Proof of concept: used malaria rapid diagnostic tests applied for parallel sequencing for surveillance of molecular markers of anti-malarial resistance in Bissau, Guinea-Bissau during 2014-2017.

BACKGROUND: Large-scale surveillance of molecular markers of anti-malarial drug resistance is an attractive method of resistance monitoring, to complement therapeutic efficacy studies in settings where the latter are logistically challenging. METHODS: Between 2014 and 2017, this study sampled malaria rapid diagnostic tests (RDTs), used in routine clinical care, from two health centres in Bissau, Guinea-Bissau. In order to obtain epidemiological insights, RDTs were collected together with patient data on age and sex. A subset of positive RDTs from one of the two sites (n = 2184) were tested for Plasmodium DNA content. Those testing positive for Plasmodium DNA by PCR (n = 1390) were used for library preparation, custom designed dual indexing and next generation Miseq targeted sequencing of Plasmodium falciparum genes pfcrt, pfmdr1, pfdhfr, pfdhps and pfk13. RESULTS: The study found a high frequency of the pfmdr1 codon 86N at 88-97%, a significant decrease of the pfcrt wildtype CVMNK haplotype and elevated levels of the pfdhfr/pfdhps quadruple mutant ranging from 33 to 51% between 2014 and 2017. No polymorphisms indicating artemisinin tolerance were discovered. The demographic data indicate a large proportion of young adults (66%, interquartile range 11-28 years) presenting with P. falciparum infections. While a total of 5532 gene fragments were successfully analysed on a single Illumina Miseq flow cell, PCR-positivity from the library preparation varied considerably from 13 to 87% for different amplicons. Furthermore, pre-screening of samples for Plasmodium DNA content proved necessary prior to library preparation. CONCLUSIONS: This study serves as a proof of concept for using leftover clinical material (used RDTs) for large-scale molecular surveillance, encompassing the inherent complications regarding to methodology and analysis when doing so. Factors such as RDT storage prior to DNA extraction and parasitaemia of the infection are likely to have an effect on whether or not parasite DNA can be successfully analysed, and are considered part of the reason the data yield is suboptimal. However, given the necessity of molecular surveillance of anti-malarial resistance in settings where poor infrastructure, poor economy, lack of educated staff and even surges of political instability remain major obstacles to performing clinical studies, obtaining the necessary data from used RDTs, despite suboptimal output, becomes a feasible, affordable and hence a justifiable method.

Evaluation and assessment of clique arrangements for the estimation of omnipolar electrograms in high density electrode arrays: an experimental animal model study.

High-density catheters combined with Orientation Independent Sensing (OIS) methods have emerged as a groundbreaking technology for cardiac substrate characterisation. In this study, we aim to assess the arrangements and constraints to reliably estimate the so-called omnipolar electrogram (oEGM). Performance was evaluated using an experimental animal model. Thirty-eight recordings from nine retrospective experiments on isolated perfused rabbit hearts with an epicardial HD multielectrode were used. We estimated oEGMs according to the classic triangular clique (4 possible orientations) and a novel cross-orientation clique arrangement. Furthermore, we tested the effects of interelectrode spacing from 1 to 4 mm. Performance was evaluated by means of several parameters that measured amplitude rejection ratios, electric field loop area, activation pulse width and morphology distortion. Most reliable oEGM estimations were obtained with cross-configurations and interelectrode spacings [Formula: see text] mm. Estimations from triangular cliques resulted in wider electric field loops and unreliable detection of the direction of the propagation wavefront. Moreover, increasing interelectrode distance resulted in increased pulse width and morphology distortion. The results prove that current oEGM estimation techniques are insufficiently accurate. This study opens a new standpoint for the design of new-generation HD catheters and mapping software.

Modeling Metastatic Colonization in a Decellularized Organ Scaffold-Based Perfusion Bioreactor.

