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Objectives Amoebiasis is caused by the most common intestinal protozoan parasite Entamoeba histolytica. This parasite causes amoebic colitis, which is manifested by diarrhea, followed by dysentery. The laboratory diagnosis of intestinal amoebiasis in most cases is by microscopic examination of stool samples. Other nonroutine methods include coproantigen enzyme-linked immunosorbent assay (ELISA) from stool samples, serum ELISA for antibodies, stool culture, isoenzyme analysis, and polymerase chain reaction (PCR). The present study aimed to comparatively analyze the different diagnostic modalities used for the detection of E. histolytica from the stool sample of patients with intestinal amoebiasis. Materials and Methods This study was undertaken with 631 patients, during a period of 3 years, from January 2017 to December 2019. Stool specimen obtained from each patient was subjected to direct microscopic wet mount examination, coproantigen ELISA, and nested multiplex PCR, respectively. Results Out of all the patients tested, 5.2% were positive for E. histolytica. Among the positive cases, stool microscopy was positive in 3.17%, coproantigen ELISA was positive in 29 (4.6%) cases, and PCR was positive in 30 (4.75%) cases. Statistical Analysis The prevalence of E. histolytica infection was summarized as percentages. The three diagnostic tests done were statistically analyzed, taking microscopy as the gold standard. The agreement between techniques (microscopy, coproantigen ELISA, and PCR) was analyzed with kappa statistics. Sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy were summarized as percentage with 95% confidence interval. Conclusion In all suspected amoebiasis cases, a combination of stool microscopy, coproantigen testing with molecular detection of the parasite offers the best approach to diagnosis of this parasitic infection.

Objectives Plasmodium vivax malaria was thought to be rare in Africans who lack the Duffy blood group antigen expression. However, recent studies indicate that P. vivax can infect Duffy-negative individuals and has spread into areas of high Duffy negativity across Africa. Our study compared epidemiological and genetic features of P. vivax between African regions. Methods A standardized approach was used to identify and quantify P. vivax from Botswana, Ethiopia, and Sudan, where Duffy-positive and Duffy-negative individuals coexist. The study involved sequencing the Duffy binding protein (DBP) gene and inferring genetic relationships among P. vivax populations across Africa. Results Among 1215 febrile patients, the proportions of Duffy negativity ranged from 20–36% in East Africa to 84% in southern Africa. Average P. vivax prevalence among Duffy-negative populations ranged from 9.2% in Sudan to 86% in Botswana. Parasite density in Duffy-negative infections was significantly lower than in Duffy-positive infections. P. vivax in Duffy-negative populations were not monophyletic, with P. vivax in Duffy-negative and Duffy-positive populations sharing similar DBP haplotypes and occurring in multiple, well-supported clades. Conclusions Duffy-negative Africans are not resistant to P. vivax, and the public health significance of this should not be neglected. Our study highlights the need for a standardized approach and more resources/training directed towards the diagnosis of vivax malaria in Africa.

Stomatopods in India are well known with 79 species recorded to date. Here I report the Odontodactylus japonicus (De Haan, 1844) and Golden Mantis Shrimp Lysiosquilla tredecimdentata Holothuis, 1941 for the first time in Puducherry coastal waters. A single specimen of Lysiosquilla tredecimdentata was collected from by-catch in the Nallavadu landing centre, Puducherry coast on 19 November 2019 and two specimens of L. tredecimdentata were recorded again in Pillaichavadi landing centre of Puducherry coast on 22 November 2019. One specimen of Odontodactylus japonicus was collected at Nallavadu landing centre, Puducherry coast on 20 December 2019. The present study was undertaken to identify the status of distribution, habitat, and ecological aspects along with the information of spread, confinement, endemism as well as rare, threatened and endangered species. The significance of these new observations is to discern the taxonomic position and characteristics for better understanding of the mantis shrimp group. The specimens were identified, described, illustrated, and measured morphometrically.

Coryneforms other than Corynebacterium diphtheriae are often ignored when isolated from clinical laboratories as in most instances they represent the normal commensals of skin. Corynebacterium jeikeium was initially considered only as commensal, later it was identified as the causative agent for severe sepsis in immunocompromised patients. Here, we present a case of meningoencephalitis caused by Corynebacterium jeikeium in a 30 year old adult male with HIV and tuberculosis co-infection. Serial cerebrospinal fluid from external ventricular drainage had grown Corynebacterium jeikeium which was identified by automated MALDI-TOF system which aided in the timely diagnosis and prompt management of the patient. Keywords: Corynebacterium jeikeium, Meningoencephalitis, HIV tuberculosis co-infection, External ventricular drainage.

The objective of the research was to discern the impact of micronutrients as a foliar spray in annual moringa (Moringa oleifera Lam.) var. PKM 1. The plants were sprayed at three different phases—vegetative, flowering and pod setting phase with micronutrients singly or in combination of ferrous sulphate + citric acid, boric acid, zinc sulphate, manganese sulphate or a mixture of all. The experimental results revealed that all treatments performed significantly better when compared to control with respect to all morphological and yield attributes. The maximum plant height, number of primary branches, number of flowers per panicle, number of panicles per tree, fruit setting percentage, pod yield per tree, protein, ascorbic acid, carotenoid content and micronutrients status in leaves and pods were recorded highest in T8 (FeSO4 1% + Citric Acid 0.1%) + ZnSO4 0.5% + MnSO4 0.5% + H3BO3 0.2%) (micronutrient mixture) than other treatment combinations. The research has proven that the foliar spray of these micronutrient combinations increased the growth, quality and yield of annual moringa var. PKM-1 significantly. The research outcome is in corroboration that the nutrient status of moringa leaves is found to be higher when compared to the pods. Hence, the consumption of moringa leaves will be more beneficial than the pods in terms of nutrient supplement to alleviate malnutrition and sustain nutritional security.

Introduction: Vancomycin-resistant enterococci (VRE) are emerging as an important multidrug-resistant pathogen causing nosocomial infections, predominantly bacteremia and urinary tract infections. VRE bacteremia has caused a significant increase in the duration of the hospital stay and mortality and had caused high public health threat due to limited treatment options. Materials and methods: Between October 2017 and September 2020, all consecutive patients with culture-proven bloodstream infection with Enterococcus species, isolated for the first time, were included in the study. A total of 427 Enterococcus species were identified, and antimicrobial susceptibility tests were performed and interpreted using Clinical and Laboratory Standard Institute guidelines. Results: Of the total 427 Enterococcus species isolated, 63 (45.6%) were VRE. Among them, 51/63 (81%) were Enterococcus faecium (E. faecium) and 5/63 (8%) were Enterococcus faecalis. There was an increased trend of VRE rate in the bloodstream infections of 6.12% (2018), 13.2% (2019), and 19.2% (2020). The majority of the VRE patients [43/63 (68%)] were admitted to the intensive care units (ICUs). Vancomycin A (VanA) is the most common phenotype isolated from 51/63(81%) patients. Conclusion: This increasing trend of VRE bacteremia is a red alert to the clinicians and the infection control practitioners, so that strict antibiotic policies and proper adherence to the infection control practices can be initiated to reduce the VRE rate. How to cite this article: Sivaradjy M, Gunalan A, Priyadarshi K, Madigubba H, Rajshekar D, Sastry AS. Increasing Trend of Vancomycin-resistant Enterococci Bacteremia in a Tertiary Care Hospital of South India: A Three-year Prospective Study. Indian J Crit Care Med 2021;25(8):881–885.

