Single-Nucleotide Polymorphisms in the Beta-Tubulin Gene and Its Relationship with Treatment Response to Albendazole in Human Soil-Transmitted Helminths in Southern Mozambique.

Soil-transmitted helminth (STH) cornerstone control strategy is mass drug administration (MDA) with benzimidazoles. However, MDA might contribute to selection pressure for anthelmintic resistance, as occurred in livestock. The aim of this study is to evaluate the treatment response to albendazole and the relationship with the presence of putative benzimidazole resistance single-nucleotide polymorphisms (SNPs) in the ß-tubulin gene of STH in Southern Mozambique. After screening 819 participants, we conducted a cohort study with 184 participants infected with STH in Manhiça district, Southern Mozambique. A pretreatment and a posttreatment stool samples were collected and the STH infection was identified by duplicate Kato-Katz and quantitative polymerase chain reaction (qPCR). Cure rate and egg reduction rates were calculated. Putative benzimidazole resistance SNPs (F167Y, F200T, and E198A) in Trichuris trichiura and Necator americanus were assessed by pyrosequencing. Cure rates by duplicate Kato-Katz and by qPCR were 95.8% and 93.6% for Ascaris lumbricoides, 28% and 7.8% for T. trichiura, and 88.9% and 56.7% for N. americanus. Egg reduction rate by duplicate Kato-Katz was 85.4% for A. lumbricoides, 34.9% for T. trichiura, and 40.5% for N. americanus. Putative benzimidazole resistance SNPs in the ß-tubulin gene were detected in T. trichiura (23%) and N. americanus (21%) infected participants at pretreatment. No statistical difference was observed between pretreatment and posttreatment frequencies for none of the SNPs. Although treatment response to albendazole was low, particularly in T. trichiura, the putative benzimidazole resistance SNPs were not higher after treatment in the population studied. New insights are needed for a better understanding and monitoring of human anthelmintic resistance.

Evaluation of antibody serology to determine current helminth and Plasmodium falciparum infections in a co-endemic area in Southern Mozambique.

BACKGROUND: Soil-transmitted helminths (STH), Schistosoma spp. and Plasmodium falciparum are parasites of major public health importance and co-endemic in many sub-Saharan African countries. Management of these infections requires detection and treatment of infected people and evaluation of large-scale measures implemented. Diagnostic tools are available but their low sensitivity, especially for low intensity helminth infections, leaves room for improvement. Antibody serology could be a useful approach thanks to its potential to detect both current infection and past exposure. METHODOLOGY: We evaluated total IgE responses and specific-IgG levels to 9 antigens from STH, 2 from Schistosoma spp., and 16 from P. falciparum, as potential markers of current infection in a population of children and adults from Southern Mozambique (N = 715). Antibody responses were measured by quantitative suspension array Luminex technology and their performance was evaluated by ROC curve analysis using microscopic and molecular detection of infections as reference. PRINCIPAL FINDINGS: IgG against the combination of EXP1, AMA1 and MSP2 (P. falciparum) in children and NIE (Strongyloides stercoralis) in adults and children had the highest accuracies (AUC = 0.942 and AUC = 0.872, respectively) as markers of current infection. IgG against the combination of MEA and Sm25 (Schistosoma spp.) were also reliable markers of current infection (AUC = 0.779). In addition, IgG seropositivity against 20 out of the 27 antigens in the panel differentiated the seropositive endemic population from the non-endemic population, suggesting a possible role as markers of exposure although sensitivity could not be assessed. CONCLUSIONS: We provided evidence for the utility of antibody serology to detect current infection with parasites causing tropical diseases in endemic populations. In addition, most of the markers have potential good specificity as markers of exposure. We also showed the feasibility of measuring antibody serology with a platform that allows the integration of control and elimination programs for different pathogens.

Plasmodium falciparum and Helminth Coinfections Increase IgE and Parasite-Specific IgG Responses.

