Longitudinal assessment of K103N mutants frequency by allele-specific PCR during the intermittent off-therapy periods.

<p>w, week; Y, yes; N, no; *, K103N mutants also detected by direct sequencing; dashes, samples not available.</p

Correlation between K103N mutants quantified by allele-specific real-time PCR and ultra-deep pyrosequencing.

<p>A. Frequencies of K103N mutants. B. Number of K103N mutant copies The correlation was estimated by calculating Spearman's rank correlation coefficient for 16 samples successfully quantified by both methods for the AAC and/or AAT mutated codons.</p

K103N mutants in 11 patients quantified by allele-specific real-time PCR and ultra-deep pyrosequencing.

<p>The frequencies of K103N mutants are shown with corresponding absolute numbers of K103N log₁₀ copies per mL of plasma in brackets.</p><p>w, week; AAC and AAT codons encode the asparagine « N » at position 103; dashes, samples not available.</p

Sensitivity of the allele-specific PCR assay for detecting minor K103N mutants and reciprocal validation with ultra-deep pyrosequencing.

<p><b>A</b>. Plot of the measured frequencies of K103N mutants versus the input template, assessed on four independent experiments of mixtures of wild-type and K103N mutants at various frequencies. The inter-assay variability is shown by error bars representing the standard deviation. The assay detects K103N mutants down to a frequency of 0.01% in all experiments. <b>B</b>. Measured frequencies of K103N mutants on mixtures of wild-type and K103N mutants with known proportions of K103N. <b>C</b>. Correlation between measurements by the two methods.</p

Detection threshold of ultra-deep pyrosequencing for detecting minor K103N variants in function of the read number.

<p>The mean error rate of pyrosequencing at codon 103 was 0.0047 [CI99, 0.00085–0.00856]. The upper confidence limit of the error rate was used to calculate the sensitivity of pyrosequencing for a given number of reads. Poisson distribution was used to distinguish authentic variants from artefactual sequences resulting from errors arising during PCR amplification and ultra-deep pyrosequencing. Only those variants whose frequency of occurrence yielded a <i>P</i> value of <0.001 according to the Poisson model were considered authentic.</p

Higher efavirenz half-life in patients in whom K103N emerged than in whom it did not.

<p>The Wilcoxon rank sum test was used for the comparison.</p

Univariate or multivariate logistic regression analysis of baseline characteristics that significantly correlated with a false negative result of the PCR assay in the blood for the diagnosis of toxoplasmic encephalitis in patients with AIDS, French West Indies and Guiana, 2008–2011.

<p>Univariate or multivariate logistic regression analysis of baseline characteristics that significantly correlated with a false negative result of the PCR assay in the blood for the diagnosis of toxoplasmic encephalitis in patients with AIDS, French West Indies and Guiana, 2008–2011.</p

Demographic, laboratory and clinical baseline characteristics of all patients, of patients with a final diagnosis of TE, and of patients without a final diagnosis of TE, French West Indies and Guiana, 2008–2011.

<p>Demographic, laboratory and clinical baseline characteristics of all patients, of patients with a final diagnosis of TE, and of patients without a final diagnosis of TE, French West Indies and Guiana, 2008–2011.</p

Neighbor-joining clustering of <i>T</i>. <i>gondii</i> strains based on 15 microsatellite markers.

<p>Squares are for the type I (GT1), II (ME49), and III (VEG) reference strains; the yellow point is for the HTI01 strain isolated in the present study; black points (BRA01–10) are for strains collected in patients infected with imported strains whose epidemiological and genotyping investigation revealed that the origin of infection was likely Brazil; red points (AMZ 1–18) are for strains collected in patients with Amazonian toxoplasmosis who had been infected with wild strains from the rainforest of French Guiana; green points (GUF01–05) are for strains from toxoplasmosis cases diagnosed in babies with congenital toxoplasmosis or immunocompromised patients living in the anthropized areas of French Guiana; blue points are for strains collected in patients infected in Guadeloupe (GLP01–05) and Martinique (MTQ01–05), both in the French West Indies.</p

Genotyping analysis with 15 microsatellite markers of 3 reference type I, II, and III strains, the HTI01 strain isolated in the present study, and 43 strains collected in human cases of toxoplasmosis from South America and the Caribbean region by the French national reference center for toxoplasmosis.

<p>Genotyping analysis with 15 microsatellite markers of 3 reference type I, II, and III strains, the HTI01 strain isolated in the present study, and 43 strains collected in human cases of toxoplasmosis from South America and the Caribbean region by the French national reference center for toxoplasmosis.</p