Multiple mutations in SARS-CoV-2 variants of concern (VOCs) may increase, transmission, disease severity, immune evasion and facilitate zoonotic or anthoprozoonotic infections. Four such mutations, ΔH69/V70, L452R, E484K and N501Y, occur in the SARS-CoV-2 spike glycoprotein in combinations that allow detection of the most important VOCs. Here we present two flexible RT-qPCR platforms for small-and large-scale screening to detect these mutations, and schemes for adapting the platforms for future mutations. The large-scale RT-qPCR platform, was validated by pair-wise matching of RT-qPCR results with WGS consensus genomes, showing high specificity and sensitivity. Detection of mutations using this platform served as an important interventive measure for the Danish public health system to delay the emergence of VOCs and to gain time for vaccine administration. Both platforms are valuable tools for WGS-lean laboratories, as well for complementing WGS to support rapid control of local transmission chains worldwide.

Context: Acute undifferentiated febrile illness (AUFI) is characterized by a sudden onset of raised body temperature and is a common cause of hospital admission though not recognized as a disease state by the World Health Organization. Epstein-Barr virus (EBV) is reported to account for a significant occurrence of AUFI cases. Aim: To know the role of EBV infection as a cause of acute undifferentiated febrile illness (AUFI). Settings and design: We have used the combination of EBV serological assays to establish the role of the Epstein-Barr virus as the cause of acute undifferentiated febrile illness. Methods and material: A total of 721 suspected cases of acute undifferentiated febrile illness which were tested negative for other common causes of acute febrile illness were selected for the study. Serum samples collected from these cases were tested for the presence of the EBV viral capsid antigen (VCA) IgM antibody. All positive serum samples were tested for the presence of EBV Epstein-Barr nuclear antigen (EBNA) IgG. Statistical analysis used: Statistical analysis was performed with the help of Microsoft Excel software (Microsoft Corporation, Redmond, USA). Results: Out of 721 suspected AUFI cases tested for EBV VCA IgM antibodies, 117 samples were positive and 604 were negative. All these 117 samples were tested for EBV EBNA IgG antibodies in which 88 were positive and 29 were negative. In our study, we found that around 4% (positive for VCA IgM and negative for EBNA IgG) of AUFI cases can be attributed to primary acute EBV infection. Conclusions: EBV infection should be considered particularly in AUFI cases of less than five years of age even in those who do not meet the typical presentation of fever, lymphadenopathy and sore throat. Our study should help to raise awareness regarding the possibility of EBV infection particularly in AUFI cases. A high index of suspicion and timely diagnosis will definitely help clinicians to avoid a battery of investigations and misuse of antibiotics in cases of AUFI.

Groupers are biologically diverse, commercially important and highly regarded of all fish species associated with reef fisheries globally. Given the huge demand and high value in live fish trade, restaurants and supermarkets around the world, the accurate identification of the closely related grouper species is crucial to prevent any adulteration. Partial nucleotide sequence data of the mitochondrial cytochrome c oxidase I (COI) gene, which is globally accepted as fish DNA barcoding gene, was generated for six commercially important grouper species belonging to the genus, Epinephelus viz. E. fuscoguttatus, E. coioides, E. logispinis, E. malabaricus, E. bleekeri and E. tauvina collected from Indian waters. Based on the analysis of 704 bp COI gene sequences, a PCR-RFLP method was developed to identify all the six species accurately. The PCR products of COI gene fragment was digested with selected restriction enzymes viz. HinfI, AseI, HindIII and BsaJI. Restriction endonuclease HinfI was enabled to produce species-specific fragments for E. coioides, E. malabaricus, E. logispinis and E. bleekeri. The result shows that AseI restriction enzyme could be used to differentiate E. fuscoguttatus from other five species. The expected and actual molecular weight of the digested products was similar indicating the authenticity of the methods followed. This is the first report on differentiating closely related grouper species using above mentioned restriction endonucleases and COI gene fragment. PCR-RFLP profiles generated could be useful for detecting seafood fraud by identifying the grouper species under the genus Epinephelus using samples from fresh, frozen, cooked and even processed products.

Growth of textile industries led to production of enormous dye varieties. These textile dyes are largely used, chemically stable and easy to synthesize. But they are recalcitrant and persist as less biodegradable pollutants when discharged into waterbodies. Potential use of enzyme-linked bioremediation of textile dyes will control their toxicity in waterbodies. Bioinformatics and Molecular docking tool provides an insight into remediation mechanism by predicting susceptibility of dye degradation using oxidoreductive enzymes. In this study, six dyes, Reactive Red F3B, Remazol Red RGB, Joyfix Red RB, Joyfix Yellow MR, Remazol Blue RGB and Turquoise CL-5B of azo, anthraquinone and phthalocyanine molecular class were identified as potential targets for degradation by laccase and azoreductase of Aeromonas hydrophila in addition to Lysinibacillus sphaericus through in silico docking tool BioSolveIT-FlexX. Azoreductase breaks azo bonds by ping-pong mechanism whereas laccase decolorizes dyes by free radical mechanism which is not specific in nature. Results were analyzed based on parameters like stability, catalytic action and selectivity for enzyme-dye interactions. Amino acids of enzymes interacted with several dyes substantiating variations in active site for enzyme-ligand binding affinity. This suggests the role of enzymes in decolorizing an extensive variety of textile dyes, thereby, aiding in understanding the enzyme mechanisms in Bioremediation.

Unlike the case in Asia and Latin America, Plasmodium vivax infections are rare in sub-Saharan Africa due to the absence of the Duffy blood group antigen (Duffy antigen), the only known erythrocyte receptor for the P. vivax merozoite invasion ligand, Duffy binding protein 1 (DBP1). However, P. vivax infections have been documented in Duffy-negative individuals throughout Africa, suggesting that P. vivax may use ligands other than DBP1 to invade Duffy-negative erythrocytes through other receptors. To identify potential P. vivax ligands, we compared parasite gene expression in Saimiri and Aotus monkey erythrocytes infected with P. vivax Salvador I (Sal I). DBP1 binds Aotus but does not bind to Saimiri erythrocytes; thus, P. vivax Sal I must invade Saimiri erythrocytes independent of DBP1. Comparing RNA sequencing (RNAseq) data for late-stage infections in Saimiri and Aotus erythrocytes when invasion ligands are expressed, we identified genes that belong to tryptophan-rich antigen and merozoite surface protein 3 (MSP3) families that were more abundantly expressed in Saimiri infections compared with Aotus infections. These genes may encode potential ligands responsible for P. vivax infections of Duffy-negative Africans.

Multiple SARS-CoV-2 variants have evolved increasing the transmissibility of the virus and/or cause its escape from immune response. Whole genome sequencing remains the gold standard and is implemented into national surveillance programs worldwide to detect these novel variants of concern. However, this technique can have a high turnaround time and it might not be possible to immediately be scaled up when fast action is required to limit the spread of SARS-CoV-2 variants every time a new local outbreak occurs. Four key mutations H69/V70 deletion, L452R, E484K and N501Y with multiple reassurance in the SARS-CoV-2 spike (S) glycoprotein were identified to have a distinct signature enabling to detect SARS-CoV-2 variants of worldwide concern (Alpha [B.1.1.7], Alpha [B.1.17 + E484K], Beta [B.1.351], Gamma [P1], Delta [B.1.617,2]) as well as variants of interest/notes. We developed RT-qPCRs to detect these key mutations and increased the sensitivity of the PCRs by using modified probes targeting the 452R, 484K and 501Y mutations, with a single nucleotide exchange present, leading to an amino acid substitution. Large scale screening of the SARS-CoV2 variants of concern was included into the national surveillance program in Denmark, where the RT-qPCRs run as multiplexed (H69/V70 and N501Y mutation) - or as a single reactions detecting all four key mutations in parallel in a 384-well format. Our RT-qPCR systems offer a fast and flexible testing strategy to detect SARS-CoV-2 variants of concern and of interest/note, especially needed in countries where whole genome sequencing in large scale is too cost- and labour intensive.

