Marija Vujanić

Comparative evaluation of three commercial Toxoplasma-specific IgG antibody avidity tests and significance in different clinical settings

Determination of the avidity of specific IgG antibodies has become a generally accepted diagnostic aid for dating Toxoplasma infection. In this study, the Labsystems, VIDAS and EUROIMMUN Toxoplasma IgG avidity assays were compared on a series of 133 Toxoplasma IgG- and IgM-positive sera from symptomatic patients (n=28), from pregnant (n=43) and non-pregnant (n=26) women, and on 18 IgG-positive and IgM-negative sera from chronically infected patients. The results showed excellent concordance between the Labsystems and VIDAS tests in both the IgM-positive (r=0.82, kappa=0.771) and IgM-negative (kappa=0.609) sera, whilst the agreement of the EUROIMMUN assay with both the Labsystems and VIDAS tests in the IgM-positive sera was moderate (kappa=0.575 and kappa=0.525, respectively) and in the IgM-negative sera was poor (kappa=0.000). Analysis of the kinetics of the maturation of avidity in 13 patients in whom follow-up sera were available showed that, despite a general trend of maturation, in two patients the avidity did not become high during 6 and 11 months of follow-up. In view of the clinical setting, in the symptomatic patients, despite one case of complete discrepancy and five cases of partial discrepancy, the Labsystems and VIDAS tests were in almost perfect agreement (kappa=0.812), whilst the agreement in pregnant and non-pregnant women was substantial (kappa=0.754 and kappa=0.708, respectively). In conclusion, the Labsystems and VIDAS tests are equally reliable for the measurement of Toxoplasma IgG avidity; the choice of test should depend on the laboratory set-up. The EUROIMMUN test may be an acceptable alternative in resource-limited settings, but should be used prudently.

Kinetics of parasite burdens in blood and tissues during murine toxoplasmosis.

A sensitive real-time PCR technique was used to examine the distribution of Toxoplasma gondii in the blood and tissues of mice during acute and chronic infection. Groups of Swiss Albino mice, inoculated i.p. with 10(2) or 10(6) tachyzoites of the RH strain as a typical type-1 strain, or fed 10 cysts of the Me49 strain as a typical type-2 strain, were killed at different time points post-infection (p.i.), and blood and organs including the lungs, brain and liver were harvested for DNA extraction. Toxoplasma DNA was quantified by a real-time PCR targeted at the 529bp gene fragment, with a detection limit of a single parasite per g/ml of tissue. The results showed a strain- and dose-dependent spread of Toxoplasma. In infection with type-1 parasites, in case of a high infective dose, Toxoplasma DNA was detected within 24h p.i. in all analyzed tissues including the brain. Conversely, in case of a low infective dose, parasitaemia was undetectable early p.i., at a time when Toxoplasma DNA was detected in the tissues, but reached very high levels as infection progressed. With both infective doses, pre-death parasite burdens were higher in the blood than in the tissues, whereas the same loads in the lungs suggest that reaching these Toxoplasma burdens may be critical for survival. In infection with Me49 parasites, steady high parasite burdens were noted up to the end of the experiment at d42 only in the brain, parasitaemia was low but detectable throughout, and Toxoplasma DNA was completely cleared only from the liver. These data are important to better understand the pathogenesis of toxoplasmosis, and also as baseline data for the experimental evaluation of novel chemotherapeutics.

Toxoplasmosis in Naturally Infected Rodents in Belgrade, Serbia

To assess the role of synanthropic rodents in the epidemiology of urban toxoplasmosis, Toxoplasma gondii infection was examined in 144 rats (Rattus norvegicus) and 12 mice (Mus musculus) captured using live animal traps in three locations in Belgrade city characterized by poor housing and degraded environment. In rats, specific IgG antibodies were detected by modified agglutination test in 22 (27.5%) of the 80 blood samples available. Toxoplasma brain cysts were microscopically detected in 11 (7.6%), and Toxoplasma DNA by real-time polymerase chain reaction was demonstrated in 15 (10.4%) animals. Of these, both cysts and Toxoplasma DNA were detected in five (3.5%) rats. In mice, cysts were observed in 3 (25%), but Toxoplasma DNA was detected in even 10 (83.3%) animals, including all 3 with morphologically recognized cysts. Being a link in the chain of Toxoplasma infection, the existence of urban rodent reservoirs of infection represents a public health risk.

