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Seroprevalence and associated risk factors of Toxoplasma gondii infection in a representative Australian human population: The Busselton health study

Published:February 24, 2020DOI:https://doi.org/10.1016/j.cegh.2020.02.005

      Abstract

      Introduction

      Despite being identified as one of the top neglected parasitic infections, Toxoplasma gondii has received little recognition in Australia with no previously published prevalence data from the general human population. The objectives of the current study were to: determine the prevalence of evidence of exposure to T. gondii infection in an Australian community dwelling population, and: identify associated risk factors.

      Methods

      Sera from 75 males and 75 age-matched females living in Busselton, Western Australia were tested for the presence of anti-T. gondii IgG and IgM antibodies using enzyme-linked immunosorbent assay. Survey derived data were also analysed to evaluate risk factors.

      Results

      IgG and IgM antibodies were detected in 99 (66.0%) and 15 (10.0%) of subjects, respectively. IgG seroprevalence increased from 44.4% (95% confidence interval (95% CI): 18.9–73.3%) in the 18–34 year-old age group to 81.0% (95% CI: 60.0–92.3%) in the 75–84 age group. The observed IgG seroprevalence increased at a rate of 0.8% with each year of age. No risk factors were identified.

      Conclusions

      The first study of its kind in Australia found T. gondii infection to be highly prevalent. Toxoplasma gondii infection has been neglected in Australian notifiable disease programs therefore Australian public health authorities should focus on improving education to raise awareness and commence longitudinal epidemiological data collection to supplement public health models targeting T. gondii transmission control.

      Keywords

      1. Introduction

      First described in 1908 by Nicolle and Manceaux,
      • Nicolle C.
      • Manceaux L.
      Sur une infection à corps de Leishman (ou organismes voisins) du gondi.
      the obligate intracellular parasite T. gondii infects approximately one third of the world's population and is considered one of the most successful human parasites.
      • Peng H.J.
      • Chen X.C.
      • Lindsay D.S.
      A review, competence compromise and concomitance-reaction of the host cell to Toxoplasma gondii infection and development.
      • Centers for Disease Control and Prevention
      Parasites - Toxoplasmosis (Toxoplasma Infection).
      • Halonen S.K.
      • Weiss L.M.
      Toxoplasmosis.
      • Pappas G.
      • Roussos N.
      • Falagas M.E.
      Toxoplasmosis snapshots, global status of Toxoplasma gondii seroprevalence and implications for pregnancy and congenital toxoplasmosis.
      The Centers for Disease Control has prioritised T. gondii as one of the top “Five Neglected Parasitic Infections” due to the severity of illness, high incidence, and potential for prevention.
      Centers for Disease Control and Prevention
      Neglected Parasitic Infections (NPIs) in the United States.
      Humans acquire T. gondii infection by the ingestion of food, water, or soil contaminated by oocysts from the definitive hosts, cats (family, Felidae; genera, Felis and Lynx). Toxoplasma gondii is also transmitted in people vertically via placenta and horizontally via blood transfusion.
      • Centers for Disease Control and Prevention
      Parasites - Toxoplasmosis (Toxoplasma Infection).
      ,
      • Tenter A.
      • Heckeroth A.
      • Weiss M.
      Toxoplasma gondii, from animals to humans.
      Capable of infecting all warm-blooded animals, T. gondii infection is one of considerable public health impact. The global prevalence rates of this parasite are remarkable, with figures ranging from 15 to 85% depending on social habits, climate conditions, hygienic standards, and geographical regions.
      • Robert-Gangneux F.
      • Dardé M.L.
      Epidemiology of and diagnostic strategies for toxoplasmosis.
      Although T. gondii has a worldwide distribution and possibly the widest host range of any parasite, there is only one species (T. gondii) in the genus Toxoplasma and the cat family are the only definitive hosts in which T. gondii sexual development is known to occur.
      • Dupey J.P.
      The history of Toxoplasma gondii – the first 100 years.
      Infection starts with an acute phase followed by a chronic phase and this parasite has three infectious stages: sporozoites, tachyzoites and bradyzoites.
      • Hunter C.A.
      • Sibley L.D.
      Modulation of innate immunity by Toxoplasma gondii virulence effectors.
      Due to their active multiplication, tachyzoites cause extensive tissue damage and may disseminate to different tissues of the host. Toxoplasma gondii tachyzoites infects circulating white blood cells and can use them as a “Trojan Horse” to gain access through protective tissues to organs such as the brain, a compartment where entry of immune cells is restricted. The tachyzoites eventually undergo conversion and become slowly multiplying bradyzoites within tissue cysts. In instances where the tissue cysts become activated, the bradyzoites can undergo further conversion and become tachyzoites.
      • Tenter A.
      • Heckeroth A.
      • Weiss M.
      Toxoplasma gondii, from animals to humans.
      ,
      • Robert-Gangneux F.
      • Dardé M.L.
      Epidemiology of and diagnostic strategies for toxoplasmosis.
      All parasitic stages can infect feline enterocytes leading to oocyst production, which restarts the life cycle of T. gondii.
      • Dupey J.P.
      The history of Toxoplasma gondii – the first 100 years.
      It should be noted that the term “toxoplasmosis” should be reserved to describe cases where pathological and/or clinical symptoms of the disease caused by T. gondii are active while the term “T. gondii infection” should be used to describe asymptomatic infection or evidence of the persistence of the parasite in the host (latent or chronic). Infection can present with various nonspecific signs and symptoms, but most have been previously described as similar to flulike indicators.
      • Tenter A.
      • Heckeroth A.
      • Weiss M.
      Toxoplasma gondii, from animals to humans.
      In immunocompetent individuals, it is thought that the disease is asymptomatic and the individual recovers without treatment because of an efficient immune system which limits the spread of the rapidly multiplying tachyzoites.
      • Tenter A.
      • Heckeroth A.
      • Weiss M.
      Toxoplasma gondii, from animals to humans.
      Consequently, people are not routinely screened for evidence of T. gondii infection unless they are clinically immunocompromised or pregnant, in which cases infection may lead to serious complications which can be lethal.
      • Robert-Gangneux F.
      • Dardé M.L.
      Epidemiology of and diagnostic strategies for toxoplasmosis.
      Acute infection during pregnancy can cause damage to the developing fetus.
      • Robert-Gangneux F.
      • Dardé M.L.
      Epidemiology of and diagnostic strategies for toxoplasmosis.
      In all circumstances, specific antibodies to antigens of this parasite remain detectable in the serum throughout the life of the host, with dormant cysts being formed in various anatomical sites, including the central nervous system, often establishing latent toxoplasmosis.
      • Halonen S.K.
      • Weiss L.M.
      Toxoplasmosis.
      Toxoplasma gondii can infect and replicate in any nucleated host cells, leading to the production of various inflammatory biomarkers via the acute inflammatory response and antigen-specific adaptive immunity, in parallel to seroconversion.
      • Halonen S.K.
      • Weiss L.M.
      Toxoplasmosis.
      This facilitates a state of chronic inflammation at various anatomical sites in the host.
      • Tenter A.
      • Heckeroth A.
      • Weiss M.
      Toxoplasma gondii, from animals to humans.
      Consequently, chronic T. gondii infection has been linked to several autoimmune disorders including thyroid disease, systemic sclerosis, rheumatoid arthritis, and inflammatory bowel syndrome.
      • Carter C.J.
      Toxoplasmosis and polygenic disease susceptibilitygenes, extensive Toxoplasma gondii host/pathogen interactome enrichment in nine psychiatric or neurological disorders.
      Several reports have demonstrated a positive correlation between T. gondii infection and numerous neurological disorders and cancers.
      • Tenter A.
      • Heckeroth A.
      • Weiss M.
      Toxoplasma gondii, from animals to humans.
      ,
      • Dupey J.P.
      The history of Toxoplasma gondii – the first 100 years.
      ,
      • Henriques S.A.
      • Brett R.
      • Alexander J.
      • Pratt J.
      • Roberts C.W.
      Neuropsychiatric disease and Toxoplasma gondii infection.
      Despite the fact that T. gondii is distributed worldwide, there is very limited information on the prevalence, incidence rate, epidemiology, and risk factors specific to the general Australian human population-because T. gondii infection is not a notifiable disease in Australia and most infections are asymptomatic. However, using T. gondii incidence data from overseas foodborne illnesses surveys, Hall and Kirk
      • Kirk M.
      • Glass K.
      calculated a conservative incidence of up to 7,150 new symptomatic cases of T. gondii infection in Australia each year. In addition, a few publications have reported prevalence rates amongst pregnant woman throughout the country: 35% in Western Australia,
      • Walpole I.R.
      • Hodgen N.
      • Bower C.
      Congenital toxoplasmosis, a large survey in western Australia.
      23% in Melbourne,
      • Karunajeewa H.
      • Siebert D.
      • Hammond R.
      • Garland S.
      • Kelly H.
      Seroprevalence of varicella zoster virus, parovirus B19 and Toxoplasma gondii in a Melbourne obstetric population, implications for management.
      23% in South Australia,
      • Berger S.
      Toxoplasmosis, Global Status 2010 Edition.
      and 26% in Queensland.
      • Berger S.
      Toxoplasmosis, Global Status 2010 Edition.
      The objectives of this study were to: 1) determine the seroprevalence of T. gondii infection in a representative Australian population as no data exist to date; and 2) identify risk factors for T. gondii infection. We undertook the first age- and gender-matched study in Australia by utilising sera and cross-sectional clinical data (respiratory and chest conditions; various disease states; anthropometric measurements; and biochemical and haematological laboratory test results) collected from a community-dwelling cohort of adults attending the 2005–2007 Busselton Health Survey (BHS) in Western Australia.