Oseltamivir-resistant influenza viruses arise due to amino-acid mutations in key residues, but these changes often reduce their replicative and transmission fitness. Widespread oseltamivir-resistance has not yet been observed in A(H1N1)pdm09 viruses. However, it is known that permissive mutations in the neuraminidase (NA) of former seasonal A(H1N1) viruses from 2007-2009 buffered the detrimental effect of the NA H275Y mutation, resulting in fit oseltamivir-resistant viruses that circulated widely. This study explored two approaches to predict permissive mutations that may enable a fit H275Y A(H1N1)pdm09 variant to arise.A computational approach used phylogenetic andin silicoprotein stability analyses to predict potentially permissive mutations, which were then evaluated byin vitroNA enzyme activity and expression analysis, followed byin vitroreplication. The second approach involved the generation of a virus library which encompassed all possible individual 2.9 × 104codon mutations in the NA whilst keeping H275Y fixed. To select for variant viruses with the greatest fitness, the virus library was serially passaged in ferrets (via contact and aerosol transmission) and resultant viruses were deep sequenced.The computational approach predicted three NA permissive mutations, and even though they only offset thein vitroimpact of H275Y on NA enzyme expression by 10%, they could restore replication fitness of the H275Y variant in A549 cells. In our experimental approach, a diverse virus library (97% of 8911 possible single amino-acid substitutions were sampled) was successfully transmitted through ferrets, and sequence analysis of resulting virus pools in nasal washes identified three mutations that improved virus transmissibility. Of these, one NA mutation, I188T, has been increasing in frequency since 2017 and is now present in 90% of all circulating A(H1N1)pdm09 viruses.Overall, this study provides valuable insights into the evolution of the influenza NA protein and identified several mutations that may potentially facilitate the emergence of a fit H275Y A(H1N1)pdm09 variant.

CONTEXT The emergence of drug resistant pathogens pose major threat to hospitalized patients as well as to the community associated with increased mortality and morbidity. The treatment of carbapenem resistant enterobacteriaceae, one of the top WHO priority pathogen remains a global issue. Combination therapy with different classes of antibiotics have been tried with the aim to reduce toxicity, to increase the efficacy of the drugs and to reduce resistance. The in-vitro synergy methods have to be carried out to determine whether the combination of those antibiotics are synergistic, antagonistic or additive. AIMS We have performed in-vitro synergy testing by checkerboard method for colistin -meropenem combination to determine whether the combination of the two antibiotics were synergistic or antagonistic. METHODS AND MATERIAL All the consecutive twenty five blood isolates of Escherichia coli and twenty five blood isolates of Klebsiella pneumoniae which were showing resistance to carbapenems by either disc diffusion or vitek 2 were collected over a period of 6 months and checkerboard method was performed. STATISTICAL ANALYSIS USED The reduction of MIC of colisin on combination with meropenem compared to MIC of colistin alone is analyzed by McNemar's chisquare test with the help of software Stata version 14 and p value

The Duffy blood group is a critical receptor for Plasmodium vivax (P. vivax) invasion of red blood cells, and consequently, P. vivax infections were considered rare in sub-Saharan Africa where the prevalence of Duffy-negativity is high. However, recently, P. vivax infections have been found in Duffy-negative Africans throughout the malaria transmission area of sub-Saharan Africa, raising important questions concerning the molecular composition of these P. vivax clones and the red blood cell receptors that facilitate their invasion. Here, we describe an unusually high number of P. vivax infections in febrile Duffy-negative Africans in Dschang, Cameroon (177 of 500 outpatients), as compared with Santchou (two of 400 outpatients) and Kyé-ossi (two of 101 outpatients), in other areas in Cameroon. In the discussion, we speculate on the possible reasons why Dschang might account for the unusually large numbers of P. vivax infections in Duffy-negative individuals living there.

Longitudinal studies of gut microbiota following specific interventions are vital for understanding how they influence host health. However, robust longitudinal sampling of gut microbiota is a major challenge, which can be addressed using in vitro fermentors hosting complex microbial communities. Here, by employing 16S rRNA gene amplicon sequencing, we investigated the adaptation and succession of human fecal microbial communities in an automated multistage fermentor. We performed two independent experiments using different human donor fecal samples, one configured with two units of three colon compartments each studied for 22 days and another with one unit of two colon compartments studied for 31 days. The fermentor maintained a trend of increasing microbial alpha diversity along colon compartments. Within each experiment, microbial compositions followed compartment-specific trajectories and reached independent stable configurations. While compositions were highly similar between replicate units, they were clearly separated between different experiments, showing that they maintained the individuality of fecal inoculum rather than converging on a fermentor-specific composition. While some fecal amplicon sequence variants (ASVs) were undetected in the fermentor, many ASVs undetected in the fecal samples flourished in vitro. These bloomer ASVs accounted for significant proportions of the population and included prominent health-associated microbes such as Bacteroides fragilis and Akkermansia muciniphila. Turnover in community compositions is likely explained by feed composition and pH, suggesting that these communities can be easily modulated. Our results suggest that in vitro fermentors are promising tools to study complex microbial communities harboring important members of human gut microbiota. IMPORTANCEIn vitro fermentors that can host complex gut microbial communities are promising tools to investigate the dynamics of human gut microbiota. In this work, using an automated in vitro gut fermentor consisting of different colon compartments, we investigated the adaptation dynamics of two different human fecal microbial communities over 22 and 31 days. By observing the temporal trends of different community members, we found that many dominant members of the fecal microbiota failed to maintain their dominance in vitro, and some of the low-abundance microbes undetected in the fecal microbiota successfully grew in the in vitro communities. Microbiome compositional changes and blooming could largely be explained by feed composition and pH, suggesting that these communities can be modulated to desired compositions via optimizing culture conditions. Thus, our results open up the possibility of modulating in vitro microbial communities to predefined compositions by optimizing feed composition and culture conditions.

Recent studies indicated that Plasmodium vivax can infect Duffy-negative individuals, but the varied diagnostic and methodological approaches have limited our ability to characterize P. vivax across Africa. Here, we utilized a standardized approach to compare epidemiological and genetic attributes of P. vivax from Botswana, Ethiopia, and Sudan, where Duffy-positive and Duffy-negative individuals coexist. Among 1,215 febrile patients, the proportions of Duffy negativity range from 20-36% in East Africa to 84% in Southern Africa. Considerable differences were observed in P. vivax prevalence among Duffy-negative populations ranging from averaged 9.2% in Sudan to 86% in Botswana. P. vivax parasite density in Duffy-negative infections is significantly lower than in Duffy-positive infections. Phylogenetic analyses of 229 PvDBP sequences indicated that Duffy-negative P. vivax were not monophyletic but occurred in multiple well-supported clades, suggesting independent origins. Duffy-negative Africans are clearly not resistant to P. vivax and the public health significance should no longer be neglected.

Environmental DNA barcoding technology is gaining innovative applications when monitoring diverse tropics of marine environment. Since previous studies have shown that most marine eDNA sequences cannot be assigned to any species and in most cases to any phyla, since only 15 percent of the animal species is barcoded and accessible in reference libraries, we propose to test the effectiveness of current DNA barcode reference libraries in identifying amphipod barcodes and/or strengthening the existing library. Over 2500 amphipod individuals were collected for morphological analysis and DNA barcoding from estuarine sediments. We barcoded 22 amphipod species belonging to 17 genera, 13 families in the order Amphipoda. We found that 13 species (59 percent) were missing from the reference barcode libraries and were first time barcoded. More than 80 percent of the species were not reported previously form the sampled area. The minimum and maximum inter-specific pair-wise distance values was found to be respectively 0.16 and 5.51 percent. We show that defining family specific species threshold values would be imperative, rather than expecting a universal barcode gap for amphipod species. The overall mean pair-wise distance, nucleotide diversity and Tajima statistics were 3.59 percent, 0.27 and 2.62, respectively. Species identification through neighbour joining tree shows that the taxa of same species clustered together indicating the efficacy of COI sequences in amphipod species delineation. There is a strong need to increase the number of amphipod species barcodes in the reference database, because 59 percent of barcoded species were found absent. For better facilitation of environmental monitoring, the sequences were published via public databases. We show that with the advent of next generation sequencing technologies, reference datasets like ours will become important for health evaluation and monitoring of different environments using amphipod barcodes.

