Laboratory development of a simple stool sample processing method diagnosis of pediatric tuberculosis using Xpert Ultra

A disadvantage of nonradioactive microsphere techniques is that the processing of samples is time-consuming and complex. We developed and validated a simplified processing method for the fluorescent microsphere (FM) technique. In seven anesthetized dogs with coronary artery stenosis up to six different FM and five different radioactivity labeled microspheres (RM) were injected. Two FM and two RM labels were injected simultaneously to enable inter- and intramethod comparison. After gamma-counting samples of blood, myocardium (n = 168), and other organs (n = 59) were digested in test tubes with 2 N ethanolic KOH (60 degrees C, 48 h), microspheres were sedimented by centrifugation, dye was extracted in the same tube, and fluorescence was measured. With this processing method, recovery of FM was approximately 100%. Good correlations for inter- and intramethod comparisons were found [r = 0.985 +/- 0.01 (mean +/- SD)]. The lower intermethod correlation for blue microspheres (r = 0.958) indicates that the use of this label is less desirable. RM and FM endocardial-to-epicardial blood flow ratios correlated well (r = 0.974). With this one-vessel centrifugal sedimentation method and at least five fluorescently labeled microspheres, blood flow can be reliably measured in various organs, including ischemic myocardium.

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

Parasites & Vectors ◽

10.1186/s13071-021-04941-w ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Javier Gandasegui ◽

Berta Grau-Pujol ◽

María Cambra-Pelleja ◽

Valdemiro Escola ◽

Maria Antonietta Demontis ◽

...

Keyword(s):

Pore Size ◽

Stool Sample ◽

Large Scale ◽

Trichuris Trichiura ◽

Sample Processing ◽

Tubulin Gene ◽

Necator Americanus ◽

Benzimidazole Resistance ◽

Extensive Evaluation ◽

Β Tubulin

Abstract 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. Graphical Abstract

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Fenitrothion and esfenvalerate stability during corn and wheat sample processing

Scientia Agricola ◽

10.1590/s0103-90162008000200008 ◽

2008 ◽

Vol 65 (2) ◽

pp. 157-160

Author(s):

Javier Alberto Vásquez-Castro ◽

Gilberto Casadei de Baptista ◽

Luiz Roberto Pimentel Trevizan ◽

Casimiro Dias Gadanha Junior

Keyword(s):

Gas Chromatography ◽

Large Scale ◽

Processing Method ◽

Stored Grain ◽

Sample Processing ◽

Wheat Sample ◽

Dry Ice ◽

Insecticide Residues ◽

Stored Grains ◽

The Stability

The presence of insecticide residues in cereals represents a risk for the consumer, because these substances are used in large scale to protect stored grains from the attack of pests. The effects of three processing methods for corn and wheat samples on the stability of stored-grain protective insecticides were here evaluated. Fenitrothion and esfenvalerate were applied so as to produce theoretical concentrations of 10 and 0.5 mg kg-1, respectively. Two hours after treatment, the grains were processed and deposition was analyzed by gas chromatography. Grain species did not influence insecticide stability. This stability was only dependent upon the processing method and insecticide type. Grains processed together with dry ice provided the greatest percentage of recovery for both insecticides. Regardless of the processing method, more esfenvalerate than fenitrothion was recovered, thus demonstrating the greater stability of the pyrethroid during this operation.

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Comparison of Sample Preparation Methods for Recovering Salmonella from Raw Fruits, Vegetables, and Herbs

Journal of Food Protection ◽

10.4315/0362-028x-64.10.1459 ◽

2001 ◽

Vol 64 (10) ◽

pp. 1459-1465 ◽

Cited By ~ 40

Author(s):

ANDREA B. BURNETT ◽

LARRY R. BEUCHAT

Keyword(s):

Sample Preparation ◽

Pathogenic Bacteria ◽

Preparation Method ◽

Morphological Characteristics ◽

Processing Method ◽

Sample Processing ◽

Processing Methods ◽

Preparation Methods ◽

Wide Range ◽

The Mean

Methods for preparing raw fruits, vegetables, and herbs for enrichment or direct plating to determine the presence and populations of pathogenic bacteria vary greatly. A study was done to compare three sample processing methods (washing in 0.1% peptone, stomaching, and homogenizing) for their influence on recovery of Salmonella inoculated onto 26 types of raw produce. The mean numbers of Salmonella recovered from 10 fruits, 11 vegetables, and 5 herbs using all three processing methods were 7.17, 7.40, and 7.27 log10 CFU/sample, respectively. Considering all 26 types of produce and all processing methods, the number of Salmonella recovered ranged from 7.24 to 7.29 log10 CFU/sample, with no significant differences attributable to a particular sample processing method. Mean percent recoveries of Salmonella from washed, stomached, and homogenized produce were 39.4, 44.7, and 42.4%, respectively. Mean percent recoveries from fruits, vegetables, and herbs, regardless of sample preparation method, were 41.7, 50.1, and 25.9%, respectively. The number of Salmonella recovered from stomached and homogenized produce, but not washed produce, with pH ≤ 4.53 was significantly less than the number recovered from produce with pH from 5.53 to 5.99, suggesting that the acidic environment in stomachates and homogenates was lethal to a portion of Salmonella. Reduced percent recoveries from herbs (pH 5.94 to 6.34) is attributed, in part, to antimicrobials released from plant cells during sample preparation. Overall, the type of processing method did not substantially affect the number of Salmonella recovered from the 26 types of raw produce representing a wide range of structural and morphological characteristics, composition, and pH. The influence of sample size, diluent composition, and processing time on efficiency of recovery of Salmonella and other pathogens needs to be evaluated before a method(s) for processing samples of raw produce can be recommended.

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