      2. Materials and methods

      The present study describes serology on blood samples and data collected from adult residents of the Western Australian town of Busselton, a centre for farming, vineyards, timber, and mineral sands industries. The cross-sectional general population BHS was conducted between 2005 and 2007 with participants recruited from the compulsory electoral role. This survey was conducted by the Busselton Population Medical Research Institute (BPMRI), a prominent biobank. Details on recruitment and study protocols from this survey are described in Musk et al.
      • Musk A.W.
      • Knuiman M.
      • Hunter M.
      • et al.
      Patterns of airway disease and the clinical diagnosis of asthma in the Busselton population.
      Ethical approval was obtained from the Edith Cowan University Human Research Ethics Committee (Project Number 16090).

      2.1 Study design and criteria for selection of participants

      Through an age- and gender-matched cross-sectional study, the seroprevalence of T. gondii was measured in 150 subjects. Sera were analysed for the presence of IgG and IgM antibodies against T. gondii using commercially available qualitative ELISA methods (Demeditec Diagnostics GmbH, Germany). These kits have claimed clinical sensitivities and specificities of 99% and 98%, respectively, for the IgG assay; 99% and 100%, respectively, for the IgM assay. Survey-derived data including respiratory and chest conditions; various disease states; together with anthropometric measurements; details of pet ownership; and biochemical and haematological laboratory test results were investigated for possible significant risk factor association. Participants were recruited by reference to the following criteria: 1) do not have a documented medical diagnosis of diabetes, 2) not taking any glucose-lowering medications, and 3) have fasting and 2-h glucose values below the diagnostic thresholds for diabetes. In addition, subjects were screened for history of treatment with psychoactive medication and were excluded. These criteria were selected because of the previously reported and established associations between positive T. gondii serology, diabetes, and numerous forms of serious mental illnesses.
      • Dupey J.P.
      The history of Toxoplasma gondii – the first 100 years.
      ,
      • Carter C.J.
      Toxoplasmosis and polygenic disease susceptibilitygenes, extensive Toxoplasma gondii host/pathogen interactome enrichment in nine psychiatric or neurological disorders.
      ,
      • Molan A.
      • Nosaka K.
      • Hunter M.
      • Wang W.
      The role of Toxoplasma gondii as a possible inflammatory agent in the pathogenesis of type 2 diabetes mellitus in humans.
      ,
      • Molan A.
      • Nosaka K.
      • Hunter M.
      • Wang W.
      A systemic review and meta-analysis of human case-control studies examining the association between Toxoplasma gondii infection and type 2 diabetes mellitus.