Plasmodium falciparum responsible for the most virulent form of malaria invades human erythrocytes through multiple ligand-receptor interactions. The P. falciparum reticulocyte binding protein homologues (PfRHs) are expressed at the apical end of merozoites and form interactions with distinct erythrocyte surface receptors that are important for invasion. Here using a range of monoclonal antibodies (mAbs) against different regions of PfRH1 we have investigated the role of PfRH processing during merozoite invasion. We show that PfRH1 gets differentially processed during merozoite maturation and invasion and provide evidence that the different PfRH1 processing products have distinct functions during invasion. Using in-situ Proximity Ligation and FRET assays that allow probing of interactions at the nanometre level we show that a subset of PfRH1 products form close association with micronemal proteins Apical Membrane Antigen 1 (AMA1) in the moving junction suggesting a critical role in facilitating junction formation and active invasion. Our data provides evidence that time dependent processing of PfRH proteins is a mechanism by which the parasite is able to regulate distinct functional activities of these large processes. The identification of a specific close association with AMA1 in the junction now may also provide new avenues to target these interactions to prevent merozoite invasion.

Babesia microti is an intraerythrocytic parasite and the primary causative agent of human babesiosis. It is transmitted by Ixodes ticks, transfusion of blood and blood products, organ donation, and perinatally. Despite its global public health impact, limited progress has been made to identify and characterize immunodominant B. microti antigens for diagnostic and vaccine use. Using genome-wide immunoscreening, we identified 56 B. microti antigens, including some previously uncharacterized antigens. Thirty of the most immunodominant B. microti antigens were expressed as recombinant proteins in E. coli. Among these, the combined use of two novel antigens and one previously described antigen provided 96% sensitivity and 100% specificity in identifying B. microti antibody containing sera in an ELISA. Using extensive computational sequence and bioinformatics analyses and cellular localization studies, we have clarified the domain architectures, potential biological functions, and evolutionary relationships of the most immunodominant B. microti antigens. Notably, we found that the BMN-family antigens are not monophyletic as currently annotated, but rather can be categorized into two evolutionary unrelated groups of BMN proteins respectively defined by two structurally distinct classes of extracellular domains. Our studies have enhanced the repertoire of immunodominant B. microti antigens, and assigned potential biological function to these antigens, which can be evaluated to develop novel assays and candidate vaccines.

The neuraminidase (NA) inhibitor (NAI) oseltamivir (OST) is the most widely used influenza antiviral drug. Several NA amino acid substitutions are reported to reduce viral susceptibility to OST in in vitro assays. However, whether there is a correlation between the level of reduction in susceptibility in vitro and the efficacy of OST against these viruses in vivo is not well understood. In this study, a ferret model was utilised to evaluate OST efficacy against circulating influenza A and B viruses with a range of in vitro generated 50% inhibitory concentrations (IC50) values for OST. OST efficacy against an A(H1N1)pdm09 and an A(H1N1)pdm09 virus with the H275Y substitution in neuraminidase was also tested in the macaque model. The results from this study showed that OST had a significant impact on virological parameters compared to placebo treatment of ferrets infected with wild-type influenza A viruses with normal IC50 values (~1 nM). However, this efficacy was lower against wild-type influenza B and other viruses with higher IC50 values. Differing pathogenicity of the viruses made evaluation of clinical parameters difficult, although some effect of OST in reducing clinical signs was observed with influenza A(H1N1) and A(H1N1)pdm09 (H275Y) viruses. Viral titres in macaques were too low to draw conclusive results. Analysis of the ferret data revealed a correlation between IC50 and OST efficacy in reducing viral shedding but highlighted that the current WHO guidelines/criteria for defining normal, reduced or highly reduced inhibition in influenza B viruses based on in vitro data are not well aligned with the low in vivo OST efficacy observed for both wild-type influenza B viruses and those with reduced OST susceptibility., Author summary Oseltamivir (Tamiflu) is an antiviral widely used for the treatment of infection due to influenza viruses and is especially useful for the treatment of severely ill high-risk patients. Antiviral resistance to oseltamivir is of concern as it can undermine the utility of the drug. Influenza viruses can become less susceptible to oseltamivir due to amino acid substitutions that arise in the viral surface protein, neuraminidase (NA). During influenza surveillance, to monitor for resistance, laboratory experiments are carried out to measure viral susceptibility to oseltamivir, whereby the quantity of drug needed to inhibit NA enzyme action is measured. However, how well these laboratory measurements predict resistance of viruses to oseltamivir in a clinical setting is not well understood. In this study, we developed an animal model to evaluate this relationship between laboratory data and clinical efficacy in more detail and demonstrated that in vivo efficacy of oseltamivir is lower against viruses that show reduced inhibition by oseltamivir in laboratory testing. A key finding from our analysis was that oseltamivir had reduced efficacy in reducing viral shedding against wild-type influenza B viruses compared to wild-type influenza A viruses, which has implications on how data from influenza B viruses with reduced susceptibility are currently being interpreted.

Motivation Due to the risk of inducing an immediate Type I (IgE-mediated) allergic response, proteins intended for use in consumer products must be investigated for their allergenic potential before introduction into the marketplace. The FAO/WHO guidelines for computational assessment of allergenic potential of proteins based on short peptide hits and linear sequence window identity thresholds misclassify many proteins as allergens. Results We developed AllerCatPro which predicts the allergenic potential of proteins based on similarity of their 3D protein structure as well as their amino acid sequence compared with a data set of known protein allergens comprising of 4180 unique allergenic protein sequences derived from the union of the major databases Food Allergy Research and Resource Program, Comprehensive Protein Allergen Resource, WHO/International Union of Immunological Societies, UniProtKB and Allergome. We extended the hexamer hit rule by removing peptides with high probability of random occurrence measured by sequence entropy as well as requiring 3 or more hexamer hits consistent with natural linear epitope patterns in known allergens. This is complemented with a Gluten-like repeat pattern detection. We also switched from a linear sequence window similarity to a B-cell epitope-like 3D surface similarity window which became possible through extensive 3D structure modeling covering the majority (74%) of allergens. In case no structure similarity is found, the decision workflow reverts to the old linear sequence window rule. The overall accuracy of AllerCatPro is 84% compared with other current methods which range from 51 to 73%. Both the FAO/WHO rules and AllerCatPro achieve highest sensitivity but AllerCatPro provides a 37-fold increase in specificity. Availability and implementation https://allercatpro.bii.a-star.edu.sg/ Supplementary information Supplementary data are available at Bioinformatics online.

Trager and Jensen established a method for culturing Plasmodium falciparum, a breakthrough for malaria research worldwide. Since then, multiple attempts to establish Plasmodium vivax in continuous culture have failed. Unlike P. falciparum, which can invade all aged erythrocytes, P. vivax is restricted to reticulocytes. Thus, a constant supply of reticulocytes is considered critical for continuous P. vivax growth in vitro. A critical question remains why P. vivax selectively invades reticulocytes? What do reticulocytes offer to P. vivax that is not present in mature erythrocytes? One possibility is protection from oxidative stress by glucose-6-phosphate dehydrogenase (G6PD). Here, we also suggest supplements to the media and procedures that may reduce oxidative stress and, as a result, establish a system for the continuous culture of P. vivax.

