Assays to Detect β-Tubulin Codon 200 Polymorphism in Trichuris trichiura and Ascaris lumbricoides

The soil-transmitted helminths Ascaris lumbricoides and Trichuris trichiura are gastrointestinal nematodes causing many disabilities in tropical parts of the developing world. Control programs, such as “The Focussing Resources on Effective School Health” (FRESH) Partnership, have been implemented to remove human soil-transmitted nematodes through large-scale use of benzimidazole anthelmintic drugs for school-aged children in developing countries. The benzimidazole drugs albendazole and mebendazole are commonly used as a single annual treatment in areas where the burden is high. In veterinary nematodes, repeated use of these anthelmintics has selected for resistant populations. Resistance to benzimidazoles is commonly associated with a single amino acid substitution from phenylalanine to tyrosine in the β-tubulin gene at position 200. In this study, we have developed pyrosequencing assays for codon 200 in A. lumbricoides and T. trichiura to screen for this single nucleotide polymorphism (SNP) in β-tubulin. The 200Tyr SNP was detected at low frequency in T. trichiura from non-treated people from Kenya and at high frequency in T. trichiura from treated people from Panama. The presence of the resistance-associated SNP may play a role in the sometimes low and variable efficacy of benzimidazole anthelmintics against T. trichiura

Prediction of child health by household density and asset-based indices in impoverished indigenous villages in rural Panamá

Chronic infection over a 16-month period and stunting of preschool children were compared between more spatially dense versus dispersed households in rural Panama´ . Chronic protozoan infection was associated with higher household density, lower household wealth index, poor household water quality, yard defecation, and the practice of not washing hands with soap before eating. Models for chronic diarrhea confirmed the importance of household wealth, water quality, sanitation, and hygiene practices. Furthermore, chronic protozoan infection was an important predictor for low height-for-age, along with low household wealth index scores, but not household density. Thus, despite better access to health related infrastructure in the more densely populated households, chronic protozoan infection was more common, and was associated with higher rates of child stunting, compared with more dispersed households

Regional, household and individual factors that influence soil transmitted helminth reinfection dynamics in preschool children from rural indigenous Panamá.

BackgroundFew studies have investigated the relative influence of individual susceptibility versus household exposure factors versus regional clustering of infection on soil transmitted helminth (STH) transmission. The present study examined reinfection dynamics and spatial clustering of Ascaris lumbricoides, Trichuris trichiura and hookworm in an extremely impoverished indigenous setting in rural Panamá over a 16 month period that included two treatment and reinfection cycles in preschool children.Methodology/principle findingsSpatial cluster analyses were used to identify high prevalence clusters for each nematode. Multivariate models were then used (1) to identify factors that differentiated households within and outside the cluster, and (2) to examine the relative contribution of regional (presence in a high prevalence cluster), household (household density, asset-based household wealth, household crowding, maternal education) and individual (age, sex, pre-treatment eggs per gram (epg) feces, height-for-age, latrine use) factors on preschool child reinfection epgs for each STH. High prevalence spatial clusters were detected for Trichuris and hookworm but not for Ascaris. These clusters were characterized by low household density and low household wealth indices (HWI). Reinfection epg of both hookworm and Ascaris was positively associated with pre-treatment epg and was higher in stunted children. Additional individual (latrine use) as well as household variables (HWI, maternal education) entered the reinfection models for Ascaris but not for hookworm.Conclusions/significanceEven within the context of extreme poverty in this remote rural setting, the distinct transmission patterns for hookworm, Trichuris and Ascaris highlight the need for multi-pronged intervention strategies. In addition to poverty reduction, improved sanitation and attention to chronic malnutrition will be key to reducing Ascaris and hookworm transmission

Comparison of regional, household and individual factors influencing STH reinfection of Panamanian preschool children¹.

1<p>Summary statistics presented are mean ±SEM or % (95% CL).</p>2<p>Asset based index, weights derived from the first component of Principle Components Analysis.</p>*<p>p<0.05;</p>**<p>p<0.001.</p

Final multiple logistic regression models predicting household presence within Cycle 2 high prevalence clusters¹^,².

1<p>Odds ratios ±95% CI.</p>2<p>Latrine access and maternal education did not enter any model.</p>3<p>Asset based index, weights derived from the first component of Principle Components Analysis.</p>4<p>NE = Variable was excluded during the stepwise process (p>0.10).</p

Sample sizes for two STH reinfection cycles among Panamanian preschool children.

<p>Reinfection epg sample sizes only include individuals who also received treatment. Cycle 1 Baseline, 2 wk and 3 mo epg estimates were calculated using Kato Katz methodology. Cycle 1 9 mo reinfection epg and all Cycle 2 epg estimates were calculated using FLOTAC.</p

Prevalence and intensity for <i>Ascaris</i>, hookworm and <i>Trichuris</i> in Panamanian preschool children.

<p>Prevalence (A, C, E) and intensity (B, D, F) for <i>Ascaris</i> (A,B), hookworm (C,D) and <i>Trichuris</i> (E,F) were assessed by Kato Katz (dark grey bar – left vertical axis) and FLOTAC (light grey bar – right vertical axis) during two reinfection cycles. Different letters indicate significant differences (P<0.05) among sample periods (lower case Kato Katz comparisons; upper case FLOTAC comparisons). Solid vertical bar separates Kato Katz from FLOTAC data. A single dose of Albendazole (200 mg: 1–2 yrs, 400 mg: 3–5 yrs) was delivered after the baseline samples in each cycle and children who remained infected after treatment in Cycle 2 received a second dose. Data presented consider each cycle independently resulting in a different sample group for the 9 mo reinfection point of Cycle 1 and the baseline of Cycle 2. Each data point represents the population estimates based on all available fecal samples at each time point for children who received treatment at baseline of the respective cycle.</p

Faecal egg count reduction rates for <i>A. lumbricoides</i>, <i>T. trichiura</i> and hookworms.

1<p>FEC, faecal egg count, (egg per gram of stool).</p>2<p>CI, confidence interval. The confidence intervals of the egg reduction rate were calculated using a bootstrap resampling method <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002247#pntd.0002247-Efron1" target="_blank">[45]</a>.</p>3<p>ERR, egg reduction rate. The ERR estimates were obtained by dividing the difference between the arithmetic mean of the pre- and post-treatment FEC (at group level, ignoring individual variability) by the mean of the pre-treatment mean count (see text).</p>4<p>NA = Not applicable.</p>5<p>HW = Hookworm.</p

Genotype frequencies of the β-tubulin gene position 200 in <i>T. trichiura</i> from Haiti and Kenya.

<p>Genotype frequencies of <i>T. trichura</i> collected in Haiti (A) and in Kenya (B); Number of individual <i>T. trichiura</i> eggs genotyped according to the available material, in Haiti was 65 in the untreated group (pre-Tx) and 38 in the treated group (post-Tx), in Kenya was 40 in the untreated group and 90 in the treated group. Sequences were diploid, TT indicates the homozygous susceptible-type TTC/TTC, TA the heterozygous TTC/TAC and AA, the homozygous resistance-type TAC/TAC; Tx = treatment, *Indicates a significant difference (p<0.001) in genotype frequency between the pre- and post treatment groups. P-values were obtained by Fisher's exact test.</p