Comparative evaluation of the <i>Schistosoma</i> PCR-ELISA system and the Kato-Katz technique for the diagnosis of <i>S. mansoni</i> infection.

<p>Comparative evaluation of the <i>Schistosoma</i> PCR-ELISA system and the Kato-Katz technique for the diagnosis of <i>S. mansoni</i> infection.</p

Analytical specificity of PCR followed by 6% polyacrylamide gel electrophoresis with silver staining and ELISA (optical density, OD) for purified DNA from adult <i>S. magrebowiei</i> (lane 2), <i>S. rhodaini</i> (lane 3), <i>S. japonicum</i> (lane 4), <i>S. intercalatum</i> (lane 5), <i>S. haematobium</i> (lane 6), <i>S. bovis</i> (lane 7) and <i>S. mansoni</i> (lane 8) worms.

<p>Lane 1, PCR negative control. The genus specificity of the <i>Schistosoma</i> PCR-ELISA system was demonstrated by the amplification of the 121-bp tandem repeat DNA sequence in all samples with the same set of primers. Absorbance readings were also consistent with the positive control (<i>S. mansoni</i> DNA).</p

Analytical sensitivity of PCR-ELISA system.

<p><b>A.</b> Analytical sensitivity of PCR followed by 6% polyacrylamide gel electrophoresis with silver staining and ELISA (optical density, OD) for 10-fold serial dilutions of genomic DNA extracted from a saline solution containing ∼2,000 <i>S. mansoni</i> eggs. The detection limit was 1.3 fg of genomic DNA. A ladder-type banding pattern was exhibited, as expected, due the amplification of the <i>Schistosoma</i> tandem-repeated unit, with the main DNA band of 110 bp present in all samples. <b>B.</b> The Pearson's correlation coefficient between PCR-ELISA OD and log[template DNA]+10 was 0.986 (<i>P</i><0.0001).</p

Correlation between log-transformed individual measurements of eggs per gram (epg) of feces and absorbance readings.

<p><b>A.</b> A Spearman's correlation coefficient of 0.616 (<i>P</i><0.0001) was found for a comparison considering all 206 samples. <b>B.</b> Considering positive and negative samples for the Kato-Katz technique and the <i>Schistosoma</i> PCR-ELISA system, a Spearman's correlation coefficient of 0.700 (<i>P</i><0.0001) was observed. The continuous line represents the linear regression, and the hatched line represents the 95% CI.</p

Results obtained with the <i>Schistosoma</i> PCR-ELISA system grouped into two levels of intensity of infection.

a<p>Intensity of infection was based on examination of one fecal sample by the Kato-Katz technique (12 slides - 500 mg feces).</p>b<p>Considering only concordant results between the Kato-Katz technique and the <i>Schistosoma</i> PCR-ELISA system; OD  =  optical density.</p

Low Parasite Load Estimated by qPCR in a Cohort of Children Living in Urban Area Endemic for Visceral Leishmaniasis in Brazil

<div><h3>Background</h3><p>An important issue associated with the control of visceral leishmaniasis is the need to identify and understand the relevance of asymptomatic infection caused by <em>Leishmania infantum</em>. The aim of this study was to follow the course of asymptomatic <em>L. infantum</em> infection in children in an area of Brazil where it is endemic. The children were assessed twice during a 12-month period.</p> <h3>Methodology</h3><p>In this population study, 1875 children, ranging from 6 months to 7 years of age, were assessed. Blood samples were collected on filter papers via finger prick and tested by ELISA (<em>L. infantum</em> soluble antigen and rk39). Seropositives samples (n = 317) and a number of seronegatives samples (n = 242) were subjected to qPCR. After 12 months, blood samples were collected from a subgroup of 199 children and tested for <em>Leishmania</em> spp. to follow the course of infection.</p> <h3>Principal Findings</h3><p>At baseline qPCR testing identified 82 positive samples. The prevalence rate, as estimated for 1875 children based on the qPCR results, was 13.9%. The qPCR testing of whole blood samples collected from a cohort of children after 12 months (n = 199) yielded the following results: of the 44 (22.1%) children with positive qPCR results at baseline, only 10 (5.0%) remained positive, and 34 (17.1%) became negative; and of the 155 (77.9%) children with negative qPCR results, 131 (65.8%) remained negative, and 24 (12.1%) became positive at the follow-up measurement. The samples with positive findings at baseline (n = 82) had a mean of 56.5 parasites/mL of blood; and at follow-up the mean positive result was 7.8 parasites/mL.</p> <h3>Conclusions</h3><p>The peripheral blood of asymptomatic children had a low and fluctuating quantity of <em>Leishmania</em> DNA and a significant decrease in parasitemia at 1-year follow-up. Quantitative PCR enables adequate monitoring of <em>Leishmania</em> infection.</p> </div

Parasite load of children remained positive tested by qPCR results at baseline and follow-up.

<p>The samples that tested positive at baseline had a mean concentration of 56.5±33.4 parasites/mL of blood. At follow-up, the mean parasitemia of children who remained positive was 7.8±7.0 parasites/mL (baseline, dark; follow-up, clear).</p

Agreement between serological and qPCR results for <i>L. infantum</i> infection in children (n = 559) living in the northwestern region of Belo Horizonte in 2010.

<p>Kappa<0.1.</p><p>Abbreviation: qPCR, quantitative polymerase chain reaction.</p

Diagnosis of asymptomatic infection in 199 subjects by qPCR at baseline and 12-month follow-up in Belo Horizonte, Minas Gerais (2009–2010).

<p>Abbreviations: follow-up; qPCR, quantitative polymerase chain reaction.</p

Estimate by qPCR of the prevalence of <i>L. infantum</i> infection in children living in the northwestern region of Belo Horizonte (n = 1875).

<p>Abbreviations: CI, confidence interval; qPCR, quantitative polymerase chain reaction.</p