Identification and characterization of microRNAs in the zoonotic fluke Fasciolopsis buski.

Fasciolopsis buski is a food-borne zoonotic parasite which is transmitted by aquatic plants, with pigs and humans as the definitive hosts. The objective of the present study was to characterize the microRNA (miRNA) profiles of this parasite by Solexa deep sequencing and bioinformatic analysis. Approximately 12 million high-quality reads were obtained from adult F. buski. A total of 286 miRNA candidates were found and 24 miRNA candidates were conserved miRNAs in the miRBase database. Three novel miRNAs were identified and confirmed by stem-loop reverse transcriptase polymerase chain reaction (RT-PCR). The miRNAs found in the present study belong to 13 families whose members showed high bias. The guanine (G) was the dominant nucleotide at the beginning and middle of the conserved miRNAs, particularly at the positions of 2nd, 6th, and 13th. To our knowledge, this is the first report of the miRNA profiles of F. buski, which would lay a foundation for further functional studies of miRNAs of F. buski.

Development of Lateral Flow Immunoassay for Antigen Detection in Human Angiostrongylus cantonensis Infection.

Angiostrongyliasis is difficult to be diagnosed for the reason that no ideal method can be used. Serologic tests require specific equipment and are not always available in poverty-stricken zone and are time-consuming. A lateral flow immunoassay (LFIA) may be useful for angiostrongyliasis control. We established a LFIA for the diagnosis of angiostrongyliasis based on 2 monoclonal antibodies (mAbs) against antigens of Angiostrongylus cantonensis adults. The sensitivity and specificity were 91.1% and 100% in LFIA, while those of commercial ELISA kit was 97.8% and 86.3%, respectively. Youden index was 0.91 in LFIA and 0.84 in commercial ELISA kit. LFIA showed detection limit of 1 ng/ml of A. cantonensis ES antigens. This LFIA was simple, rapid, highly sensitive and specific, which opened an alternative approach for the diagnosis of human angiostrongyliasis.

[Application of DNA Microarray Technology in Human Trypanosomiasis Research].

With the rapid development of molecular biological techniques, DNA microarray has shown great advantage in biology and medicine as it allows high-throughput measurement of various biological parameters. Trypanosomiasis remains the focus among numerous human blood parasitic diseases. The DNA microarray is a useful technique for studying the trypanosome genome and parasite-host interaction, as well as for vaccine screening and drug development. This review gives an update on the application of DNA microarray in human research on Trypanosoma brucei and Trypanosoma cruzi.

[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.

[Cloning, Expression and Immunodiagnostic Evaluation of the Fasciola gigantica Thioredoxin Peroxidase].

OBJECTIVE: To immunoscreen the gene encoding thioredoxin peroxidase (TPx) from a cDNA library made from adult Fasciola gigantica worms, clone and express the gene, and evaluate the immunodiagnostic value of TPx recombinant protein. METHODS: The A ZAP cDNA library was immunoscreened with pooled serum of fascioliasis gigantica patients. The obtained positive clones were sequenced and analyzed by multiple sequence alignment. The full-length (rFgTPx) and N-termianal truncated (rFgTPx_nt) sequence of FgTPx was subcloned into prokaryotic plasmid pET28a(+) with a non-fusion expression technique, respectively. The recombinant proteins of rFgTPx and rFgTPx_nt were purified by His-bind affinity column (Ni-NTA). rFgTPx and rFgTPx_nt were used in indirect ELISA to test the antibody response of the serum samples. Sera of 27 fascioliasis gigantica patients, 15 patients with schistosomaisis japonica, 15 clonorchiasis sinensis patients, and 32 healthy donors were tested by using the recombinant protein based ELISA. RESULTS: The TPx recombinant proteins were obtained through expression, purification and renaturation, the relative molecular mass of rFgTPx and rFgTPx_nt were Mr 30,000 and Mr 26,000, respectively. The total diagnostic coincidence rate, sensitivity and specificity of rFgTPx_nt-based ELISA was 87.6% (78/89), 66.7% (18/27), and 96.8% (60/62), respectively. The cross reaction with Schistosoma japonicum and Clonorchis sinensis was 0 and 1/15 for rFgTPx_nt, respectively. Before and after treatment, A450 value of the serum samples from fascioliasis patients was 0.233 ± 0.088 and 0.129 ± 0.072, respectively (t = 4.27, P < 0.01). CONCLUSION: The gene encoding TPx is expressed in the prokaryotic expression system. The recombinant protein shows proper sensitivity and high specificity for the serodiagnosis of Fasciola gigantica infection.