Plasmodium vivax malaria is much less common in Africa than the rest of the world because the parasite relies primarily on the Duffy antigen/chemokine receptor (DARC) to invade human erythrocytes, and the majority of Africans are Duffy negative. Recently, there has been a dramatic increase in the reporting of P. vivax cases in Africa, with a high number of them being in Duffy negative individuals, potentially indicating P. vivax has evolved an alternative invasion mechanism that can overcome Duffy negativity. Here, we analyzed single nucleotide polymorphism (SNP) and copy number variation (CNV) in Whole Genome Sequence (WGS) data from 44 P. vivax samples isolated from symptomatic malaria patients in southwestern Ethiopia, where both Duffy positive and Duffy negative individuals are found. A total of 123,711 SNPs were detected, of which 22.7% were nonsynonymous and 77.3% were synonymous mutations. The largest number of SNPs were detected on chromosomes 9 (24,007 SNPs; 19.4% of total) and 10 (16,852 SNPs, 13.6% of total). There were particularly high levels of polymorphism in erythrocyte binding gene candidates including merozoite surface protein 1 (MSP1) and merozoite surface protein 3 (MSP3.5, MSP3.85 and MSP3.9). Two genes, MAEBL and MSP3.8 related to immunogenicity and erythrocyte binding function were detected with significant signals of positive selection. Variation in gene copy number was also concentrated in genes involved in host-parasite interactions, including the expansion of the Duffy binding protein gene (PvDBP) on chromosome 6 and MSP3.11 on chromosome 10. Based on the phylogeny constructed from the whole genome sequences, the expansion of these genes was an independent process among the P. vivax lineages in Ethiopia. We further inferred transmission patterns of P. vivax infections among study sites and showed various levels of gene flow at a small geographical scale. The genomic features of P. vivax provided baseline data for future comparison with those in Duffy-negative individuals and allowed us to develop a panel of informative Single Nucleotide Polymorphic markers diagnostic at a micro-geographical scale., Author summary Plasmodium vivax is the most geographically widespread parasite species that causes malaria in humans. Although it occurs in Africa as a member of a mix of Plasmodium species, P. vivax is dominant in other parts of the world outside of Africa (e.g., Brazil). It was previously thought that most African populations were immune to P. vivax infections due to the absence of Duffy antigen chemokine receptor (DARC) gene expression required for erythrocyte invasion. However, several recent reports have indicated the emergence and potential spread of P. vivax across human populations in Africa. Compared to Southeast Asia and South America where P. vivax is highly endemic, data on polymorphisms in erythrocyte binding gene candidates of P. vivax from Africa is limited. Filling this knowlege gap is critical for identifying functional genes in erythrocyte invasion, biomarkers for tracking the P. vivax isolates from Africa, as well as potential gene targets for vaccine development. This paper examined the level of genetic polymorphisms in a panel of 43 potential erythrocyte binding protein genes based on whole genome sequences and described transmission patterns of P. vivax infections from different study sites in Ethiopia based on the genetic variants. Our analyses showed that chromosomes 9 and 10 of the P. vivax genomes isolated in Ethiopia had the most high-quality genetic polymorphisms. Among all erythrocyte binding protein gene candidates, the merozoite surface proteins 1 and merozoite surface protein 3 showed high levels of polymorphism. MAEBL and MSP3.8 related to immunogenicity and erythrocyte binding function were detected with significant signals of positive selection. The expansion of the Duffy binding protein and merozoite surface protein 3 gene copies was an independent process among the P. vivax lineages in Ethiopia. Various levels of gene flow were observed even at a smaller geographical scale. Our study provided baseline data for future comparison with P. vivax in Duffy negative individuals and help develop a panel of genetic markers that are informative at a micro-geographical scale.

Background High frequency (∼130 Hz) deep brain stimulation (DBS) of the subthalamic region is an established clinical therapy for the treatment of late stage Parkinson's disease (PD). Direct modulation of the hyperdirect pathway, defined as cortical layer V pyramidal neurons that send an axon collateral to the subthalamic nucleus (STN), has emerged as a possible component of the therapeutic mechanisms. However, numerous questions remain to be addressed on the basic biophysics of hyperdirect pathway stimulation. Objective Quantify action potential (AP) initiation, propagation, and cortical invasion in hyperdirect neurons during subthalamic stimulation. Methods We developed an anatomically and electrically detailed computational model of hyperdirect neuron stimulation with explicit representation of the stimulating electric field, axonal response, AP propagation, and synaptic transmission. Results We found robust AP propagation throughout the complex axonal arbor of the hyperdirect neuron. Even at therapeutic DBS frequencies, stimulation induced APs could reach all of the intracortical axon terminals with ∼100% fidelity. The functional result of this high frequency axonal driving of the thousands of synaptic connections made by each directly stimulated hyperdirect neuron is a profound synaptic suppression that would effectively disconnect the neuron from the cortical circuitry. Conclusions The synaptic suppression hypothesis integrates the fundamental biophysics of electrical stimulation, axonal transmission, and synaptic physiology to explain a generic mechanism of DBS.

Plasmodium vivax is the main cause of malarial disease in Asia and South America. Plasmodium vivax infection was thought to be absent in African populations who are Duffy blood group antigen negative (Duffy-negative). However, many cases of P. vivax infection have recently been observed in Duffy-negative Africans. This raises the question: were P. vivax infections in Duffy-negative populations previously missed or has P. vivax adapted to infect Duffy-negative populations? This review focuses on recent P. vivax findings in Africa and reports views on the parasite ligands that may play a role in Duffy-negative P. vivax infections. In addition, clues gained from studying P. vivax infection of reticulocytes are presented, which may provide possible avenues for establishing P. vivax culture in vitro.

Significance We have shown in this study that the malaria parasite can rapidly evolve to adapt for loss of an “essential” kinase, PfCDPK1. PfCDPK1 could not be disrupted in the wild-type parasite. However, we were able to disrupt CDPK1 in the transgenic parasites adapted for reduced kinase activity of mutant PfCDPK1. Strategic disruption of PfCDPK1 highlights the importance of understanding the compensatory mechanisms, especially for targets belonging to multigene families. Our study unequivocally demonstrates that PfCDPK1 is critical for mosquito infections and its disruption leads to defective gametogenesis. Our study also suggests involvement of CDPK1 in regulation of sexual stage-specific genes during the asexual proliferation. Targeting PfCDPK1 may be a good strategy for developing transmission-blocking drugs., Efforts to knock out Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) from asexual erythrocytic stage have not been successful, indicating an indispensable role of the enzyme in asexual growth. We recently reported generation of a transgenic parasite with mutant CDPK1 [Bansal A, et al. (2016) MBio 7:e02011-16]. The mutant CDPK1 (T145M) had reduced activity of transphosphorylation. We reasoned that CDPK1 could be disrupted in the mutant parasites. Consistent with this assumption, CDPK1 was successfully disrupted in the mutant parasites using CRISPR/Cas9. We and others could not disrupt PfCDPK1 in the WT parasites. The CDPK1 KO parasites show a slow growth rate compared with the WT and the CDPK1 T145M parasites. Additionally, the CDPK1 KO parasites show a defect in both male and female gametogenesis and could not establish an infection in mosquitoes. Complementation of the KO parasite with full-length PfCDPK1 partially rescued the asexual growth defect and mosquito infection. Comparative global transcriptomics of WT and the CDPK1 KO schizonts using RNA-seq show significantly high transcript expression of gametocyte-specific genes in the CDPK1 KO parasites. This study conclusively demonstrates that CDPK1 is a good target for developing transmission-blocking drugs.