      2.2 Sample and data collection

      This study utilised 20 μL of serum aliquoted from banked samples (stored at −80 °C) that had been previously collected by the BPMRI from volunteers aged 18–80 years during the 2005–2007 BHS. Each participant was required to complete a standard self-administered questionnaire that obtained information on respiratory and chest conditions; various disease states; and pet ownership. Each participant had anthropometric and body composition measurements taken in addition to blood samples collected from the cubital fossa in 10 mL Red top-clot, 2 mL Purple top-EDTA, and 2 mL Grey Top -FlOx Vacutainer tubes (BD Biosciences, Franklin Lakes, New Jersey, USA) for laboratory biochemical and haematological analyses which were conducted by PathWest Laboratory Medicine- QEII Medical Centre, Nedlands, Western Australia. Serum for the present study was aliquoted and stored at −80 °C until used.

      2.3 Sample integrity and laboratory analysis

      Lipemic, haemolysed, icteric, or turbid (bacterially contaminated) samples were excluded as they may cause false positive or false negative results. Test samples were diluted 1:101 with ready-to-use sample diluent (5 μL serum + 500 μL sample diluent). The total volume of serum required from each subject to perform the IgG and IgM ELISAs was 20 μL (5 μL for IgG ELISA, 5 μL for IgM ELISA, and 10 μL for repeat/s in the event of equivocal result/s). The sera were tested for anti-T. gondii IgG and anti-T. gondii IgM antibodies using the Demeditec Diagnostics DETOX01 (IgG ELISA) and DETOX03 (IgM ELISA) kits according to the manufacturers’ instructions. Briefly, these assays have been designed for the qualitative evaluation of specific IgG and IgM antibodies against Toxoplasma in serum. The analysis was performed double-blind to avoid result bias. Samples from the female and male groups were randomly sorted/selected, and the analyst performing the analysis was not aware of the source of samples. Then,100 μL of the diluted (1:101) sample and the ready-to-use calibrators (IgG [IU/mL]; A, 0; B, 10; C, 40; D, 100; E, 250; IgM [U/mL]; A, 1; B, 10; C, 30; D, 120) were pipetted into each test well (coated with T. gondii strain RH antigens, isolated from infected mice, common to both the IgG and IgM assays) leaving one well empty for the substrate blank. The plate was covered and incubated for 60 min at room temperature. The wells were then washed three times with 300 μL of diluted washing solution using a Bio-Plex Pro II Microplate Wash Station (Bio-Rad Laboratories, Berkeley, California). Subsequently, 100 μL of ready-to-use conjugate was added into each well except the substrate blank well. The plate was covered and incubated at room temperature for 30 min. This was followed by another washing procedure as outlined above, after which 100 μL of the ready-to-use substrate was pipetted into each well including the substrate blank well. A final incubation phase for 20 min at room temperature in the dark was performed before terminating the substrate reaction with the addition of 100 μL of the ready-to-use stop solution into each well. The plate was then mixed and the wiped in preparation for reading. This was performed using a FLUOstar Omega microplate reader (BMG Labtech, Offenburg, Germany) at an absorption of 450 nm. A standard curve was generated by plotting the mean absorbance at 450 nm for each standard concentration (x axis) against the target antibody concentration (y axis). This was used to determine the qualitative concentration of target antibody in each sample. By the criteria of the assays used, values higher than the cut-offs (IgG, 10 IU/mL; IgM, 10 U/mL) were considered positive, values less than the cut-offs were considered negative, while values falling within a grayzone of+/-20% of the cut-off values were considered equivocal and retested once.

      2.4 Statistical analysis

      Basic descriptive statistics were calculated for the seropositive and seronegative groups linking gender and age variables. Seroprevalence of T. gondii along with the odds ratios (OR) and corresponding 95% confidence intervals (95% CI) were calculated. The Chi-squared test was used to evaluate the seroprevalence values between positive and negative subjects with respect to demographic categorical variables (health and diseases states, pet ownership) to examine possible significant risk factor associations. For continuous quantitative variables (anthropometric and body composition measurements, laboratory results), the normality of the data distribution was assessed using the Shapiro-Wilk test in addition to Q-Q plots and skewness and kurtosis values. As the laboratory and anthropometric were normally distributed, the Student's t-test was used to compare selected parameters between the study groups. Probability values were calculated on the basis of two-tailed tests. For all analyses, a p value less than 0.05 was considered statistically significant. Data was analysed using IBM® SPSS® Statistics version 25.0 software (SPSS Inc., Armonk, New York, USA).

      3. Results

      In this study, the 150 selected subjects were matched for age, gender, and age group distribution. The mean age was 58.8 ± 15.8 years. Most of patients were in the 55–64 (n = 32) and 65–74 year-old (n = 32) age groups; followed by the 45–54 year-old (n = 31) age group; then the 35–44 (n = 21) and 75–84 year-old (n = 21) age groups. The basic descriptive and biometric statistics are summarised in Table 1.
      Table 1Basic descriptive and biometric statistics for the study group (n = 150) at baseline including age group stratification. BHS, Western Australia.
      ParameterOverallMalesFemales
      Number of subjects1507575
      Average age (±SD)58.8 ± 15.863.5 ± 14.554.0 ± 15.8
      Height (cm ± SD)168.4 ± 9.3173.9 ± 7.7163.0 ± 7.5
      Weight (Kg ± SD)77.2 ± 15.685.8 ± 13.868.6 ± 12.2
      Waist (cm ± SD)91.3 ± 13.299.5 ± 10.283.3 ± 10.7
      BMI (±SD)27.1 ± 4.528.4 ± 3.825.9 ± 4.9
      Cat owners20.0%16.0%24.0%
      Dog owners37.3%40.0%34.7%
      Other pet owners22.7%21.3%24.0%
      Average age is recorded in years; cm, centimeter; Kg, kilogram; SD, standard deviation; BMI, body mass index.