Assessing the association between the circulating levels of inflammatory cytokines and the risk of tuberculosis: A bidirectional two-sample mendelian randomization study.

BACKGROUND: Numerous observational studies have previously reported an association between inflammatory cytokines and tuberculosis (TB). However, the causal relationship between these factors remains unclear. Consequently, we conducted two-sample Mendelian randomization (MR) analyses to ascertain the causal link between levels of inflammatory cytokines and the risk of TB. METHODS: Single nucleotide polymorphisms (SNPs) robustly associated with the cytokines, located in or close to their coding gene. SNP was obtained from genome-wide association studies (GWAS) of 8293 individuals of Finnish. TB data was obtained from the UK Biobank, which included 46,293 individuals of European ancestry (comprising 2277 TB cases and 46,056 controls). Two-sample, bi-directional MR analyses using inverse-variance weighted (IVW) method as the primary analysis. Followed by comprehensive sensitivity analyses to validate the robustness of results. RESULT: The study showed that the causal relationship between circulating levels of interleukin (IL)-7 and risk of TB (odds ratio [OR] = 1.001, 95% confidence intervals [CIs]: 1.000, 1.003. p = 0.047). No causal associations were observed between other influencing factors and the occurrence of TB. Furthermore, the analysis revealed that TB infection exhibited negative causal associations with macrophage inflammatory protein 1 alpha ([MIP-1α], OR = 0.007, 95% CI: 0.000, 0.192. p = 0.004), IL-2 (OR = 0.014, 95% CI: 0.010, 0.427. p = 0.014), interleukin-2 receptor alpha chain([IL-2rα], OR = 0.019, 95% CI: 0.001, 0.525. p = 0.019) and basic fibroblast growth factor ([bFGF], OR = 0.066, 95% CI: 0.006, 0.700. p = 0.024). CONCLUSION: The study has illuminated the causal link between inflammatory cytokines and TB, thereby enhancing our comprehension of the potential mechanisms underlying TB pathogenesis. This discovery offers promising avenues for the identification of novel therapeutic targets in TB treatment. These insights may ultimately pave the way for more effective treatment approaches, thereby improving patient outcomes.

Impact factors of Blastocystis hominis infection in persons living with human immunodeficiency virus: a large-scale, multi-center observational study from China.

BACKGROUND: Blastocystis hominis (Bh) is zoonotic parasitic pathogen with a high prevalent globally, causing opportunistic infections and diarrhea disease. Human immunodeficiency virus (HIV) infection disrupts the immune system by depleting CD4+ T lymphocyte (CD4+ T) cell counts, thereby increasing Bh infection risk among persons living with HIV (PLWH). However, the precise association between Bh infection risk and HIV-related biological markers and treatment processes remains poorly understood. Hence, the purpose of the study was to explore the association between Bh infection risk and CD4+ T cell counts, HIV viral load (VL), and duration of interruption in antiviral therapy among PLWH. METHODS: A large-scale multi-center cross-sectional study was conducted in China from June 2020 to December 2022. The genetic presence of Bh in fecal samples was detected by real-time fluorescence quantitative polymerase chain reaction, the CD4+ T cell counts in venous blood was measured using flowcytometry, and the HIV VL in serum was quantified using fluorescence-based instruments. Restricted cubic spline (RCS) was applied to assess the non-linear association between Bh infection risk and CD4+ T cell counts, HIV VL, and duration of interruption in highly active antiretroviral therapy (HARRT). RESULTS: A total of 1245 PLWH were enrolled in the study, the average age of PLWH was 43 years [interquartile range (IQR): 33, 52], with 452 (36.3%) being female, 50.4% (n = 628) had no immunosuppression (CD4+ T cell counts > 500 cells/µl), and 78.1% (n = 972) achieved full virological suppression (HIV VL < 50 copies/ml). Approximately 10.5% (n = 131) of PLWH had interruption. The prevalence of Bh was found to be 4.9% [95% confidence interval (CI): 3.8-6.4%] among PLWH. Significant nonlinear associations were observed between the Bh infection risk and CD4+ T cell counts (Pfor nonlinearity < 0.001, L-shaped), HIV VL (Pfor nonlinearity < 0.001, inverted U-shaped), and duration of interruption in HARRT (Pfor nonlinearity < 0.001, inverted U-shaped). CONCLUSIONS: The study revealed that VL was a better predictor of Bh infection than CD4+ T cell counts. It is crucial to consider the simultaneous surveillance of HIV VL and CD4+ T cell counts in PLWH in the regions with high level of socioeconomic development. The integrated approach can offer more comprehensive and accurate understanding in the aspects of Bh infection and other opportunistic infections, the efficacy of therapeutic drugs, and the assessment of preventive and control strategies.