Coinfection with Plasmodium falciparum and helminths may impact the immune response to these parasites because they induce different immune profiles. We studied the effects of coinfections on the antibody profile in a cohort of 715 Mozambican children and adults using the Luminex technology with a panel of 16 antigens from P. falciparum and 11 antigens from helminths (Ascaris lumbricoides, hookworm, Trichuris trichiura, Strongyloides stercoralis, and Schistosoma spp.) and measured antigen-specific IgG and total IgE responses. We compared the antibody profile between groups defined by P. falciparum and helminth previous exposure (based on serology) and/or current infection (determined by microscopy and/or qPCR). In multivariable regression models adjusted by demographic, socioeconomic, water, and sanitation variables, individuals exposed/infected with P. falciparum and helminths had significantly higher total IgE and antigen-specific IgG levels, magnitude (sum of all levels) and breadth of response to both types of parasites compared to individuals exposed/infected with only one type of parasite (P ≤ 0.05). There was a positive association between exposure/infection with P. falciparum and exposure/infection with helminths or the number of helminth species, and vice versa (P ≤ 0.001). In addition, children coexposed/coinfected tended (P = 0.062) to have higher P. falciparum parasitemia than those single exposed/infected. Our results suggest that an increase in the antibody responses in coexposed/coinfected individuals may reflect higher exposure and be due to a more permissive immune environment to infection in the host. IMPORTANCE Coinfection with Plasmodium falciparum and helminths may impact the immune response to these parasites because they induce different immune profiles. We compared the antibody profile between groups of Mozambican individuals defined by P. falciparum and helminth previous exposure and/or current infection. Our results show a significant increase in antibody responses in individuals coexposed/coinfected with P. falciparum and helminths in comparison with individuals exposed/infected with only one of these parasites, and suggest that this increase is due to a more permissive immune environment to infection in the host. Importantly, this study takes previous exposure into account, which is particularly relevant in endemic areas where continuous infections imprint and shape the immune system. Deciphering the implications of coinfections deserves attention because accounting for the real interactions that occur in nature could improve the design of integrated disease control strategies.

Towards soil-transmitted helminths transmission interruption: The impact of diagnostic tools on infection prediction in a low intensity setting in Southern Mozambique.

World Health Organization goals against soil-transmitted helminthiases (STH) are pointing towards seeking their elimination as a public health problem: reducing to less than 2% the proportion of moderate and heavy infections. Some regions are reaching WHO goals, but transmission could rebound if strategies are discontinued without an epidemiological evaluation. For that, sensitive diagnostic methods to detect low intensity infections and localization of ongoing transmission are crucial. In this work, we estimated and compared the STH infection as obtained by different diagnostic methods in a low intensity setting. We conducted a cross-sectional study enrolling 792 participants from a district in Mozambique. Two stool samples from two consecutive days were collected from each participant. Samples were analysed by Telemann, Kato-Katz and qPCR for STH detection. We evaluated diagnostic sensitivity using a composite reference standard. By geostatistical methods, we estimated neighbourhood prevalence of at least one STH infection for each diagnostic method. We used environmental, demographical and socioeconomical indicators to account for any existing spatial heterogeneity in infection. qPCR was the most sensitive technique compared to composite reference standard: 92% (CI: 83%- 97%) for A. lumbricoides, 95% (CI: 88%- 98%) for T. trichiura and 95% (CI: 91%- 97%) for hookworm. qPCR also estimated the highest neighbourhood prevalences for at least one STH infection in a low intensity setting. While 10% of the neighbourhoods showed a prevalence above 20% when estimating with single Kato-Katz from one stool and Telemann from one stool, 86% of the neighbourhoods had a prevalence above 20% when estimating with qPCR. In low intensity settings, STH estimated prevalence of infection may be underestimated if based on Kato-Katz. qPCR diagnosis outperformed the microscopy methods. Thus, implementation of qPCR based predictive maps at STH control and elimination programmes would disclose hidden transmission and facilitate targeted interventions for transmission interruption.

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 ß-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 ß-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 ß-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.