      3.1 Seroprevalence of T. gondii infection and interaction with gender

      From the 150 subjects tested during the study period, 99 (66.0%) were IgG seropositive and 15 (10.0%) were IgM seropositive. Female subjects had an overall lower IgG seroprevalence than their male counterparts, 61.3% and 70.7%, respectively (p = 0.228). However, the opposite was seen with IgM seroprevalence with the difference being significant, 17.3% and 2.7%, respectively (p = 0.003). Univariate analysis identified male gender as a possible risk factor for the presence of IgG antibodies (odds ratio (OR): 1.52; 95% CI: 0.77–3.00, p = 0.228) and female gender for the presence of IgM antibodies (OR: 0.13; 95% CI: 0.0–0.60, p = 0.003) [Table 2].
      Table 2Stratified seroprevalence of IgG and IgM antibodies against T. gondii detected by ELISA in adults aged 18 to 84+ years by gender and age group. BHS, Western Australia.
      Toxoplasma IgG/IgM serology
      POS (n)NEG (n)PrevalenceOR95% CIp-value
      Gender (IgG)
      Female462961.3%refRefref
      Male532270.7%1.520.77–3.000.228
      Total995166.0%
      Gender (IgM)
      Female136217.3%refRefref
      Male2732.7%0.130.03–0.600.003
      Total1513510.0%
      Age Group (IgG)
      18–344544.4%refrefref
      35–4491242.9%0.940.19–4.520.936
      45–5424777.4%4.290.90–20.420.057
      55–64201262.5%2.080.47–9.310.331
      65–74221068.8%2.750.61–12.480.181
      75–8417481.0%5.310.96–29.300.046
      >843175.0%3.750.27–51.380.308
      Total995166.0%
      n, number of subjects; POS, number of subjects in which anti-IgG antibodies were detected; NEG, number of subjects in which anti-IgG antibodies were not detected; OR, odds ratio; 95% CI, 95% confidence interval; age is recorded in years.

      3.2 Seroprevalence of T. gondii infection and interaction with age

      IgG seroprevalence increased from 44.4% (95% CI: 18.9–73.3%) in the 18–34 year-old age group to 81.0% (95% CI: 60.0–92.3%) in the 75–84 year-old age group. Therefore, the observed IgG seroprevalence increased at a rate of 0.8% with each year of age (excluding the 18–34 and > 84 year-old age groups due to low numbers of participants and gender skew). IgM was excluded from further analysis due to the low numbers of seropositive subjects. A relationship was observed between age and T. gondii IgG seropositivity as higher IgG seroprevalences were observed with increasing age. IgG seropositivity increased rapidly in all age groups remaining at over 60% in the 45 year-old and above age groups [Table 2].

      3.3 Toxoplasma gondii seroprevalence and associated demographic factors

      Regarding risk factors associated with T. gondii infection, univariate analysis showed that the various respiratory and disease conditions (asthma, arthritis, bronchitis, cancer, eczema, food allergies, hay fever, other chest conditions, pleurisy, pneumonia, sinusitis), pet ownership, and anthropometric measurements were not significantly associated with T. gondii infection in IgG-seropositive subjects. However, seropositive were more likely than the controls to own a dog (OR = 1.54, p = 0.343) while the seronegative subjects reported higher ownership of pets other than cats or dogs (OR = 0.57, p = 0.157) [Table 3].
      Table 3Univariate analysis of the variables associated with the seroprevalence of anti-Toxoplasma gondii IgG antibodies among subjects. BHS, Western Australia.
      Toxoplasma gondii IgG serology
      POS (n = 99)ND (n = 51)Prev. (%)OR95% CIp-value
      Health State
      SOB at rest535.3%0.850.20–3.710.830
      Chronic cough231022.0%1.240.54–2.860.612
      Chronic phlegm9610.0%0.750.25–2.240.605
      Chronic rhinitis462547.3%0.900.46–1.780.767
      Wheeze321833.3%0.880.43–1.780.715
      Chest tightness401838.7%1.240.62–2.500.543
      Disease State
      Asthma201221.3%0.820.37–1.850.637
      Arthritis362138.0%0.850.44–1.640.620
      Bronchitis221524.7%0.690.32–1.480.333
      Cancer466.7%0.320.08–1.180.072
      Eczema10812.0%0.600.22–1.640.319
      Food allergies858.7%0.810.25–2.610.722
      Hay fever262030.7%0.550.27–1.130.103
      Other chest1049.3%1.320.39–4.440.652
      Pleurisy736.7%1.220.30–4.920.782
      Pneumonia181320.7%0.650.29–1.460.295
      Sinusitis191623.3%0.520.24–1.130.095
      Pet Ownership
      Cat ownership371937.3%1.010.50–2.020.989
      Dog ownership22820.0%1.540.63–3.740.343
      Other pet ownership191522.7%0.570.26–1.250.157
      Body Mass Index (BMI)
      Underweight: <18.5100.7%0.471
      Normal: 18.5–24.9302033.3%0.670.33–1.370.273
      Overweight: 25.0–29.9441640.0%1.750.86–3.570.122
      Obese: >30.0241526.0%0.770.36–1.640.494
      Waist Circumference
      M > 102 cm, F > 88 cm411839.3%1.300.64–2.610.467
      n, number of subjects; POS, number of subjects in which anti-IgG antibodies were detected; ND, number of subjects in which anti-IgG antibodies were not detected; Prev, prevalence of risk factor in the entire cohort (n = 150); OR, odds ratio; 95% CI, 95% confidence interval; SOB, shortness of breath.

