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Observational Study
. 2016 Nov;18(11):687-695.
doi: 10.1016/j.micinf.2016.06.009. Epub 2016 Jul 8.

Adaptive Immune Response in Symptomatic and Asymptomatic Enteric Protozoal Infection: Evidence for a Determining Role of Parasite Genetic Heterogeneity in Host Immunity to Human Giardiasis

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Free PMC article
Observational Study

Adaptive Immune Response in Symptomatic and Asymptomatic Enteric Protozoal Infection: Evidence for a Determining Role of Parasite Genetic Heterogeneity in Host Immunity to Human Giardiasis

Zahra Babaei et al. Microbes Infect. .
Free PMC article

Abstract

The genetic basis of the ultimate clinical outcomes of human giardiasis has been the subject of numerous investigations. We previously demonstrated roles for both host and parasite factors in determining the outcome of enteric infection in a murine model of Giardia duodenalis infection. In the current study, fecal and serum specimens from healthy controls and human subjects infected with the intestinal parasite G. duodenalis were assessed. Using a semi-nested PCR method, clinical isolates were genetically characterized based on the gdh and tpi loci, and the phylogenetic trees were constructed. Using a sandwich ELISA method, the serum levels of representative TH1 and TH2 cytokines were measured in infected human subjects and healthy controls. Here we showed that symptomatic human giardiasis was characterized by significantly elevated serum levels of the TH1 cytokine IFN-γ compared to healthy controls, whereas asymptomatic human subjects and healthy controls had comparable levels of serum IFN-γ. Further analyses showed that human subjects infected with G. duodenalis genotype AI had significantly elevated levels of serum IFN-γ and IL-10, but not IL-5, whereas human subjects infected with AII had similar levels of those cytokines compared to healthy controls. These data demonstrate roles for both host and parasite factors in the determination of the outcome of enteric infections and may further broaden our understanding of host-parasite interaction during enteric protozoal infections.

Keywords: Cytokines; Giardia duodenalis; Human; Protozoa.

Figures

Fig 1
Fig 1
Flow diagram deciphering the subject selection process. This diagram outlines the study recruitment of 40 subjects who were stratified into different groups based on the absence or the presence of clinical manifestations and the presence of infection with a particular genotype of G. duodenalis (AI/AII). Genotypes analyzed do not add up to 40 as some genotypes (n= 3) were excluded from cytokine analysis because the results obtained between the gdh and tpi loci were inconsistent.
Fig 2
Fig 2
Breakdown of clinical symptoms in human subjects (n = 28) with symptomatic giardiasis (A). Fecal consistently in human subjects with human giardiasis was determined by a scoring system, where scores of 1 to 4 denoted formed, soft, loose, and watery stool consistency, respectively (B).
Fig 3
Fig 3
Agarose gel electrophoresis of the semi-nested PCR amplification of a 432-bp of the gdh gene of G. duodenalis. Lanes 1, negative control (no DNA); 2, positive control; 3-5, patient's samples; M, DNA ladder (A). Digestion of PCR product targeting the gdh gene using BspLI (NlaIV) to distinguish AI (90-, 120-, 150-bp) and AII (70-, 80-, 90-, 120-bp) genotypes. Lane 1, undigested PCR product; 2 and 3, AI genotype; 4 and 5, AII genotype; 6, mixed infections (AI+AII); M, DNA ladder (B). Agarose gel electrophoresis of the semi-nested PCR amplification of a 476-bp of the tpi gene of the G. duodenalis assemblage A. Lanes 1, positive control; 2-6, patient's samples; 7, negative control (no DNA); M, DNA ladder (C). Digestion of PCR product targeting the tpi gene using RsaI to distinguish AI (437-, 15-bp) and AII (232-, 235-, 15-bp) genotypes. Lane 1, undigested PCR product of the G. duodenalis assemblage A; 2-4; AI genotype; 5, AII genotype; 6 and 7, mixed infections (AI+AII); M, DNA ladder (D). Sizes in base pairs are denoted by the numbers at left.
Fig 4
Fig 4
Cytokine profiles in asymptomatic and symptomatic human giardiasis and in human subjects infected with genetically-distinct G. duodenalis genotypes. Human subjects infected with G. duodenalis were stratified into asymptomatic and symptomatic patients based on the absence or the presence of clinical symptoms. Serum levels of the cytokines IFN-γ (A), IL-5 (B) and IL-10 (C) were measured using a sandwich ELISA method in human subjects with asymptomatic (n = 12) and symptomatic (n = 28) giardiasis as well as in healthy controls (n = 38). *p < 0.05 (a one-way ANOVA, followed by Bonferroni post-hoc adjustment test). Bar graphs represent Mean±SEM. Clinical isolates of G. duodenalis from asymptomatic and symptomatic patients were genetically characterized by a semi-nested PCR targeting the gdh or tpi loci, followed by a restriction fragment length polymorphism (RFLP) method. Serum levels of IFN-γ (D), IL-5 (E), and IL-10 (F) were measured in human subjects singly infected with AI (n= 18) or AII (n= 10) genotypes as well as in healthy controls (n= 38) by ELISA. *p < 0.05; **p < 0.01 (A one-way ANOVA, followed by Bonferroni post-hoc adjustment test). Bar graphs represent Mean±SEM.

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