Metastatic cancer spread is responsible for most cancer-related deaths. To colonize a new organ, invading cells adapt to, and remodel, the local extracellular matrix (ECM), a network of proteins and proteoglycans underpinning all tissues, and a critical regulator of homeostasis and disease. However, there is a major lack in tools to study cancer cell behavior within native 3D ECM. Here, an in-house designed bioreactor, where mouse organ ECM scaffolds are perfused and populated with cells that are challenged to colonize it, is presented. Using a specialized bioreactor chamber, it is possible to monitor cell behavior microscopically (e.g., proliferation, migration) within the organ scaffold. Cancer cells in this system recapitulate cell signaling observed in vivo and remodel complex native ECM. Moreover, the bioreactors are compatible with co-culturing cell types of different genetic origin comprising the normal and tumor microenvironment. This degree of experimental flexibility in an organ-specific and 3D context, opens new possibilities to study cell-cell and cell-ECM interplay and to model diseases in a controllable organ-specific system ex vivo.

Lewis x is highly expressed in normal tissues: a comparative immunohistochemical study and literature revision.

An immunohistochemical analysis was employed to determine the expression of carbohydrate antigens associated to mucins in normal epithelia. Tissue samples were obtained as biopsies from normal breast (18), colon (35) and oral cavity mucosa (8). The following carbohydrate epitopes were studied: sialyl-Lewis x, Lewis x, Lewis y, Tn hapten, sialyl-Tn and Thomsen-Friedenreich antigen. Mucins were also studied employing antibodies against MUC1, MUC2, MUC4, MUC5AC, MUC6 and also normal colonic glycolipid. Statistical analysis was performed and Kendall correlations were obtained. Lewis x showed an apical pattern mainly at plasma membrane, although cytoplasmic staining was also found in most samples. TF, Tn and sTn haptens were detected in few specimens, while sLewis x was found in oral mucosa and breast tissue. Also, normal breast expressed MUC1 at a high percentage, whereas MUC4 was observed in a small number of samples. Colon specimens mainly expressed MUC2 and MUC1, while most oral mucosa samples expressed MUC4 and MUC1. A positive correlation between MUC1VNTR and TF epitope (r=0.396) was found in breast samples, while in colon specimens MUC2 and colonic glycolipid versus Lewis x were statistically significantly correlated (r=0.28 and r=0.29, respectively). As a conclusion, a defined carbohydrate epitope expression is not exclusive of normal tissue or a determined localization, and it is possible to assume that different glycoproteins and glycolipids may be carriers of carbohydrate antigens depending on the tissue localization considered.

High throughput resistance profiling of Plasmodium falciparum infections based on custom dual indexing and Illumina next generation sequencing-technology.

Genetic polymorphisms in P. falciparum can be used to indicate the parasite's susceptibility to antimalarial drugs as well as its geographical origin. Both of these factors are key to monitoring development and spread of antimalarial drug resistance. In this study, we combine multiplex PCR, custom designed dual indexing and Miseq sequencing for high throughput SNP-profiling of 457 malaria infections from Guinea-Bissau, at the cost of 10 USD per sample. By amplifying and sequencing 15 genetic fragments, we cover 20 resistance-conferring SNPs occurring in pfcrt, pfmdr1, pfdhfr, pfdhps, as well as the entire length of pfK13, and the mitochondrial barcode for parasite origin. SNPs of interest were sequenced with an average depth of 2,043 reads, and bases were called for the various SNP-positions with a p-value below 0.05, for 89.8-100% of samples. The SNP data indicates that artemisinin resistance-conferring SNPs in pfK13 are absent from the studied area of Guinea-Bissau, while the pfmdr1 86 N allele is found at a high prevalence. The mitochondrial barcodes are unanimous and accommodate a West African origin of the parasites. With this method, very reliable high throughput surveillance of antimalarial drug resistance becomes more affordable than ever before.

The use of juvenile Solea solea as sentinel in the marine platform of the Ebre Delta: in vitro interaction of emerging contaminants with the liver detoxification system.