      3.4 Laboratory test results and anthropometric measurements

      Findings from the laboratory and anthropometric studies are summarised in Table 4. It was found that IgG seropositive subjects had a significantly lower red cell distribution width (RDW), a measure of the variation in the size and volume of erythrocytes and forms part of the complete blood count (reference range: 11.5–14.6%), than seronegative subjects (average of 3.1 and 5.8, respectively; p < 0.01). Also, seropositive subjects had significantly lower absolute eosinophil counts (adult reference range: 4.0–5.5 x 106 cells/mL) when compared to the seronegative subjects (average of 3.3 and 4.0, respectively, p < 0.025). Furthermore, a significantly higher neutrophil count was observed for seropositive subjects when compared to the seronegative subjects (average of 58.5 and 55.6, respectively, p < 0.033). The remaining anthropometric examinations and laboratory results were comparable and no significant association with T. gondii seropositivity was found.
      Table 4Association between Toxoplasma gondii IgG seropositivity, and selected biochemical and haematological laboratory parameters and anthropometric examinations, raw and after adjustment for gender. BHS, Western Australia.
      ParameterIgG +IgG -PFemale IgG + vs IgG-T2DM IgG-pMale IgG + vs IgG-CTRL IgG-p
      Mean ± SDMean ± SDMean ± SDMean ± SDMean ± SDMean ± SD
      Height (cm)167.8 ± 9.8169.7 ± 8.20.234161.5 ± 7.9165.4 ± 6.10.025173.3 ± 7.9175.3 ± 7.20.313
      Weight (Kg)77.1 ± 16.177.3 ± 14.50.96368.3 ± 12.669.0 ± 11.60.78784.9 ± 14.988.1 ± 10.30.353
      BMI (Kg/m2)27.3 ± 4.626.8 ± 4.40.49026.2 ± 5.025.3 ± 4.70.43728.3 ± 4.028.7 ± 3.10.669
      Waist (cm)92.3 ± 12.889.4 ± 13.90.19784.6 ± 11.281.3 ± 9.60.18699.3 ± 9.8100.1 ± 11.20.761
      SBP (mmHg)126.1 ± 17.9120.8 ± 16.50.080123.0 ± 18.9114.5 ± 14.50.043128.9 ± 16.7129.1 ± 15.40.949
      DBP (mmHg)78.3 ± 10.775.7 ± 9.50.14076.4 ± 9.873.5 ± 8.80.20580.0 ± 11.378.5 ± 9.70.592
      Hb (g/L)145.8 ± 13.2144.5 ± 12.80.547137.9 ± 10.4137.8 ± 9.20.953152.8 ± 11.3153.0 ± 11.70.958
      Hct (L/L)0.4 ± 0.00.4 ± 0.00.6660.4 ± 0.00.4 ± 0.00.9590.4 ± 0.00.4 ± 0.00.773
      RCC
      Red cell, lymphocyte, monocyte, eosinophil, basophil, and neutrophil counts are recorded as absolute counts (x106 cells/mL).
      4.7 ± 0.54.7 ± 0.40.5774.5 ± 0.44.5 ± 0.30.9914.9 ± 0.44.9 ± 0.40.921
      RDW (%)3.1 ± 5.45.8 ± 6.3<0.013.2 ± 5.36.3 ± 6.30.0273.1 ± 5.55.3 ± 6.30.143
      Lymph
      Red cell, lymphocyte, monocyte, eosinophil, basophil, and neutrophil counts are recorded as absolute counts (x106 cells/mL).
      30.7 ± 7.832.6 ± 7.00.14032.0 ± 8.233.7 ± 7.30.37129.5 ± 7.331.3 ± 6.50.335
      Mono
      Red cell, lymphocyte, monocyte, eosinophil, basophil, and neutrophil counts are recorded as absolute counts (x106 cells/mL).
      6.9 ± 1.87.3 ± 1.40.1906.5 ± 1.87.0 ± 1.20.2107.3 ± 1.67.7 ± 1.50.330
      Eosin
      Red cell, lymphocyte, monocyte, eosinophil, basophil, and neutrophil counts are recorded as absolute counts (x106 cells/mL).
      3.3 ± 1.54.0 ± 1.90.0253.1 ± 1.54.0 ± 2.20.0543.5 ± 1.44.0 ± 1.50.220
      Baso
      Red cell, lymphocyte, monocyte, eosinophil, basophil, and neutrophil counts are recorded as absolute counts (x106 cells/mL).
      0.6 ± 0.50.5 ± 0.50.1330.5 ± 0.50.3 ± 0.50.1000.7 ± 0.50.7 ± 0.50.950
      Neutro
      Red cell, lymphocyte, monocyte, eosinophil, basophil, and neutrophil counts are recorded as absolute counts (x106 cells/mL).
      58.5 ± 7.955.6 ± 7.60.03358.0 ± 8.655.0 ± 8.10.14259.0 ± 7.456.4 ± 7.00.160
      Platelets244.4 ± 62.1254.5 ± 61.00.348271.6 ± 57.6266.8 ± 52.80.723220.4 ± 56.3238.9 ± 68.10.229
      MCV (fL)89.7 ± 4.090.0 ± 3.90.69989.2 ± 3.289.1 ± 3.30.99090.2 ± 4.591.0 ± 4.40.468
      TP (g/L)74.8 ± 3.974.2 ± 3.50.41374.3 ± 3.574.0 ± 3.70.79075.2 ± 4.274.5 ± 3.30.482
      Alb (g/L)45.5 ± 2.646.3 ± 1.90.05545.7 ± 2.345.9 ± 1.90.71645.3 ± 2.946.8 ± 1.80.025
      Bili (μmol/L)10.8 ± 6.09.2 ± 3.70.0878.9 ± 4.18.6 ± 3.30.76412.5 ± 6.910.0 ± 4.10.124
      Gluc (mmol/L)5.2 ± 0.55.1 ± 0.40.2235.0 ± 0.45.0 ± 0.40.6545.3 ± 0.55.3 ± 0.30.465
      Insulin (mU/L)8.1 ± 6.06.9 ± 4.20.1757.0 ± 4.16.0 ± 2.90.2489.1 ± 7.18.0 ± 5.20.500
      CRP (mg/L)2.6 ± 2.82.4 ± 3.70.7702.6 ± 3.52.1 ± 2.00.4832.5 ± 2.12.8 ± 5.30.742
      Chol (mmol/L)5.5 ± 0.95.4 ± 1.00.4005.6 ± 1.25.4 ± 1.00.3165.4 ± 1.05.3 ± 1.10.710
      HDL mmol/L)1.6 ± 0.51.6 ± 0.50.8981.8 ± 0.51.7 ± 0.40.3061.4 ± 0.41.4 ± 0.50.887
      LDL (mmol/L)3.3 ± 0.83.2 ± 0.90.6243.3 ± 0.93.2 ± 0.90.7273.3 ± 0.93.3 ± 1.00.772
      Trig (mmol/L)1.4 ± 0.71.3 ± 0.50.2301.2 ± 0.51.1 ± 1.40.4181.6 ± 0.71.5 ± 0.70.586
      ALP (U/L)66.8 ± 19.170.0 ± 25.70.39063.1 ± 19.167.4 ± 25.20.40770.0 ± 17.873.5 ± 26.60.524
      ALT (U/L)14.2 ± 7.813.7 ± 6.40.73612.6 ± 7.613.0 ± 6.70.81115.5 ± 7.714.7 ± 6.10.659
      Creat (μmol/L)77.7 ± 20.376.0 ± 14.60.76661.1 ± 9.066.3 ± 9.00.17998.0 ± 5.989.0 ± 9.50.029
      +, anti-IgG antibodies not detected; -, anti-IgG antibodies not detected SD, standard deviation; BMI. Body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; mmHg, millimeters of mercury; Hb, haemoglobin; Hct, haematocrit; RCC, red cell count; RDW, red cell distribution width; WCC, white cell count; MCV, mean cell volume; TP, total protein; Alb, albumin; Bili, bilirubin; Gluc; glucose; Chol, cholesterol; Trig, triglycerides; CRP, c-reactive protein; HDL, high-density lipoproteins: LDL, low-density lipoproteins; ALP, alkaline phosphatase; ALT, alanine transaminase; Creat, creatinine.
      a Red cell, lymphocyte, monocyte, eosinophil, basophil, and neutrophil counts are recorded as absolute counts (x106 cells/mL).
      When examining for gender, seropositive females where significantly shorter in height (average of 161.5 and 165.4, respectively, p = 0.025) and had significantly higher systolic blood pressure (123.0 and 114.5 mmHg, respectively, p = 0.043) when compared to their seronegative counterparts. These findings were not found in the male subjects except for creatinine levels where seropositive subjects had significantly higher levels of serum creatinine than seronegative males (98.0 and 89.0 μmol/L, respectively, p = 0.029; reference range for males, up to 110 μmol/L).