Intestinal parasitic infection (IPI) constitute a global health burden causing clinical morbidity in 450 million people. Many of these are women of reproductive age and children in developing countries. Mass deworming programmes with improvement in lifestyle are likely to reduce the intensity and prevalence of infection over the years. Hence, we aimed to assess the prevalence of intestinal parasitic infections among patients in a tertiary healthcare setting and to examine its time trends. A descriptive cross-sectional study was done using routinely collected data in a tertiary care hospital in South India. Details of examination of stool samples for the presence of intestinal helminth and protozoan ova/cysts, over the period of 5 years (2014–2019) were extracted from laboratory register and hospital information system. The presence of intestinal parasitic infection was determined by stool microscopy (direct wet mount and concentration techniques). Of the total 3267 stool samples, 303 (9.3%) had at least one parasite; 3.9% (93/3267) with helminths and 2.5% (81/3267) Entamoeba and multi-parasitism was seen in 0.14%. Stool samples from more than 18 years age had high positivity rate than others. Majority of the helminth infections were caused by Ascaris (57%) followed by hookworm (42%). Initially IPI which was 10.9% in 2014 declined to 10% in 2016 and attained a peak of 12.4% in 2017 then decreased to 6.7% in 2018. Nearly one out of ten patients had a parasitic infection. Prevalence surveys in the community followed by strengthening the deworming procedures will reduce the burden of IPIs.

Plasmodium vivax invasion of human erythrocytes depends on the Duffy Binding Protein (PvDBP) which interacts with the Duffy antigen. PvDBP copy number has been recently shown to vary between P. vivax isolates in Sub-Saharan Africa. However, the extent of PvDBP copy number variation, the type of PvDBP multiplications, as well as its significance across broad samples are still unclear. We determined the prevalence and type of PvDBP duplications, as well as PvDBP copy number variation among 178 Ethiopian P. vivax isolates using a PCR-based diagnostic method, a novel quantitative real-time PCR assay and whole genome sequencing. For the 145 symptomatic samples, PvDBP duplications were detected in 95 isolates, of which 81 had the Cambodian and 14 Malagasy-type PvDBP duplications. PvDBP varied from 1 to >4 copies. Isolates with multiple PvDBP copies were found to be higher in symptomatic than asymptomatic infections. For the 33 asymptomatic samples, PvDBP was detected with two copies in two of the isolates, and both were the Cambodian-type PvDBP duplication. PvDBP copy number in Duffy-negative heterozygotes was not significantly different from that in Duffy-positives, providing no support for the hypothesis that increased copy number is a specific association with Duffy-negativity, although the number of Duffy-negatives was small and further sampling is required to test this association thoroughly., Author summary Plasmodium vivax invasion of human erythrocytes relies on interaction between the Duffy antigen and P. vivax Duffy Binding Protein (PvDBP). Whole genome sequences from P. vivax field isolates in Madagascar identified a duplication of the PvDBP gene and PvDBP duplication has also been detected in non-African P. vivax-endemic countries. Two types of PvDBP duplications have been reported, termed Cambodian and Malagasy-type duplications. Our study used a combination of PCR-based diagnostic method, a novel quantitative real-time PCR assay, and whole genome sequencing to determine the prevalence and type of PvDBP duplications, as well as PvDBP copy number on a broad number of P. vivax samples in Ethiopia. We found that over 65% of P. vivax isolated from the symptomatic infections were detected with PvDBP duplications and PvDBP varied from 1 to >4 copies. The majority of PvDBP duplications belongs to the Cambodian-type while the Malagasy-type duplications was also detected. For the asymptomatic infections, despite a small sample size, the majority of P. vivax were detected with a single-copy based on both PCR and qPCR assays. There was no significant difference in PvDBP copy number between Duffy-null heterozygote and Duffy-positive homozygote/heterozygote. Further investigation is needed with expanded Duffy-null homozygotes to examine the functional significance of PvDBP expansion.

Summary Coronaviruses are a diverse group of viruses that infect mammals and birds. Bats are reservoirs for several different coronaviruses in the Alphacoronavirus and Betacoronavirus genera. They also appear to be the natural reservoir for the ancestral viruses that generated the severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus outbreaks. Here, we detected coronavirus sequences in next‐generation sequence data created from Eonycteris spelaea faeces and urine. We also screened by PCR urine samples, faecal samples and rectal swabs collected from six species of bats in Singapore between 2011 and 2014, all of which were negative. The phylogenetic analysis indicates this novel strain is most closely related to lineage D Betacoronaviruses detected in a diverse range of bat species. This is the second time that coronaviruses have been detected in cave nectar bats, but the first coronavirus sequence data generated from this species. Bat species from which this group of coronaviruses has been detected are widely distributed across SE Asia, South Asia and Southern China. They overlap geographically, often share roosting sites and have been witnessed to forage on the same plant. The addition of sequence data from this group of viruses will allow us to better understand coronavirus evolution and host specificity.

Erythrocyte invasion by merozoite is a multistep process involving multiple ligand-receptor interactions. The Plasmodium falciparum reticulocyte binding protein homologues (PfRHs) consists of five functional members. The differential expression of PfRHs has been linked to the utilization of different invasion pathways by the merozoites as well as a mechanism of immune evasion. PfRHs are expressed at the apical end of merozoite and form interactions with distinct red blood cell (RBC) surface receptors that are important for successful invasion. Here we show that PfRH2b undergoes processing before and during merozoite invasion. The different processed fragments bind to chymotrypsin sensitive RBC surface receptors. We also show that PfRH2b follows the merozoite tight junction during invasion. Monoclonal antibodies (mAbs) inhibit merozoites invasion by blocking tight junction formation. mAbs binding to PfRH2b block merozoites intracellular Ca2+ signal necessary for EBA175 surface expression. The data suggests that a conserved function of PfRHs, where their interaction with RBC surface receptors facilitated recruitment of EBA175 and other tight junction proteins necessary for merozoite invasion by modulating merozoite intracellular Ca2+ signals.

Influenza is a ubiquitous infection with a spectrum ranging from mild to severe. The mystery regarding such variability in the clinical spectrum has not been fully unravelled, although a role for the complex interplay among virus characteristics, host immune response and environmental factors has been suggested. Antivirals and current vaccines have a limited role in prophylaxis and treatment because they primarily target surface glycoproteins which undergo antigenic/genetic changes under host immune pressure. Targeting conserved internal proteins could lead the way to a universal vaccine which can be used against various types/subtypes. However, this is on the distant horizon, so in the meantime, developing improved vaccines should be given high priority. In this review, we discuss where the current influenza research stands in terms of vaccines, adjuvants, and how we can better predict the vaccine strains for upcoming influenza seasons by understanding complex phenomena which drive the continuous antigenic evolution.

Objective: To study the echinoderms diversity in Mudasalodai and Pazhayar landing centers during November 2012 to December 2014. Methods: All the collected echinoderm samples were stored at 4 °C until further use. Results: As many as 19 species of echinoderms were recorded in both stations and it belongs to 10 families, three classes such as Asteroidea, Echinoidea and Ophiuroidea. The percentage composition varied from 0.15% to 73%. The species diversity ranged from 1.031 to 2.6; species richness was from 1.02 to 4.06 and species evenness ranged from 0.93 to 0.99. K-dominance curve drawn paralleled the trend of diversity indices. Cluster and multidimensional scaling showed the similarity in echinoderms composition within the stations. Conclusions: The present investigation is evident that the echinoderm occurrence was predominant during the summer season and proved statistically by using SPSS Primer 6.0.