[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.

Optimal control strategies of SARS-CoV-2 Omicron supported by invasive and dynamic models.

BACKGROUND: There is a raising concern of a higher infectious Omicron BA.2 variant and the latest BA.4, BA.5 variant, made it more difficult in the mitigation process against COVID-19 pandemic. Our study aimed to find optimal control strategies by transmission of dynamic model from novel invasion theory. METHODS: Based on the public data sources from January 31 to May 31, 2022, in four cities (Nanjing, Shanghai, Shenzhen and Suzhou) of China. We segmented the theoretical curves into five phases based on the concept of biological invasion. Then, a spatial autocorrelation analysis was carried out by detecting the clustering of the studied areas. After that, we choose a mathematical model of COVID-19 based on system dynamics methodology to simulate numerous intervention measures scenarios. Finally, we have used publicly available migration data to calculate spillover risk. RESULTS: Epidemics in Shanghai and Shenzhen has gone through the entire invasion phases, whereas Nanjing and Suzhou were all ended in the establishment phase. The results indicated that Rt value and public health and social measures (PHSM)-index of the epidemics were a negative correlation in all cities, except Shenzhen. The intervention has come into effect in different phases of invasion in all studied cities. Until the May 31, most of the spillover risk in Shanghai remained above the spillover risk threshold (18.81-303.84) and the actual number of the spillovers (0.94-74.98) was also increasing along with the time. Shenzhen reported Omicron cases that was only above the spillover risk threshold (17.92) at the phase of outbreak, consistent with an actual partial spillover. In Nanjing and Suzhou, the actual number of reported cases did not exceed the spillover alert value. CONCLUSIONS: Biological invasion is positioned to contribute substantively to understanding the drivers and mechanisms of the COVID-19 spread and outbreaks. After evaluating the spillover risk of cities at each invasion phase, we found the dynamic zero-COVID strategy implemented in four cities successfully curb the disease epidemic peak of the Omicron variant, which was highly correlated to the way to perform public health and social measures in the early phases right after the invasion of the virus.

Specific detection of Angiostrongylus cantonensis in the snail Achatina fulica using a loop-mediated isothermal amplification (LAMP) assay.

Angiostrongylus cantonensis, a rat lungworm, can cause eosinophilic meningitis and angiostrongyliasis in humans following ingestion of contaminated foods or intermediate/paratenic hosts with infective larvae. The snail Achatina fulica is one of the important intermediate hosts of A. cantonensis and is commonly eaten by humans in some countries. In the present study, we developed a loop-mediated isothermal amplification (LAMP) method for the specific detection of A. cantonensis in Ac. fulica. Primers for LAMP were designed based on the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA (rDNA) of A. cantonensis. Specificity tests showed that only the products of A. cantonensis were detected when DNA samples of A. cantonensis and the heterologous control samples Anisakis simplex s.s, Trichuris trichiura, Toxocara canis, Trichinella spiralis and Ascaris lumbricoides were amplified by LAMP. Sensitivity evaluation indicated that the LAMP assay is 10 times more sensitive than the conventional polymerase chain reaction (PCR) assay. The established LAMP assay is rapid, inexpensive and easy to be performed. It can be used in clinical applications for rapid and sensitive detection of A. cantonensis in snails, which has implications for the effective control of angiostrongyliasis.