      4. Discussion

      We conducted the first seroprevalence study for T. gondii infection in a representative Australian population and found high a seroprevalence rate (IgG: 66%) compared to other countries.
      • Centers for Disease Control and Prevention
      Parasites - Toxoplasmosis (Toxoplasma Infection).
      • Halonen S.K.
      • Weiss L.M.
      Toxoplasmosis.
      • Pappas G.
      • Roussos N.
      • Falagas M.E.
      Toxoplasmosis snapshots, global status of Toxoplasma gondii seroprevalence and implications for pregnancy and congenital toxoplasmosis.
      ,
      • Wilking H.
      • Thamm M.
      • Stark K.
      • Aebischer T.
      • Seeber F.
      Prevalence, incidence estimations, and risk factors of Toxoplasma gondii infection in Germany, a representative, cross-sectional, serological study.
      • Fiedler K.
      • Hulsse C.
      • Straube W.
      • Briese V.
      Toxoplasmosis-antibody seroprevalence in mecklenburg-western pomerania (in German).
      • Beringer T.
      Is diagnosis of toxoplasmosis within the scope of prenatal care meaningful? (In German).
      • Nogareda F.
      • Le Strat Y.
      • Villena I.
      • De Valk H.
      • Goulet V.
      Incidence and prevalence of Toxoplasma gondii infection in women in France, 1980–2020, model-based estimation.
      • Hofhuis A.
      • van Pelt Y.T.
      • Nijhuis C.D.
      • et al.
      Decreased prevalence and age-specific risk factors for Toxoplasma gondii IgG antibodies in The Netherlands between 1995/1996 and 2006/2007.
      • Jones J.L.
      • Kruszon-Moran D.
      • Sanders-Lewis K.
      • Wilson M.
      Toxoplasma gondii infection in the United States, 1999 2004, decline from the prior decade.
      • Nagle C.M.
      • Wilson L.F.
      • Hughes M.C.B.
      • et al.
      Cancers in Australia in 2010 attributable to the consumption of red and processed meat.
      Comparing our findings to the earlier Australian studies is hindered by earlier focus on congenital cohorts, but lower seroprevalences were found in these studies among pregnant woman throughout the country: 23% in Melbourne,
      • Karunajeewa H.
      • Siebert D.
      • Hammond R.
      • Garland S.
      • Kelly H.
      Seroprevalence of varicella zoster virus, parovirus B19 and Toxoplasma gondii in a Melbourne obstetric population, implications for management.
      23% in South Australia,
      • Berger S.
      Toxoplasmosis, Global Status 2010 Edition.
      and 26% in Queensland.
      • Berger S.
      Toxoplasmosis, Global Status 2010 Edition.
      The only previously published study from Western Australia conducted by Walpole et al.
      • Walpole I.R.
      • Hodgen N.
      • Bower C.
      Congenital toxoplasmosis, a large survey in western Australia.
      in 1991, investigating the birth prevalence of congenital toxoplasmosis, found that 35% of the tested women were seropositive for anti-T. gondii IgG antibodies. Thus, our findings reveal a significant increase in the seroprevalence of T. gondii infection in this part of Australia over the last two decades. This high rate of seroprevalence confirms that this disease is widespread among the population which is notable and warrants mitigation.
      Conversely, many other countries have observed a decrease in T. gondii prevalence over the last 30 years. A 2016 nationwide all-cohort seroprevalence study from Germany
      • Wilking H.
      • Thamm M.
      • Stark K.
      • Aebischer T.
      • Seeber F.
      Prevalence, incidence estimations, and risk factors of Toxoplasma gondii infection in Germany, a representative, cross-sectional, serological study.
      reported a lower T. gondii IgG seroprevalence (55%; n = 6,564; age: 18–79 years) than previous similar studies among blood donors in North-East Germany between 1994 and 1996 (59%; n = 4,854; age 20–40),
      • Fiedler K.
      • Hulsse C.
      • Straube W.
      • Briese V.
      Toxoplasmosis-antibody seroprevalence in mecklenburg-western pomerania (in German).
      and from pregnant women from South-West Germany in 1992 (39%; n = 5,670; age 15–47).
      • Beringer T.
      Is diagnosis of toxoplasmosis within the scope of prenatal care meaningful? (In German).
      Likewise, data from France has shown decreasing T. gondii seropositivity, particularly in women of childbearing age, from 54.2% in 1995 to 43.8% in 2003, and 36.7% in 2010.
      • Nogareda F.
      • Le Strat Y.
      • Villena I.
      • De Valk H.
      • Goulet V.
      Incidence and prevalence of Toxoplasma gondii infection in women in France, 1980–2020, model-based estimation.
      In The Netherlands, T. gondii IgG seroprevalence declined from 41% in 1995–1996 to 26% in 2006–2007 (n = 5,541; age 0–79).
      • Hofhuis A.
      • van Pelt Y.T.
      • Nijhuis C.D.
      • et al.
      Decreased prevalence and age-specific risk factors for Toxoplasma gondii IgG antibodies in The Netherlands between 1995/1996 and 2006/2007.
      Furthermore, in the USA from 1999 to 2004, seroprevalence was significantly lower than our study (9.0%; n = 10,477; age 12–49), decreasing from 14.1% in 1988–1994.
      • Nagle C.M.
      • Wilson L.F.
      • Hughes M.C.B.
      • et al.
      Cancers in Australia in 2010 attributable to the consumption of red and processed meat.
      These differences show that improvements in T. gondii infection awareness, monitoring, and prevention in Australia are warranted. Detailed characterisation of Australian national prevalence and risk factors of infection with T. gondii is required to adopt universally appropriate prevention measures to avoid exposure and infection. It is well known that being aware of the ways in which T. gondii are transmitted and strategies to avoid infection is a key way in which the infection can be mitigated.
      • Wilking H.
      • Thamm M.
      • Stark K.
      • Aebischer T.
      • Seeber F.