Understanding the dual inhibition mechanism of food derivative peptides targeting the enzymes (Renin and Angiotensin Converting enzyme) in the Renin Angiotensin System. Two peptides RALP and WYT were reported to possess antihypertensive activity targeting both renin and ACE, and we have used molecular docking and molecular dynamics simulation, in order to understand the underlying mechanism. The selected peptides (RALP and WYT) from the series of peptides reported were docked to renin and ACE and two binding modes were selected based on the binding energy, interaction pattern and clusters of docking simulation. The enzyme-peptide complexes for renin and ACE (Renin/RALP1,2; ACE/RALP1,2; Renin/WYT1,2 and ACE/WYT1,2) were subjected to molecular dynamics simulation. Our results identified that the peptides inhibiting renin, tends to move out of the binding pockets (S1’ S2’) which is critical for potent binding and occupies the less important pockets (S4 and S3). This could possibly be the reason for its low potency. Whereas, the same peptides targeting ACE, tends to be intact in the pocket because of the metal ion coordination and there is an ample room to improve on its efficacy. Our results further pave way for the biochemist, medicinal chemist to design dual peptides targeting the RAS effectively. Communicated by Ramaswamy H. Sarma

Highly pathogenic avian influenza (H5N8) virus, like the recently described H5N8 strain from Korea, was detected in November 2014 in farmed turkeys and in a healthy common teal (Anas crecca) in northeastern Germany. Infected wild birds possibly introduced this virus. Published version

Little is known about the cellular mechanisms of innate immunity against dengue virus (DV) infection. Specifically, the γδ T cell response to DV has not been characterized in detail. In this article, we demonstrate that markers of activation, proliferation, and degranulation are upregulated on γδ T cells in PBMC isolated from individuals with acute dengue fever. Primary γδ T cells responded rapidly in vitro to autologous DV-infected dendritic cells by secreting IFN-γ and upregulating CD107a. The anti-DV IFN-γ response is regulated by type I IFN and IL-18 in a TCR-independent manner, and IFN-γ secreting γδ T cells predominantly expressed IL-18Rα. Antagonizing the ATP-dependent P2X7 receptor pathway of inflammasome activation significantly inhibited the anti-DV IFN-γ response of γδ T cells. Overnight priming with IL-18 produced effector γδ T cells with significantly increased ability to lyse autologous DV-infected dendritic cells. Monocytes were identified as accessory cells that augmented the anti-DV IFN-γ response of γδ T cells. Lack of monocytes in culture is associated with lower IL-18 levels in culture supernatant and diminished production of IFN-γ by γδ T cells, whereas addition of exogenous IL-18 restored the IFN-γ response of γδ T cells in monocyte-depleted cocultures with DV-infected DC. Our results indicate that primary γδ T cells contribute to the immune response during DV infection by providing an early source of IFN-γ, as well as by killing DV-infected cells, and suggest that monocytes participate as accessory cells that sense DV infection and amplify the cellular immune response against this virus in an IL-18–dependent manner.

Background : Acute pancreatitis is an inflammation of pancreas associated with reversible pancreatic parenchymal injury. Studies in several countries indicate that the levels of amylase and lipase are usually elevated among patients with acute pancreatitis. Furthermore, hyperlipidemia, mainly high levels of triglycerides, may present in acute pancreatitis. The aim of this study was to determine the levels of serum amylase and lipase as well as their correlation with serum triglyceride level in acute pancreatitis patients. Methods : A retrospective study was conducted on medical records of 48 acute pancreatitis patients in Dr. Hasan Sadikin General Hospital Bandung, Indonesia from 2007to 2011. Data collected from the medical records were age, sex, levels of serum amylase, lipase and triglyceride. The distribution of data was determined using Shapiro-Wilk test. The correlation between serum pancreatic enzyme and triglyceride was analyzed using Spearman-rank test. Results : Most patients had increased levels of serum amylase and lipase in this study. However, no correlation between serum amylase and triglyceride (p-value = 0.312) was found. Furthermore, there was no correlation between serum lipase and triglyceride (p-value = 0.241). Conclusions : The levels of serum amylase and lipase increase in most patients with acute pancreatitis with no significant correlation between serum pancreatic enzymes (amylase and lipase) and triglyceride. Keywords : Acute pancreatitis, amylase, lipase, triglyceride DOI : 10.15850/amj.v2n1.420

Background The transmission of fungi via transplant, although well-known, has not often been molecularly proven. We describe a case of donor-derived candidiasis verified by whole genome sequencing. Case Description The multiorgan donor was a 42-year-old woman with subdural hemorrhage. Procurement of the thoracic organs was performed followed by the abdominal organs. Tissue from the left bronchus grew Candida dubliniensis. The liver recipient was a 63-year-old woman with cryptogenic liver cirrhosis. She was noted to have worsening leukocytosis on postoperative day (POD) 9. Computed tomography of the abdomen and pelvis showed multiple rim-enhancing collections around the graft. Percutaneous drainage was performed. Fluid cultures grew C dubliniensis. C dubliniensis isolated from the donor's left bronchus and the liver recipient's abscesses were verified to be related by whole genome sequencing. We postulate that C dubliniensis colonizing the donor's transected trachea could have contaminated the inferior vena cava when the former was left open after explant of the donor's lungs. A portion of the donor's contaminated inferior vena cava was transplanted along with the liver graft, resulting in the infected collections in the recipient. Conclusions Our case report highlights the importance of maintaining a sterile field during organ procurement, especially in a multiorgan donor whose organs are explanted in succession.

Background Avian and swine influenza viruses circulate worldwide and pose threats to both animal and human health. The design of global surveillance strategies is hindered by information gaps on the geospatial variation in virus emergence potential and existing surveillance efforts. Methods We developed a spatial framework to quantify the geographic variation in outbreak emergence potential based on indices of potential for animal-to-human and secondary human-to-human transmission. We then compared our resultant raster model of variation in emergence potential with the global distribution of recent surveillance efforts from 359105 reports of surveillance activities. Results Our framework identified regions of Southeast Asia, Eastern Europe, Central America, and sub-Saharan Africa with high potential for influenza virus spillover. In the last 15 years, however, we found that 78.43% and 49.01% of high-risk areas lacked evidence of influenza virus surveillance in swine and domestic poultry, respectively. Conclusions Our work highlights priority areas where improved surveillance and outbreak mitigation could enhance pandemic preparedness strategies.

Plasmodium vivax was thought to infect only the erythrocytes of Duffy blood group positive people. In the last decade, P. vivax has appeared throughout Africa, both in areas where Duffy positive and negative people live side by side as in Madagascar and Ethiopia and in areas where people are primarily Duffy negative, such as in western Kenya. We performed quantitative polymerase chain reaction on blood samples dried onto filter paper to determine the prevalence of P. vivax and Plasmodium falciparum in a cohort of 300 children (newborn to 6 years of age) in Bandiagara, a Sahelian area of Mali, west Africa, where the people are Duffy negative. We report 1-3 occurrences of P. vivax in each of 25 Duffy-negative children at six time points over two rainy seasons and the beginning of the third season. The prevalence of P. vivax infection was 2.0-2.5% at every time point (June 2009 to June 2010). All children with P. vivax infections were asymptomatic and afebrile, and parasite densities were extremely low. Anemia, however, was the main burden of infection. Plasmodium vivax could become a burden to sub-Saharan Africa, and the evidence of P. vivax existence needs to be taken into consideration in designing malaria control and elimination strategies in Africa.