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.

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.

A novel recombinase polymerase amplification (RPA) assay for the rapid isothermal detection of Neospora caninum in aborted bovine fetuses.

The development of a method to rapidly diagnose Neospora caninum infection is highly desirable. Recombinase polymerase amplification (RPA), combined with lateral flow (LF) strips, is a novel approach to rapidly amplify and visualize DNA. We have developed a prototype LF-RPA assay, using primers and a probe that targeted a specific sequence in the N. caninum NC-5 gene. The N. caninum-specific LF-RPA assay was first tested on purified DNA from oocysts and amplified N. caninum DNA to detectable levels in 10 min, at a constant temperature and without the need for an expensive thermocycler. The designed RPA primers and probe displayed 100% specificity for detecting N. caninum without any cross-reaction with DNA from nine related protozoan spp. (eg Toxoplasma gondii, Sarcocystis gigantean, Sarcocystis zuoi, Hammondia hammondi, Hammondia heydorni, Eimeria cylindrica, Plasmodium falciparum, Theileria annulata and Babesia bigemina). Although, LF-RPA assay detected amounts as low as 50 fg of N. caninum DNA, it was nearly 5-fold less sensitive than previously published qPCR and nested PCR assays. We tested the diagnostic performance of the LF-RPA assay for the detection of N. caninum DNA in aborted bovine fetal tissue samples, and compared the results with those obtained from nested PCR. Out of the 75 samples examined, 18 (24%) and 17 (22.6%) tested positive using LF-RPA and nested PCR, respectively. Our results indicate that LF-RPA is a suitable assay for the rapid and reliable detection of N. caninum.

Trypanosoma brucei rhodesiense infection in a Chinese traveler returning from the Serengeti National Park in Tanzania.

BACKGROUND: Human African trypanosomiasis (HAT) is one of the most complex parasitic diseases known to humankind. It usually occurs in endemic areas in Africa, but is occasionally detected in returning travelers and migrants in non-endemic countries. CASE PRESENTATION: In August 2017, a case of HAT was diagnosed in China in a traveler returning from the Masai Mara area in Kenya and the Serengeti area in Tanzania. The traveler visited Africa from 23 July to 5 August, 2017. Upon return to China, she developed a fever (on 8 August), and Trypanosoma brucei rhodesiense infection was confirmed by laboratory tests (on 14 August) including observation of parasites in blood films and by polymerase chain reaction. She was treated with pentamidine followed by suramin, and recovered 1 month later. CONCLUSIONS: This is the first imported rhodesiense HAT case reported in China. This case alerts clinical and public health workers to be aware of HAT in travelers, and expatriates and migrants who have visited at-risk areas in Africa.

Fasciolopsis buski (Digenea: Fasciolidae) from China and India may represent distinct taxa based on mitochondrial and nuclear ribosomal DNA sequences.

BACKGROUND: Fasciolopsis buski is a zoonotic intestinal fluke infecting humans and pigs, but it has been seriously neglected. It is yet to know whether there is any genetic diversity among F. buski from different geographical locations, particularly in sequences of nuclear ribosomal DNA (rDNA) and mitochondrial (mt) DNA. Therefore, we determined the sequences of partial 18S, the complete internal transcribed spacer (ITS) rDNA and the complete mt genome of F. buski from China, compared the rDNA and mtDNA sequences with those of isolates from India and Vietnam, and assessed the phylogenetic relationships of this fluke and related fasciolid trematodes based on the mtDNA dataset. RESULTS: The complete mt genome sequence of F. buski from China is 14,833 bp, with 36 genes, including 12 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes (rrnL and rrnS). The AT content of F. buski from China is 65.12%. The gene content and arrangement of the F. buski mt genome is similar to that of Fascioloides magna. Genetic distances between isolates of F. buski from China and India were high (28.2% in mtDNA, 13.2% in ITS-1 and 9.8% in ITS-2) and distinctly higher than the interspecific differences between Fasciola hepatica and Fasciola gigantica. The rDNA and mtDNA datasets for F. buski from China (isolate from pigs) and Vietnam (isolates from humans) were identical. The intergeneric differences in amino acid and nucleotide sequences among the genera Fasciolopsis, Fascioloides and Fasciola ranged between 24.64-25.56% and 26.35-28.46%, respectively. CONCLUSIONS: Our results indicate that F. buski from China and India may represent distinct taxa, while F. buski in Vietnam and China represent the same species. These findings might have implications for the implementation of appropriate control strategies in different regions. Further studies are needed to decode mtDNA and rDNA sequences of F. buski from various geographical isolates for the better understanding of the species complex of F. buski.

