BackgroundLoop-mediated isothermal amplification (LAMP) is a high performance method for detecting DNA and holds promise for use in the molecular detection of infectious pathogens, including Plasmodium spp. However, in most malaria-endemic areas, which are often resource-limited, current LAMP methods are not feasible for diagnosis due to difficulties in accurately interpreting results with problems of sensitive visualization of amplified products, and the risk of contamination resulting from the high quantity of amplified DNA produced. In this study, we establish a novel visualized LAMP method in a closed-tube system, and validate it for the diagnosis of malaria under simulated field conditions.MethodsA visualized LAMP method was established by the addition of a microcrystalline wax-dye capsule containing the highly sensitive DNA fluorescence dye SYBR Green I to a normal LAMP reaction prior to the initiation of the reaction. A total of 89 blood samples were collected on filter paper and processed using a simple boiling method for DNA extraction, and then tested by the visualized LAMP method for Plasmodium vivax infection.ResultsThe wax capsule remained intact during isothermal amplification, and released the DNA dye to the reaction mixture only when the temperature was raised to the melting point following amplification. Soon after cooling down, the solidified wax sealed the reaction mix at the bottom of the tube, thus minimizing the risk of aerosol contamination. Compared to microscopy, the sensitivity and specificity of LAMP were 98.3% (95% confidence interval (CI): 91.1-99.7%) and 100% (95% CI: 88.3-100%), and were in close agreement with a nested polymerase chain reaction method.ConclusionsThis novel, cheap and quick visualized LAMP method is feasible for malaria diagnosis in resource-limited field settings.

Background Vivax malaria is an important public health problem in the Greater Mekong Subregion (GMS), including the China-Myanmar border. Previous studies have found that Plasmodium vivax has decreased sensitivity to antimalarial drugs in some areas of the GMS, but the sensitivity of P. vivax to antimalarial drugs is unclear in the China-Myanmar border. Here, we investigate the drug sensitivity profile and genetic variations for two drug resistance related genes in P. vivax isolates to provide baseline information for future drug studies in the China-Myanmar border. Methodology/Principal findings A total of 64 P. vivax clinical isolates collected from the China-Myanmar border area were assessed for ex vivo susceptibility to eight antimalarial drugs by the schizont maturation assay. The medians of IC 50 (half-maximum inhibitory concentrations) for chloroquine, mefloquine, pyronaridine, piperaquine, quinine, artesunate, artemether, dihydroartemisinin were 84.2 nM, 34.9 nM, 4.0 nM, 22.3 nM, 41.4 nM, 2.8 nM, 2.1 nM and 2.0 nM, respectively. Twelve P. vivax clinical isolates were found over the cutoff IC 50 value (220 nM) for chloroquine resistance. In addition, sequence polymorphisms in pvmdr1 (P. vivax multidrug resistance-1), pvcrt-o (P. vivax chloroquine resistance transporter-o), and difference in pvmdr1 copy number were studied. Sequencing of the pvmdr1 gene in 52 samples identified 12 amino acid substitutions, among which two (G698S and T958M) were fixed, M908L were present in 98.1% of the isolates, while Y976F and F1076L were present in 3.8% and 78.8% of the isolates, respectively. Amplification of the pvmdr1 gene was only detected in 4.8% of PLOS NEGLECTED TROPICAL DISEASES

BackgroundMany malaria-related studies depend on infected red blood cells (iRBCs) as fundamental material; however, infected blood samples from human or animal models include leukocytes (white blood cells or WBCs), especially difficult to separate from iRBCs in cases involving Plasmodium vivax. These host WBCs are a source of contamination in biology, immunology and molecular biology studies, requiring their removal. Non-woven fabric (NWF) has the ability to adsorb leukocytes and is already used as filtration material to deplete WBCs for blood transfusion and surgery. The present study describes the development and evaluation of a prototype NWF filter designed for purifying iRBCs from malaria-infected blood.MethodsBlood samples of P. vivax patients were processed separately by NWF filter and CF11 column methods. WBCs and RBCs were counted, parasite density, morphology and developing stage was checked by microscopy, and compared before and after treatment. The viability of filtrated P. vivax parasites was examined by in vitro short-term cultivation.ResultsA total of 15 P. vivax-infected blood samples were treated by both NWF filter and CF11 methods. The WBC removal rate of the NWF filter method was 99.03%, significantly higher than the CF11 methods (98.41%, P < 0.01). The RBC recovery rate of the NWF filter method was 95.48%, also significantly higher than the CF11 method (87.05%, P < 0.01). Fourteen in vitro short-term culture results showed that after filter treatment, P. vivax parasite could develop as normal as CF11 method, and no obvious density, developing stage difference were fund between two methods.ConclusionsNWF filter filtration removed most leukocytes from malaria-infected blood, and the recovery rate of RBCs was higher than with CF11 column method. Filtrated P. vivax parasites were morphologically normal, viable, and suitable for short-term in vitro culture. NWF filter filtration is simple, fast and robust, and is ideal for purification of malaria-infected blood.