Juveniles of Solea solea were sampled during the spring season in three consecutive years at a marine site by the mouth of the Ebre river. The aim was to assess if the extractive works from the toxic load upstream the river could be reflected on the health status of the fish living at the immediate sea. The biomarkers selected for the in vivo field study are commonly used as indicators of chemical exposures. They include activities of energy metabolism: lactate dehydrogenase (LDH) and citrate synthase (CS); neurotoxicity: cholinesterases (ChE); xenobiotic metabolism: cytochrome P450 (CYP)-dependent: EROD and BFCOD, carboxylesterase (CbE), glutathione S-transferase (GST) and uridine diphosphate glucuronyltransferase (UDPGT); and oxidative stress parameters such as catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPX) as well as levels of lipid peroxidation (LPO). These biomarkers were mostly analysed in liver but also in gills and muscle depending on their particular tissue distribution and role. A complementary in vitro approach was also sought to see the capacity of common emerging contaminants (pharmaceuticals and personal care products; PPCPs) to interact with the liver microsomal detoxification system of the fish (EROD, BFCOD and CbE activities). The results indicated that in fish sampled in 2015 there was an enhancement in detoxification parameters (EROD, BFCOD and gill GR), muscular ChEs and gill CS, but a decrease in CbE activity and a marked oxidative stress situation (increased LPO and decreased CAT activity). Also, 4 out of the 10 PPCPs tested in vitro were able to interact with the CYP3A4 (BFCOD) enzymatic system while the lipid regulators simvastatin and fenofibrate inhibited CbE activity, as it occurs in higher vertebrates. The in vivo results support the use of a multibiomarker approach when assessing the disturbances due to chemical exposures, not only spatially but also over time, once the influence of other variables has been taken into consideration. The in vitro results highlight the importance of the CYP3A4 and CbE pathway in pharmaceutical metabolism, also in fish.

Breast cancer humoral immune response: involvement of Lewis y through the detection of circulating immune complexes and association with Mucin 1 (MUC1).

BACKGROUND: In cancer patients, MUC1 glycoprotein may carry Lewis y which could be involved in immune response. PURPOSES: 1- to evaluate the presence of Lewis y and MUC1 in circulating immune complexes (Lewis y/CIC and MUC1/CIC, respectively) and their correlation; 2- to analyze the possible presence of Lewis y in carbohydrate chains of tumoral MUC1 glycoprotein and 3- to correlate serum and tissue parameters considered. METHODS: Pretreatment serum and tissue breast samples from 76 adenocarcinoma, 34 benign and 36 normal specimens were analyzed. Anti-MUC1 and anti-Lewis y MAbs were employed. To detect Lewis y/CIC and MUC1/CIC, ELISA tests were developed; serum samples containing MUC1 were previously selected by Cancer Associated Serum Antigen (CASA). Immunoprecipitation (IP) was performed in 9 malignant, benign and normal samples and analyzed by SDS-PAGE and Western blot. Lewis y and MUC1 expression was studied by immunohistochemistry (IHC). Statistical analysis was performed employing principal component analysis (PCA), ANOVA, Tukey HSD, Chi square test and classical correlation (p < 0.05). RESULTS: By ELISA, Lewis y/IgM/CIC levels showed statistically significant differences between breast cancer versus benign and normal samples; mean +/- SD values expressed in OD units were: 0.525 +/- 0.304; 0.968 +/- 0.482 and 0.928 +/- 0.447, for breast cancer, benign disease and normal samples, respectively, p < 0.05. Lewis y/IgG/CIC did not show any statistically significant difference. MUC1/IgM/CIC correlated with Lewis y/IgM/CIC. By CASA, 9 samples with MUC1 values above the cut off were selected and IP was performed, followed by SDS-PAGE and Western blot; bands at 200 kDa were obtained with each MAb in all the samples. By IHC, with C14 MAb, 47.5%, 31% and 35% of malignant, benign and normal samples, respectively, showed positive reaction while all the samples were positive with anti-MUC1 MAb; in both cases, with a different pattern of expression between malignant and non malignant samples. CONCLUSION: Our findings support that in breast cancer there was a limited humoral immune response through Lewis y/IgM/CIC levels detection which correlated with MUC1/IgM/CIC. We also found that Lewis y might be part of circulating MUC1 glycoform structure and also that Lewis y/CIC did not correlate with Lewis y expression.