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.

DNA Microarray Detection of 18 Important Human Blood Protozoan Species.

BACKGROUND: Accurate detection of blood protozoa from clinical samples is important for diagnosis, treatment and control of related diseases. In this preliminary study, a novel DNA microarray system was assessed for the detection of Plasmodium, Leishmania, Trypanosoma, Toxoplasma gondii and Babesia in humans, animals, and vectors, in comparison with microscopy and PCR data. Developing a rapid, simple, and convenient detection method for protozoan detection is an urgent need. METHODOLOGY/PRINCIPAL FINDINGS: The microarray assay simultaneously identified 18 species of common blood protozoa based on the differences in respective target genes. A total of 20 specific primer pairs and 107 microarray probes were selected according to conserved regions which were designed to identify 18 species in 5 blood protozoan genera. The positive detection rate of the microarray assay was 91.78% (402/438). Sensitivity and specificity for blood protozoan detection ranged from 82.4% (95%CI: 65.9% ~ 98.8%) to 100.0% and 95.1% (95%CI: 93.2% ~ 97.0%) to 100.0%, respectively. Positive predictive value (PPV) and negative predictive value (NPV) ranged from 20.0% (95%CI: 2.5% ~ 37.5%) to 100.0% and 96.8% (95%CI: 95.0% ~ 98.6%) to 100.0%, respectively. Youden index varied from 0.82 to 0.98. The detection limit of the DNA microarrays ranged from 200 to 500 copies/reaction, similar to PCR findings. The concordance rate between microarray data and DNA sequencing results was 100%. CONCLUSIONS/SIGNIFICANCE: Overall, the newly developed microarray platform provides a convenient, highly accurate, and reliable clinical assay for the determination of blood protozoan species.

[Establishment of A1E3 and B1C4 monoclonal antibody-based ELISA for detecting circulating antigen of Schistosoma japonicum and its preliminary application].

OBJECTIVE: To establish A1E3 and B1C4 monoclonal antibody-based ELISA for detecting circulating antigen of Schistosoma japonicum and explore its application value in the field. METHODS: The characteristics of A1E3 and B1C4 monoclonal antibodies were analyzed by SDS-PAGE and Western blotting. The SEA-based ELISA was used to evaluate the titers of A1E3 and B1C4. The orthogonal test was used to determine the best concentration of coating antibody B1C4 and optimal working concentration of A1E3-HRP. Under the optimal conditions, the serum samples of 20 acute schistosomiasis cases, 46 chronic schistosomiasis cases, and 20 control sera were tested to evaluate its detection sensitivity and specificity. Seventy-two antibody positive serum samples from Jiangling County of Hubei Province were detected and compared to a commercially available ELISA kit, to evaluate the detection effects of this method. RESULTS: The results of SDS-PAGE demonstrated that the purified A1E3 and B1C4 contained a clear heavy chain with molecular weight of 88,000 and 52,000 respectively and had the same light chain with molecular weight of 20,000; while Western blotting demonstrated that A1E3 and B1C4 could be recognized by SEA and serum samples of acute schistosomiasis cases. The SEA-based ELISA demonstrated the titers of B1C4 and A1E3 were 1:10(5) and 1:30,000, respectively. The serum samples from all the acute cases and 86.9% of the chronic cases showed a positive reaction. All of the control sera from healthy persons gave a negative response. The positive rates of the double monoclonal antibody ELISA and commercial ELISA for detecting the circulating antigen were 45.8% and 43.1% respectively, and there was no significant difference between the results of the two methods. CONCLUSION: A1E3 and B1C4 monoclonal antibody-based ELISA is established successfully. It exhibits a high sensitivity and specificity in detecting circulating antigen of Schistosoma japonicum.