Improving stool sample processing and pyrosequencing for quantifying benzimidazole resistance alleles in Trichuris trichiura and Necator americanus pooled eggs

Background: There is an urgent need for an extensive evaluation of benzimidazole efcacy in humans. In veterinary science, benzimidazole resistance has been mainly associated with three single-nucleotide polymorphisms (SNPs) in the isotype-1 β-tubulin gene. In this study, we optimized the stool sample processing methodology and resistance allele frequency assessment in Trichuris trichiura and Necator americanus anthelmintic-related SNPs by pyrosequenc‑ ing, and standardized it for large-scale benzimidazole efcacy screening use. Methods: Three diferent protocols for stool sample processing were compared in 19 T. trichiura-positive samples: fresh stool, egg concentration using metallic sieves with decreasing pore size, and egg concentration followed by fotation with saturated salt solution. Yield of each protocol was assessed by estimating the load of parasite DNA by real-time PCR. Then, we sequenced a DNA fragment of the β-tubulin gene containing the putative benzimidazole resistance SNPs in T. trichiura and N. americanus. Afterwards, resistant and susceptible-type plasmids were produced and mixed at diferent proportions, simulating diferent resistance levels. These mixtures were used to compare previ‑ ously described pyrosequencing assays with processes newly designed by our own group. Once the stool sample processing and the pyrosequencing methodology was defned, the utility of the protocols was assessed by measur‑ ing the frequencies of putative resistance SNPs in 15 T. trichiura- and 15 N. americanus-positive stool samples. Results: The highest DNA load was provided by egg concentration using metallic sieves with decreasing pore size. Sequencing information of the β-tubulin gene in Mozambican specimens was highly similar to the sequences previ‑ ously reported, for T. trichiura and N. americanus, despite the origin of the sample. When we compared pyrosequenc‑ ing assays using plasmids constructs, primers designed in this study provided the most accurate SNP frequencies. When pooled egg samples were analysed, none of resistant SNPs were observed in T. trichiura, whereas 17% of the resistant SNPs at codon 198 were found in one N. americanus sample

Long Terminal Repeat Circular DNA as Markers of Active Viral Replication of Human T Lymphotropic Virus-1 in Vivo

Abstract: Clonal expansion of human T-lymphotropic virus type-1 (HTLV-1) infected cells in vivo is well documented. Unlike human immunodeficiency virus type 1 (HIV-1), HTLV-1 plasma RNA is sparse. The contribution of the “mitotic” spread of HTLV-1 compared with infectious spread of the virus to HTLV-1 viral burden in established infection is uncertain. Since extrachromosomal long terminal repeat (LTR) DNA circles are indicators of viral replication in HIV-1 carriers with undetectable plasma HIV RNA, we hypothesised that HTLV-1 LTR circles could indicate reverse transcriptase (RT) usage and infectious activity. 1LTR and 2LTR DNA circles were measured in HTLV-1 cell lines and peripheral blood mononuclear cells (PBMC) of asymptomatic carriers (ACs) and patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T cell leukaemia/lymphoma (ATLL). 1LTR DNA circles were detected in 14/20 patients at a mean of 1.38/100 PBMC but did not differentiate disease status nor correlate with HTLV-1 DNA copies. 2LTR DNA circles were detected in 30/31 patients and at higher concentrations in patients with HTLV-1-associated diseases, independent of HTLV-1 DNA load. In an incident case the 2LTR DNA circle concentration increased 2.1 fold at the onset of HAM/TSP compared to baseline. Detectable and fluctuating levels of HTLV-1 DNA circles in patients indicate viral RT usage and virus replication. Our results indicate HTLV-1 viral replication capacity is maintained in chronic infection and may be associated with disease onset

Rapid dissemination of human T-lymphotropic virus type 1 during primary infection in transplant recipients

Abstract Background: Human T-lymphotropic virus type 1 (HTLV-1) infects an estimated 10 million persons globally with transmission resulting in lifelong infection. Disease, linked to high proviral load, occurs in a minority. In established infection HTLV-1 replicates through infectious spread and clonal expansion of infected lymphocytes. Little is known about acute HTLV-1 infection. The kinetics of early HTLV-1 infection, following transplantation-acquired infection in three recipients from one HTLV-1 infected donor, is reported. The recipients were treated with two HTLV-1 enzyme inhibitors 3 weeks post exposure following the detection of HTLV-1 provirus at low level in each recipient. HTLV-1 infection was serially monitored by serology, quantification of proviral load and HTLV-1 2LTR DNA circles and by HTLV-1 unique integration site analysis. Results: HTLV-1 antibodies were first detected 16–39 days post-transplantation. HTLV-1 provirus was detected by PCR on day 16–23 and increased by 2–3 log by day 38–45 with a peak proviral doubling time of 1.4 days, after which steady state was reached. The rapid proviral load expansion was associated with high frequency of HTLV-1 2LTR DNA circles. The number of HTLV-1 unique integration sites was high compared with established HTLV-1 infection. Clonal expansion of infected cells was detected as early as day 37 with high initial oligoclonality index, consistent with early mitotic proliferation. Conclusions: In recipients infected through organ transplantation HTLV-1 disseminated rapidly despite early antiHTLV-1 treatment. Proviral load set point was reached within 6 weeks. Seroconversion was not delayed. Unique integration site analysis and HTLV-1 2LTR DNA circles indicated early clonal expansion and high rate of infectious spread

Improving stool sample processing and pyrosequencing for quantifying benzimidazole resistance alleles in Trichuris trichiura and Necator americanus pooled eggs.