      Prevalence, incidence estimations, and risk factors of Toxoplasma gondii infection in Germany, a representative, cross-sectional, serological study.
      Because toxoplasmosis is not a notifiable disease in Australia, information on national disease incidence from the general population is missing. As discussed above, previous serosurveys for T. gondii are limited with respect to representativeness and sample sizes. Available reports are based on convenience sampling, mainly from pregnant females, and lack a randomised participant selection protocol. We therefore recommend Australian health authorities consider toxoplasmosis as a notifiable disease to: improve education and raise awareness; lower the number the frequency of primary infections; and commence longitudinal epidemiological data collection. In Germany, congenital toxoplasmosis is a mandatory notifiable disease to the Robert Koch Institute which implements data collection and processing of case data for the German infectious disease notification system.
      • Wilking H.
      • Thamm M.
      • Stark K.
      • Aebischer T.
      • Seeber F.
      Prevalence, incidence estimations, and risk factors of Toxoplasma gondii infection in Germany, a representative, cross-sectional, serological study.
      The observed IgG seroprevalence increased at an average rate of 0.8% with each year of age. A similar rate of 1.09% has been previously reported from Germany.
      • Wilking H.
      • Thamm M.
      • Stark K.
      • Aebischer T.
      • Seeber F.
      Prevalence, incidence estimations, and risk factors of Toxoplasma gondii infection in Germany, a representative, cross-sectional, serological study.
      Seropositivity increased rapidly in all age groups remaining at over 70% in the 65–74, 75–84, and >84 year-old age groups. High seroprevalence in these groups is particularly problematic since immunosuppression becomes more prominent with age. With reference to gender, men had a 1.59-times higher chance of being IgG seropositive. Since Australian males consume almost 50% more processed meat than females,
      • Nagle C.M.
      • Wilson L.F.
      • Hughes M.C.B.
      • et al.
      Cancers in Australia in 2010 attributable to the consumption of red and processed meat.
      and several studies have identified consumption of undercooked or raw meat and meat products as risk factors for foodborne transmission to humans, the higher seroprevalence observed in men in the current study may be partly explained by certain eating habits. In particular, kangaroo meat has been recognised as a source of T. gondii infection in Western Australia and due to its popularity as a lean low-fat meat, it is usually served rare to humans and raw as pet meat.
      • Parameswaran N.
      • O'Handley R.M.
      • Grigg M.E.
      • Fenwick S.G.
      • Thompson R.C.A.
      Seroprevalence of Toxoplasma gondii in wild kangaroo using an ELISA.
      Toxoplasma gondii bradyzoites remain infective when meat is undercooked, making ingestion a risk factor.
      • Parameswaran N.
      • O'Handley R.M.
      • Grigg M.E.
      • Fenwick S.G.
      • Thompson R.C.A.
      Seroprevalence of Toxoplasma gondii in wild kangaroo using an ELISA.
      Robson et al.
      • Robson J.M.B.
      • Wood R.N.
      • Sullivan J.J.
      • Nicolaides N.J.
      • Lewis B.R.
      A probable foodborne outbreak of toxoplasmosis.
      reported an outbreak of 13 cases in Australia that were attributable to the consumption of undercooked kangaroo meat. With respect to the seroprevalence of T. gondii IgM antibodies, it is difficult to draw conclusions due to the low numbers of seropositive subjects in the sampling of the current study.
      In the present study, ownership of cats, dogs, or other pets was not identified to be a risk factor associated with T. gondii seropositivity. Previous epidemiological studies have reported similar observations.
      • Petersen E.
      • Vesco G.
      • Villari S.
      • Buffolano W.
      What do we know about risk factors for infection in humans with Toxoplasma gondii and how can we prevent infections?.
      ,
      • Boyer K.
      • Hill D.
      • Mui E.
      • et al.
      Unrecognized ingestion of Toxoplasma gondii oocysts leads to congenital toxoplasmosis and causes epidemics in North America.
      Although the lack of an association of cat ownership may be surprising, due to the cat family being biologically essential to the life cycle of T. gondii as the only definitive hosts, contact with cats appears to be a less important risk factor when compared to other well established risk factors such as contact with contaminated foods.
      • Wilking H.
      • Thamm M.
      • Stark K.
      • Aebischer T.
      • Seeber F.
      Prevalence, incidence estimations, and risk factors of Toxoplasma gondii infection in Germany, a representative, cross-sectional, serological study.
      While data from other studies support cat contact as a risk factor,
      • Yad M.J.
      • Jomehzadeh N.
      • Sameri M.J.
      • Noorshahi N.
      Seroprevalence of anti-Toxoplasma gondii antibodies among pregnant woman in South khuzestan, Iran.
      • Khalil M.
      • Baothman M.
      • Alserhan F.
      • et al.
      Prevalence of Toxoplasma gondii infection in diabeticpatients in makkah AL mukarramah, Saudi arabia.
      • Avelino M.M.
      • Castro A.M.
      Risk factors for Toxoplasma gondii infection in women of childbearing age.
      prevention of T. gondii infection via cat exposure may be possible as cats only shed oocysts for up to three periods in a lifetime. In addition, oocyst sporulation can be avoided by regular removal of cat litter.
      • Dubey J.P.
      • Ferreira L.R.
      • Martins J.
      • Jones J.L.
      Sporulation and survival of Toxoplasma gondii oocysts in different types of commercial cat litter.
      Hence, the results from the current study may indicate that Australian cat owners are maintaining the hygiene of their animals safely.
