen 5-1-1, a member of the BMN1 protein family, is expressed on the outer surface of B. microti and is a promising candidate antigen for the early diagnosis of babesiosis. rBmSA5-1-1 ELISA and ICT methods show good potential for detecting specific antibodies in mice at different stages of infection.

Screening for biomarkers reflecting the progression of Babesia microti infection.

BACKGROUND: Babesiosis is caused by the invasion of erythrocytes by parasites of the Babesia spp. Babesia microti is one of the primary causative agents of human babesiosis. To better understand the status of the disease, discovering key biomarkers of the different infection stages is crucial. RESULTS: This study investigated B. microti infection in the mouse model from 0 to 270 days post-infection (dpi), using blood smears, PCR assays and ELISA. PCR assays showed a higher sensitivity when compared to microscopic examination. Specific IgG antibodies could be detected from 7 days to 270 dpi. Two-dimensional electrophoresis was combined with western blotting and mass spectrometric analysis to screen for specific reactive antigens during both the peak parasitaemia period (7 dpi) and IgG antibody response peak period (30 dpi) by the infected mice plasma. The 87 positive reactive proteins were identified and then expressed with the wheat germ cell-free system. Protein microarrays of all 87 targeted proteins were produced and hybridized with the serial plasma of infected mice model. Based on the antigen reaction profile during the infection procedure, 6 antigens were selected and expressed in Escherichia coli. Due to an early response to IgM, lower immunoreactivity levels of IgG after two months and higher immunoreactivity level IgG during nine months, four recombinant proteins were selected for further characterization, namely rBm2D97(CCF75281.1), rBm2D33(CCF74637.1), rBm2D41(CCF75408.1) and rBm7(CCF73510.1). The diagnostic efficacy of the four recombinant protein candidates was evaluated in a clinical setting using babesiosis patient plasma. The rBm2D33 showed the highest sensitivity with a positive rate of 62.5%. Additional characterization of the two candidate proteins using a mouse vaccination assay, demonstrated that rBm2D41 could reduce peak parasitaemia by 37.4%, indicating its efficacy in preventing severe babesiosis. CONCLUSIONS: The detection technologies of microscopic examination, PCR assays and antibody tests showed different sensitivities and accuracy during the different stages of B. microti infection. Antibody detection has a unique significance for B. microti infection in the asymptomatic stages. Using immunoreactivity profiles, biomarkers for disease progression were identified and represent useful information for future the diagnosis and vaccine development for this serious disease of public health significance.

Urgent needs in fostering neglected tropical diseases (NTDs) laboratory capacity in WHO Western Pacific Region: results from the external quality assessment on NTDs diagnosis in 2012-2015.

BACKGROUND: Neglected tropical diseases (NTDs) are a heterogeneous group of mainly chronic, debilitating and often stigmatizing diseases that largely affects low-income and politically marginalized populations, causing a large burden of public health, social and economies in the NTDs endemic countries. NTDs are caused by infections with a range of pathogen, including bacteria, parasites, protozoa and viruses. The accurate diagnosis of NTDs is important for reducing morbidity, preventing mortality and for monitoring of control programs. External Quality Assessment (EQA), a component of laboratory quality assurance, aims to assess the performance of participating laboratories in detecting parasitic infections. The aim of this paper is to report the findings and put forward the recommendations on capacity build from the EQA results of participating NTDs laboratories in selected countries in the WHO Western Pacific Region from 2012 to 2015. METHODS: Reference or public health laboratories at national level working on NTDs in 6 countries participated in EQAs organized by the National Institute of Parasitic Diseases (NIPD) of Chinese Center for Disease Control and Prevention (CDC) based in Shanghai, China. Two representatives of each participating laboratory were invited to NIPD to detect NTDs' parasitic infections using the same prepared samples for serological tests (IHA and ELISA) and helminth eggs' morphological tests (Direct smear and Kato-Katz). All of the results were scored and analyzed by using SPSS statistics 19.0 software. RESULTS: The percentage of participants who had EQA score ≥ 60 during 2012-2015 for direct smear test were 80.00% (2012), 71.43% (2013), 100% (2014) and 75.00% (2015), whereas for Kato-Katz test were 80.00% (2012), 57.14% (2013), 100% (2014) and 37.50% (2015), respectively. The detection rate of helminth eggs varied in different species, with Ascaris lumbricoides being the highest at 94.07% in average. All laboratories did very well with ELISA tests as shown by the high scores in all four years except Lab A in the first and last EQA. For the positive or negative judgments of serum samples, the total coincidence rates of ELISA between 2012 and 2015 were 90.00%, 99.29%, 94.29% and 98.75%, respectively. While the total coincidence rates of IHA were respectively 100%, 95.00%, 90.00% and 97.50%. However, detecting low levels of serum antibody remained problematic for IHA when the titres of samples were taken into consideration. CONCLUSION: This study demonstrate that EQA scheme have been beneficial to the participating laboratories. The EQA programme identifies certain deficiencies which were needed to overcome and improved the laboratories' performance in helminthiasis diagnosis. However, further optimization of accuracy and uniformity in NTDs diagnosis remains a big challenge.