Seasonal influenza A viruses (IAV) originate from pandemic IAV and have undergone changes in antigenic structure, including addition of glycans to the hemagglutinin (HA) glycoprotein. The viral HA is the major target recognized by neutralizing antibodies and glycans have been proposed to shield antigenic sites on HA, thereby promoting virus survival in the face of widespread vaccination and/or infection. However, addition of glycans can also interfere with the receptor binding properties of HA and this must be compensated for by additional mutations, creating a fitness barrier to accumulation of glycosylation sites. In addition, glycans on HA are also recognized by phylogenetically ancient lectins of the innate immune system and the benefit provided by evasion of humoral immunity is balanced by attenuation of infection. Therefore, a fine balance must exist regarding the optimal pattern of HA glycosylation to offset competing pressures associated with recognition by innate defenses, evasion of humoral immunity and maintenance of virus fitness. In this review, we examine HA glycosylation patterns of IAV associated with pandemic and seasonal influenza and discuss recent advancements in our understanding of interactions between IAV glycans and components of innate and adaptive immunity. Published version

Bats are unique mammals, exhibit distinctive life history traits and have unique immunological approaches to suppression of viral diseases upon infection. High-throughput next-generation sequencing has been used in characterizing the virome of different bat species. The cave nectar bat, Eonycteris spelaea, has a broad geographical range across Southeast Asia, India and southern China, however, little is known about their involvement in virus transmission. Here we investigate the diversity and abundance of viral communities from a colony of Eonycteris spelaea residing in Singapore. Our results detected 47 and 22 different virus families from bat fecal and urine samples, respectively. Among these, we identify a large number of virus families including Adenoviridae, Flaviviridae, Reoviridae, Papillomaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, and Polyomaviridae. In most cases, viral sequences from Eonycteris spelaea are genetically related to a group of bat viruses from other bat genera (e.g., Eidolon, Miniopterus, Rhinolophus and Rousettus). The results of this study improve our knowledge of the host range, spread and evolution of several important viral pathogens. More significantly, our findings provide a baseline to study the temporal patterns of virus shedding and how they correlate with bat phenological trends.

The successful invasion of Plasmodium is an essential step in their life cycle. The parasite reticulocyte-binding protein homologues (RHs) and erythrocyte-binding like proteins are two families involved in the invasion leading to merozoite-red blood cell (RBC) junction formation. Ca2+ signaling has been shown to play a critical role in the invasion. RHs have been linked to Ca2+ signaling, which triggers the erythrocyte-binding like proteins release ahead of junction formation, consistent with RHs performing an initial sensing function in identifying suitable RBCs. RH5, the only essential RHs, is a highly promising vaccine candidate. RH5-basigin interaction is essential for merozoite invasion and also important in determining host tropism. Here, we show that RH5 has a distinct function from the other RHs. We show that RH5-Basigin interaction on its own triggers a Ca2+ signal in the RBC resulting in changes in RBC cytoskeletal proteins phosphorylation and overall alterations in RBC cytoskeleton architecture. Antibodies targeting RH5 that block the signal prevent invasion before junction formation consistent with the Ca2+ signal in the RBC leading to rearrangement of the cytoskeleton required for invasion. This work provides the first time a functional context for the essential role of RH5 and will now open up new avenues to target merozoite invasion. Ministry of Education (MOE) Ministry of Health (MOH) National Medical Research Council (NMRC) Accepted version This research is supported by the Singapore Ministry of Health's National Medical Research Council under its Individual Research Grant (NMRC/1308/2011) and the Singapore Ministry of Education Academic Research Fund Tier 1 grant (RG 152/14).This work is also supported by MoE Tier 1 Grant (SUTDT12015006) from Singapore University of Technology and Design.

SUMMARYAn outbreak of gastroenteritis affected 453 attendees (attack rate 28·5%) of six separate events held at a hotel in Singapore. Active case detection, case-control studies, hygiene inspections and microbial analysis of food, environmental and stool samples were conducted to determine the aetiology of the outbreak and the modes of transmission. The only commonality was the food, crockery and cutlery provided and/or handled by the hotel's Chinese banquet kitchen. Stool specimens from 34 cases and 15 food handlers were positive for norovirus genogroup II. The putative index case was one of eight norovirus-positive food handlers who had worked while they were symptomatic. Several food samples and remnants tested positive forEscherichia colior high faecal coliforms, aerobic plate counts and/or total coliforms, indicating poor food hygiene. This large common-source outbreak of norovirus gastroenteritis was caused by the consumption of contaminated food and/or contact with contaminated crockery or cutlery provided or handled by the hotel's Chinese banquet kitchen.

Erythrocyte invasion by malaria parasites is essential for blood-stage development and an important determinant of host range. In Plasmodium vivax, the interaction between the Duffy binding protein (DBP) and its cognate receptor, the Duffy antigen receptor for chemokines (DARC), on human erythrocytes is central to blood-stage infection. Contrary to this established pathway of invasion, there is growing evidence of P. vivax infections occurring in Duffy blood group-negative individuals, suggesting that the parasite might have gained an alternative pathway to infect this group of individuals. Supporting this concept, a second distinct erythrocyte binding protein (EBP2), representing a new member of the DBP family, was discovered in P. vivax and may be the ligand in an alternate invasion pathway. Our study characterizes this novel ligand and determines its potential role in reticulocyte invasion by P. vivax merozoites. EBP2 binds preferentially to young (CD71high) Duffy-positive (Fy+) reticulocytes and has minimal binding capacity for Duffy-negative reticulocytes. Importantly, EBP2 is antigenically distinct from DBP and cannot be functionally inhibited by anti-DBP antibodies. Consequently, our results do not support EBP2 as a ligand for invasion of Duffy-negative blood cells, but instead, EBP2 may represent a novel ligand for an alternate invasion pathway of Duffy-positive reticulocytes., IMPORTANCE For decades, P. vivax infections in humans have been defined by a unique requirement for the interaction between the Duffy binding protein ligand of the parasite and the Duffy blood group antigen receptor (DARC). Recent reports of P. vivax infections in Duffy-negative individuals challenge this paradigm and suggest an alternate pathway of infection, potentially using the recently discovered EBP2. However, we demonstrate that EBP2 host cell specificity is more restricted than DBP binding and that EBP2 binds preferentially to Duffy-positive, young reticulocytes. This finding indicates that this DBP paralog does mediate a Duffy-independent pathway of infection.

The ability of the malaria parasite Plasmodium vivax to invade erythrocytes is dependent on the expression of the Duffy blood group antigen on erythrocytes. Consequently, Africans who are null for the Duffy antigen are not susceptible to P. vivax infections. Recently, P. vivax infections in Duffy-null Africans have been documented, raising the possibility that P. vivax, a virulent pathogen in other parts of the world, may expand malarial disease in Africa. P. vivax binds the Duffy blood group antigen through its Duffy-binding protein 1 (DBP1). To determine if mutations in DBP1 resulted in the ability of P. vivax to bind Duffy-null erythrocytes, we analyzed P. vivax parasites obtained from two Duffy-null individuals living in Ethiopia where Duffy-null and -positive Africans live side-by-side. We determined that, although the DBP1s from these parasites contained unique sequences, they failed to bind Duffy-null erythrocytes, indicating that mutations in DBP1 did not account for the ability of P. vivax to infect Duffy-null Africans. However, an unusual DNA expansion of DBP1 (three and eight copies) in the two Duffy-null P. vivax infections suggests that an expansion of DBP1 may have been selected to allow low-affinity binding to another receptor on Duffy-null erythrocytes. Indeed, we show that Salvador (Sal) I P. vivax infects Squirrel monkeys independently of DBP1 binding to Squirrel monkey erythrocytes. We conclude that P. vivax Sal I and perhaps P. vivax in Duffy-null patients may have adapted to use new ligand-receptor pairs for invasion.

Malaria remains a serious public health problem with significant morbidity and mortality accounting for nearly 20% of all childhood deaths in Africa. The cyclical invasion, cytoadherence and destruction of the host's erythrocyte by the parasite are responsible for the observed disease pathology. The invasive form of the parasite, the merozoite, uses a complex set of interactions between parasite ligands and erythrocyte receptors that leads to the formation of a tight junction and ultimately successful erythrocyte invasion. Understanding the molecular mechanism underlying host cell recognition and invasion is crucial for the development of a targeted intervention strategy. Two parasite protein families termed reticulocyte-binding-like protein homologues (RBL) and the erythrocyte-binding-like (EBL) protein family are conserved in all Plasmodium species and have been shown to play an important role in host cell recognition and invasion. Over the last few years significant new insights have been gained in understanding the function of the RBL family and this review attempts to provide an update with a specific focus on the role of RBL in signal transduction pathways during invasion.