BACKGROUND: There is an urgent need for an extensive evaluation of benzimidazole efficacy in humans. In veterinary science, benzimidazole resistance has been mainly associated with three single-nucleotide polymorphisms (SNPs) in the isotype-1 β-tubulin gene. In this study, we optimized the stool sample processing methodology and resistance allele frequency assessment in Trichuris trichiura and Necator americanus anthelmintic-related SNPs by pyrosequencing, and standardized it for large-scale benzimidazole efficacy screening use. METHODS: Three different protocols for stool sample processing were compared in 19 T. trichiura-positive samples: fresh stool, egg concentration using metallic sieves with decreasing pore size, and egg concentration followed by flotation with saturated salt solution. Yield of each protocol was assessed by estimating the load of parasite DNA by real-time PCR. Then, we sequenced a DNA fragment of the β-tubulin gene containing the putative benzimidazole resistance SNPs in T. trichiura and N. americanus. Afterwards, resistant and susceptible-type plasmids were produced and mixed at different proportions, simulating different resistance levels. These mixtures were used to compare previously described pyrosequencing assays with processes newly designed by our own group. Once the stool sample processing and the pyrosequencing methodology was defined, the utility of the protocols was assessed by measuring the frequencies of putative resistance SNPs in 15 T. trichiura- and 15 N. americanus-positive stool samples. RESULTS: The highest DNA load was provided by egg concentration using metallic sieves with decreasing pore size. Sequencing information of the β-tubulin gene in Mozambican specimens was highly similar to the sequences previously reported, for T. trichiura and N. americanus, despite the origin of the sample. When we compared pyrosequencing assays using plasmids constructs, primers designed in this study provided the most accurate SNP frequencies. When pooled egg samples were analysed, none of resistant SNPs were observed in T. trichiura, whereas 17% of the resistant SNPs at codon 198 were found in one N. americanus sample. CONCLUSIONS: We optimized the sample processing methodology and standardized pyrosequencing in soil-transmitted helminth (STH) pooled eggs. These protocols could be used in STH large-scale screenings or anthelmintic efficacy trials

An LC—MS—MS method for quantitation of four new phenethylamines (BOX series) in plasma: in vivo application

The appearance of new “designer drugs” in the illicit market poses a serious health risk because they have unknown safety profiles, have a high potential for abuse, high potency, and can lead to devastating health consequences. For this reason, it is desirable to develop validated and reliable analytical screening tests that allow detection of amphetamines and related designer drugs in biological samples. We report a method for separation and quantitation of four new phenethylamines, 4-bromo-2,5-beta-trimethoxyphenethylamine (BOB), 4-methyl-2,5-beta-trimethoxyphenethylamine (BOD), 3,4-methylenedioxy-beta-methoxyphenethylamine (BOH), and 4-methyl-2,5-dimethoxy-beta-hydroxyphenethylamine (BOHD), in plasma. Quantitation was achieved via liquid chromatography—tandem mass spectrometry (LC—MS—MS) in the multiple reaction monitoring mode, using 2,3-dimethoxyphenethylamine-d 3 as internal standard. The method was validated according to international guidelines. The parameters determined were selectivity, sensitivity, matrix effect, linearity, precision, recovery, and stability. All parameters were satisfactory. To remove matrix interference, solid-phase extraction was introduced in the method as clean-up step. The same method was applied in a pharmacokinetic study to monitor the target compounds in rat plasma after a single oral administration. The developed and validated LC—MS—MS method is the first available for quantitation of BOB, BOH, BOD, and BOHD in a biological matrix. This method is recommended for use in forensic and clinical toxicology, because of its sensitivity, selectivity, and simplicity. An important extension of this method could involve its application to other complex matrices

FLO11-based model for air-liquid interfacial biofilm formation by <i>Saccharomyces cerevisiae</i>

Sardinian wine strains of Saccharomyces cerevisiae used to make sherry-like wines form a biofilm at the air-liquid interface at the end of ethanolic fermentation, when grape sugar is depleted and further growth becomes dependent on access to oxygen. Here, we show that FLO11, which encodes a hydrophobic cell wall glycoprotein, is required for the air-liquid interfacial biofilm and that biofilm cells have a buoyant density greater than the suspending medium. We propose a model for biofilm formation based on an increase in cell surface hydrophobicity occurring at the diauxic shift. This increase leads to formation of multicellular aggregates that effectively entrap carbon dioxide, providing buoyancy. A visible biofilm appears when a sufficient number of hydrophobic cell aggregates are carried to and grow on the liquid surface

Network and classification tree analysis of plasma cytokine concentration in non-ATL HTLV-1 infection and ATL.

<p>Network analysis of absolute T-cell subset count, HTLV-1 PVL and plasma cytokine concentration using at least significant Spearman correlation trends is shown for patients with ATL (A), HAM (B) and AC (C). The green and red lines denote positive and negative correlations respectively. The continuous and broken line denote statistically significant and trend correlations. The prune classification tree to classify diagnosis of AC, HAM and ATL on the basis of IL-10 and IL-17 concentration is shown in figure D. The percentage shows the distribution of all patients into different arms whilst the three decimal numbers shown specificity of each classification for diagnosis of AC, ATL and HAM respectively.</p

T-cell subsets and proviral load in AC, HAM and ATL.

<p>T-cell subsets and proviral load in AC, HAM and ATL.</p