      Although previous studies have identified some factors such as BMI and some diseases like cancer as risk factors associated with the infection with T. gondii,
      • Wilking H.
      • Thamm M.
      • Stark K.
      • Aebischer T.
      • Seeber F.
      Prevalence, incidence estimations, and risk factors of Toxoplasma gondii infection in Germany, a representative, cross-sectional, serological study.
      ,
      • Yad M.J.
      • Jomehzadeh N.
      • Sameri M.J.
      • Noorshahi N.
      Seroprevalence of anti-Toxoplasma gondii antibodies among pregnant woman in South khuzestan, Iran.
      ,
      • Khalil M.
      • Baothman M.
      • Alserhan F.
      • et al.
      Prevalence of Toxoplasma gondii infection in diabeticpatients in makkah AL mukarramah, Saudi arabia.
      ,
      • Molan A.L.
      • Rasheed I.H.
      Study the possible link between Toxoplasmosis and different kinds of cancer.
      the results of the present study did not find any association between the selected respiratory and chest conditions; various other disease states; anthropometric and body composition measurements, and T. gondii seropositivity in Busselton, Western Australia. With respect to the biochemical and hematological laboratory analyses, all parameters except red cell distribution width (RDW) were typically normal and no association with T. gondii seropositivity was found among the cohorts of the present study. Significantly higher RDW values were found in seronegative subjects when compared to seropositive subjects (p < 0.01). The RDW forms part of the standard laboratory full blood count measuring the range of variation in red blood cell width and recently, higher values have been associated with various disease states including various forms of cancer
      • Ellingsen T.S.
      • Lappegård J.
      • Skjelbakken T.
      • Brækkan S.K.
      • Hansen J.B.
      Impact of red cell distribution width on future risk of cancer and all-cause mortality among cancer patients – the Tromsø Study.
      ; carotid artery atherosclerosis
      • Wen Y.
      High red blood cell distribution width is closely associated with risk of carotid artery atherosclerosis in patients with hypertension.
      ; and metabolic syndrome.
      • Sánchez-Chaparro M.A.
      • Calvo-Bonacho E.
      • González-Quintela A.
      • et al.
      Higher red blood cell distribution width is associated with the metabolic syndrome.
      Moreover, Patel at al.
      • Patel K.V.
      • Ferrucci L.
      • Ershler W.B.
      • Longo D.L.
      • Guralnik J.M.
      Red cell distribution width and the risk of death in middle-aged and older adults.
      conducted a mortality follow up study of 8,175 adults whose RDW values had been previously recorded. They found that higher RDW values were associated with an increased risk of death and RDW is a strong predictor of mortality. Further investigation is warranted to confirm our findings, and to explore the causes of lower RDW values from seropositive subjects. Likewise, the significantly lower absolute eosinophil counts observed in the seropositive subjects when compared to the seronegative subjects (average of 3.3 and 4.0, respectively, p < 0.025) should be investigated further. Increased eosinophil count (eosinophilia) is a hallmark of parasitic infections
      • Forman R.
      • Bramhall M.
      • Logunova L.
      • Svensson-Frej M.
      • Cruickshank S.M.
      • Else K.J.
      Eosinophils may play regionally disparate roles in influencing IgA+ plasma cell numbers during large and small intestinal inflammation.
      and T. gondii has been shown to cause mild eosinophilia.
      • Matowicka-Karna J.
      • Dymicka-Piekarska V.
      • Kemona H.
      Does Toxoplasma gondii infection affect the levels of IgE and cytokines (IL-5, IL-6, IL-10, IL-12, and TNF-alpha)?.
      The strengths of the present study included a systematic health screening process leading to a characterisation of a representative study population including adjustment for gender and age. The analyses of the present study were based on diagnostic seroprevalence data, however, seroconversion is not equivalent to clinical presentation of the disease. This weakness should be considered in future studies. Lastly, specific environmental variables like lifetime cat exposure or lifetime undercooked meat consumption, that were not available from the BHS survey, should also be considered in future studies.

      5. Conclusion

      Toxoplasma gondii infection in Western Australia is prevalent. Pet ownership, amongst other variables, was not identified as significant risk factor associated with T. gondii infection in both male and female subjects. Toxoplasma gondii infection has been neglected in Australian notifiable disease programs therefore Australian public health authorities should focus on improving education to raise awareness of T. gondii and commence longitudinal epidemiological data collection to contribute and supplement public health models targeting T. gondii transmission control.

      Ethics approval and consent to participate

      approved by the Human Research Ethics Committee of Edith Cowan University on 31 January 2017, approval number: 16090. Written informed consent was obtained from the study participants.

      Funding

      This work was supported by the Joint Project of the Australian National Health and Medical Research Council (NHMRC) and the National Natural Science Foundation of China (NSFC) ( NHMRC APP1112767 - NSFC 81561128020 ). Funds from the grant were used to purchase the ELISA kits used in this study.

      Declaration of competing interest

      The authors declare no conflict of interest.

      Acknowledgements

      this work was supported by the School of Medical and Health Sciences , Edith Cowan University, Joondalup, Western Australia .

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