Advocacy of the use of facemasks by the public as a measure against the spread of COVID‐19 is controversial, with some healthcare professionals arguing that the use of a face mask may increase the rate at which people touch their faces, due to readjusting the mask. We assessed the facial touching behaviour of bus passengers in China before and after the outbreak of COVID‐19 and found that wearing a face mask does not increase the number of hand‐face contacts and is likely, therefore, to have a positive beneficial effect on suppressing the spread of COVID‐19 within populations when used in conjunction with social distancing measures.

BackgroundThe rodent malaria parasite Plasmodium yoelii is an important animal model for studying host-parasite interaction and molecular basis of malaria pathogenesis. Although a draft genome of P. yoeliiyoelii YM is available, and RNA sequencing (RNA-seq) data for several rodent malaria species (RMP) were reported recently, variations in coding regions and structure of mRNA transcript are likely present between different parasite strains or subspecies. Sequencing of cDNA libraries from additional parasite strains/subspecies will help improve the gene models and genome annotation.MethodsHere two directional cDNA libraries from mixed blood stages of a subspecies of P. yoelii (P. y. nigeriensis NSM) with or without mefloquine (MQ) treatment were sequenced, and the sequence reads were compared to the genome and cDNA sequences of P. y. yoelii YM in public databases to investigate single nucleotide polymorphisms (SNPs) in coding regions, variations in intron–exon structure and differential splicing between P. yoelii subspecies, and variations in gene expression under MQ pressure.ResultsApproximately 56 million of 100 bp paired-end reads were obtained, providing an average of ~225-fold coverage for the coding regions. Comparison of the sequence reads to the YM genome revealed introns in 5′ and 3′ untranslated regions (UTRs), altered intron/exon boundaries, alternative splicing, overlapping sense-antisense reads, and potentially new transcripts. Interestingly, comparison of the NSM RNA-seq reads obtained here with those of YM discovered differentially spliced introns; e.g., spliced introns in one subspecies but not the other. Alignment of the NSM cDNA sequences to the YM genome sequence also identified ~84,000 SNPs between the two parasites.ConclusionThe discoveries of UTR introns and differentially spliced introns between P. yoelii subspecies raise interesting questions on the potential role of these introns in regulating gene expression and evolution of malaria parasites.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-1081-9) contains supplementary material, which is available to authorized users.

Avian paramyxovirus serotype 4 (APMV-4) is found sporadically in wild birds worldwide, and it is an economically important poultry pathogen. Despite the existence of several published strains, very little is known about the distribution, host species, and transmission of APMV-4 strains. To better understand the relationships among these factors, we conducted an APMV-4 surveillance of wild birds and domestic poultry in six provinces of China suspected of being intercontinental flyways and sites of interspecies transmission. APMV-4 surveillance was conducted in 9,160 wild birds representing seven species, and 1,461 domestic poultry in live bird markets (LMBs) from December 2013 to June 2016. The rate of APMV-4 isolation was 0.10% (11/10,621), and viruses were isolated from swan geese, bean geese, cormorants, mallards, and chickens. Sequencing and phylogenetic analyses of the 11 isolated viruses indicated that all the isolates belonging to genotype I were epidemiologically connected with wild bird-origin viruses from the Ukraine and Italy. Moreover, chicken-origin APMV-4 strains isolated from the LBMs were highly similar to wild bird-origin viruses from nearby lakes with free-living wild birds. In additional, a hemagglutination-negative APMV-4 virus was identified. These findings, together with recent APMV-4 studies, suggest potential virus interspecies transmission between wild birds and domestic poultry, and reveal possible epidemiological intercontinental connections between APMV-4 transmission by wild birds.

