Title

Involvement of Toxoplasma gondii and associated inflammatory markers in the pathogenesis of type 2 diabetes mellitus

Author Identifiers

Aus Molan
ORCID: 0000-0003-3219-0972

Date of Award

2020

Degree Type

Thesis

Degree Name

Doctor of Philosophy

School

School of Medical and Health Sciences

First Advisor

Professor Wei Wang

Second Advisor

Professor Ken Nosaka

Third Advisor

Dr Michael Hunter

Abstract

Type 2 diabetes mellitus (T2DM) continues to be a major challenge for public health authorities worldwide due to its growing prevalence rates. This chronic disorder is characterised by persistent hyperglycaemia with disturbances in fat, carbohydrate, and protein metabolism resulting from abnormalities in insulin secretion, action, or both. While potential causes such as obesity, physical inactivity, and dietary patterns have been proposed to explain the growing epidemic, there may also be additional unidentified novel risk factors, such as subclinical inflammation caused by infectious agents, that contribute to this rising prevalence. The intracellular protozoan parasite Toxoplasma gondii has been identified as an understudied pathogen of interest. Infecting approximately one third of the world’s population, T. gondii is considered one of the most successful human parasites. However, while more than 25,000 original research articles, several books, and book chapters have been published on this parasite, there are still many aspects of its biology, natural life cycle, and epidemiology of which we know relatively little about, including the prevalence rate in the general Australian human population. An emerging field of research is beginning to investigate the potential of this pathogen to cause low-grade inflammation that may subsequently increase the risk and development of various metabolic conditions, particularly T2DM.

The main objectives of the present study were to: 1) determine seroprevalence of T. gondii infection in a representative Australian human population [Study One] ; 2) determine the seroprevalence of T. gondii in subjects with T2DM compared with control subjects [Study One]; 3) identify additional risk factors for T. gondii infection [Study One]; 4) examine cat ownership as an alternative risk factor for T2DM [Study Two]; 5) determine if T. gondii infection participates in the inflammatory process leading to the development of T2DM [Study Three]; and, 6) determine whether an inflammatory 11-biomarker panel could discriminate between controls, subjects with T. gondii infection, and T2DM patients, independently of other diagnostic indicators [Study Three].

Our group undertook the first set of case-control studies of their kind in Australia by utilising sera samples and survey data collected by the Busselton Population Medical Research Institute during the 2005-2007 Busselton Health Survey. The cohort comprised of 150 participants with T2DM and 150 age- and gender- matched control subjects. Commercial in-vitro diagnostic enzyme-linked immunosorbent assays were used to measure the IgG and IgM seroprevalence rates for study one and high-sensitivity multiplex methods were used to simultaneously measure the 11 biomarkers in study three.

From the 300 subjects tested, 64% had T. gondii IgG antibodies detected and 7.7% had IgM antibodies detected. IgG seropositivity increased rapidly in all age groups remaining at over 70% in the 65–74, 75–84, and >84 age groups (p <0.05). No significant difference was observed in the T. gondii IgG seroprevalence rates between the T2DM group (62%) and the healthy control group (66%) (OR: 0.84; p = 0.471). Toxoplasma gondii IgM antibodies were detected in 5% of the T2DM patients and in 10% of the control subjects (OR: 0.51; p = 0.135). There were no significant differences between male and female IgG seroprevalence rates for both the T2DM and the control groups (OR: 0.88 and 0.80; p = 0.723 and p = 0.507, respectively). The IgG seropositivity rate increased significantly in T2DM patients aged 45 to 84 years in comparison to those aged 18 to 44 years (p <0.05). This trend was not observed in the healthy non-T2DM subjects. No risk factors were associated with T. gondii seropositivity in both T2DM patients and healthy controls. IL-β, IL-4, IL-6, and IL-12(p40) were not being produced or were below the levels of detection in the majority of subjects. Serum levels of leptin, IL-4, IL-10 and PAI-1 (total) were positively associated with age in the control T. gondii seronegative group. In contrast, decreasing levels of leptin, IL-10, and PAI-1 (total) was observed in the control seropositive and T2DM seronegative groups. Age was also positively associated with IFN-γ levels in control subjects and negatively associated in the T2DM group. Increased weight, BMI, waist circumference, glucose, insulin, triglycerides, white cell count, leptin, and PAI-1 (total) results were significantly associated with T2DM (p <0.05), while no significant associations with T. gondii infection were identified (p >0.05). Strong positive associations between IL-6 and IL-4; and IL-1RA and IL-β were observed in T. gondii seropositive subjects regardless of which group they belonged to. Likewise, IL-10 showed a strong positive correlation with IL-1RA only in seronegative subjects. High-density lipoprotein displayed significant correlation (p <0.01) with: IL-6 (r = 0.506); IL-4 (r = 0.492); IL-1β (r = 0.456); IL-1RA (r = 0.507); and, IFN-γ (r = 0.456) exclusively in seropositive subjects belonging to the T2DM group.

We conclude that T. gondii infection in Western Australia is highly prevalent. Our results also showed that there is no serological evidence of an association between T. gondii infection and T2DM in the studied subjects. Although some biomarkers displayed strong association with T. gondii and/or T2DM, the differences were not significant enough in terms of discriminatory power hence the 11-biomarker panel selected for the present study is not suited for use in clinical practice independently of other diagnostic indicators. In addition, no evidence of an inflammatory association between T. gondii infection and T2DM was found. Further research is warranted to evaluate the potential of additional biomarkers as alternative and/or additional diagnostic targets to provide results with better clinical applicability. It is recommended that Australian health authorities should focus on raising awareness of toxoplasma infection and target T. gondii transmission control.

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