Newcastle disease (ND), caused by infections with virulent strains of Newcastle disease virus (NDV), is one of the most important infectious disease affecting wild, peridomestic, and domestic birds worldwide. Vaccines constructed from live, low-virulence (lentogenic) viruses are the most accepted prevention and control strategies for combating ND in poultry across the globe. Avian macrophages are one of the first cell lines of defense against microbial infection, responding to signals in the microenvironment. Although macrophages are considered to be one of the main target cells for NDV infection in vivo, very little is known about the ability of NDV to infect chicken macrophages, and virulence mechanisms of NDV as well as the polarized activation patterns of macrophages and correlation with viral infection and replication. In the present study, a cell culture model (chicken bone marrow macrophage cell line HD11) and three different virulence and genotypes of NDV (including class II virulent NA-1, class II lentogenic LaSota, and class I lentogenic F55) were used to solve the above underlying questions. Our data indicated that all three NDV strains had similar replication rates during the early stages of infection. Virulent NDV titers were shown to increase compared to the other lentogenic strains, and this growth was associated with a strong upregulation of both pro-inflammatory M1-like markers/cytokines and anti-inflammatory M2-like markers/cytokines in chicken macrophages. Virulent NDV was found to block toll-like receptor (TLR) 7 expression, inducing higher expression of type I interferons in chicken macrophages at the late stage of viral infection. Only virulent NDV replication can be inhibited by pretreatment with TLR7 ligand. Overall, this study demonstrated that virulent NDV activates a M1-/M2-like mixed polarized activation of chicken macrophages by inhibition of TLR7, resulting in enhanced replication compared to lentogenic viruses.

BackgroundIsolation of Plasmodium-infected red blood cells (iRBCs) from clinical blood samples is often required for experiments, such as ex vivo drug assays, in vitro invasion assays and genome sequencing. Current methods for removing white blood cells (WBCs) from malaria-infected blood are time-consuming or costly. A prototype non-woven fabric (NWF) filter was developed for the purification of iRBCs, which showed great efficiency for removing WBCs in a pilot study. Previous work was performed with prototype filters optimized for processing 5–10 mL of blood. With the commercialization of the filters, this study aims to evaluate the efficiency and suitability of the commercial NWF filter for the purification of Plasmodium vivax-infected RBCs in smaller volumes of blood and to compare its performance with that of Plasmodipur® filters.MethodsForty-three clinical P. vivax blood samples taken from symptomatic patients attending malaria clinics at the China–Myanmar border were processed using the NWF filters in a nearby field laboratory. The numbers of WBCs and iRBCs and morphology of P. vivax parasites in the blood samples before and after NWF filtration were compared. The viability of P. vivax parasites after filtration from 27 blood samples was examined by in vitro short-term culture. In addition, the effectiveness of the NWF filter for removing WBCs was compared with that of the Plasmodipur® filter in six P. vivax blood samples.ResultsFiltration of 1–2 mL of P. vivax-infected blood with the NWF filter removed 99.68% WBCs. The densities of total iRBCs, ring and trophozoite stages before and after filtration were not significantly different (P > 0.05). However, the recovery rates of schizont- and gametocyte-infected RBCs, which were minor parasite stages in the clinical samples, were relatively low. After filtration, the P. vivax parasites did not show apparent morphological changes. Culture of 27 P. vivax-infected blood samples after filtration showed that parasites successfully matured into the schizont stage. The WBC removal rates and iRBC recovery rates were not significantly different between the NWF and Plasmodipur® filters (P > 0.05).ConclusionsWhen tested with 1–2 mL of P. vivax-infected blood, the NWF filter could effectively remove WBCs and the recovery rates for ring- and trophozoite-iRBCs were high. P. vivax parasites after filtration could be successfully cultured in vitro to reach maturity. The performance of the NWF and Plasmodipur® filters for removing WBCs and recovering iRBCs was comparable.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-017-1855-3) contains supplementary material, which is available to authorized users.