Seasonal Variations in Human Toxoplasma Infection in Serbia

BACKGROUND Seasonal variations in the occurrence of toxoplasmosis have been studied only sporadically. We thus examined the seasonal distribution of acute toxoplasmosis in Serbia during a 4-year period (2004-2008). MATERIALS AND METHODS A total of 391 consecutive symptomatic (recent lymphadenopathy) and 715 asymptomatic (women tested for obstetric reasons) patients were tested for Toxoplasma immunoglobulin G (IgG) (including IgG avidity) and IgM antibodies. The distribution of patients with acute infection, and of all patients from both groups, was analyzed for seasonality. In addition, factors (including undercooked meat consumption, contact with cats and with soil) possibly contributing to seasonality were analyzed in patients with acute infection (cases) matched by age, sex, and time (month and year) of infection (symptomatic) or presentation (asymptomatic) with seronegative patients (controls). RESULTS Acute toxoplasmosis was serologically (IgG avidity low, IgM positive) diagnosed in 39 (10.0%) symptomatic and 38 (5.3%) asymptomatic patients. In both groups, monthly distribution of acute infections showed significant (p < 0.0001) seasonality, which was related to the four seasons of the year (p < 0.0001). Importantly, the observed seasonality was not related to the distribution of all examined patients in either group (p < 0.001). In the symptomatic patients, acute infections occurred more often between October and March (p = 0.0486). Although more asymptomatic acute infections were diagnosed between February and July (p = 0.0037), low IgG avidity suggests that infection had occurred within the previous trimester (between November and April). Undercooked meat consumption was shown as a risk factor for symptomatic infection in the October-March period (odds ratio 7.67, 95% confidence interval 1.61-36.45). CONCLUSION Seasonality patterns should be taken into account in the health education guidelines for the prevention of toxoplasmosis in pregnant women.

Molecular Detection and Genotyping of Toxoplasma gondii from Clinical Samples

Over the past two decades, molecular diagnosis of toxoplasmosis, which is based on the detection of T. gondii DNA in clinical samples, became an indispensable laboratory test. This method is independent of the immune response, and depending on methodological approach, may facilitate more accurate diagnosis, especially in cases in which inadequacy of conventional methods is faced with deteriorating and potentially severe clinical outcome (congenital, ocular toxoplasmosis and cases of immunosuppression).

Human giardiasis in Serbia: asymptomatic vs symptomatic infection

Despite the public health importance of giardiasis in all of Europe, reliable data on the incidence and prevalence in Western Balkan Countries (Serbia, Bosnia and Herzegovina, Croatia, Montenegro and FYR Macedonia) are scarce, and the relative contribution of waterborne and food-borne, or person-to-person and/or animalto- person, transmission of human giardiasis is not yet clear. To provide baseline data for the estimation of the public health risk caused by Giardia, we here review the information available on the epidemiological characteristics of asymptomatic and symptomatic human infection in Serbia. Although asymptomatic cases of Giardia represent a major proportion of the total cases of infection, high rates of Giardia infection were found in both asymptomatic and symptomatic populations. No waterborne outbreaks of giardiasis have been reported, and it thus seems that giardiasis mostly occurs sporadically in our milieu. Under such circumstances, control measures to reduce the high prevalence of giardiasis in Serbia have focused on person-to-person transmission, encouraging proper hygiene, but for more targeted intervention measures, studies to identify other risk factors for asymptomatic and symptomatic infections are needed.

Toxoplasma gondii infection induces lipid metabolism alterations in the murine host

Host lipids have been implicated in the pathogenesis of Toxoplasma gondiiinfection. To determine if Toxoplasmainfection influences the lipid status in the normal host, we assessed serum lipids of Swiss-Webster mice during infection with the BGD-1 strain (type-2) at a series of time points. Mice were bled at days zero and 42 post-infection, and subgroups were additionally bled on alternating weeks (model 1), or sacrificed at days zero, 14 and 42 (model 2) for the measurement of total cholesterol (Chl), high density lipoproteins (HDL), low density lipoproteins (LDL) and triglycerides and adiponectin. At day 42, brains were harvested for cyst enumeration. A significant decrease (p = 0.02) in HDL and total Chl was first noted in infected vs. control mice at day 14 and persisted to day 42 (p = 0.013). Conversely, LDL was unaltered until day 42, when it increased (p = 0.043). Serum LDL levels at day 42 correlated only with cyst counts of above 300 (found in 44% mice), while the change in HDL between days zero and 42 correlated with both the overall mean cyst count (p = 0.041) and cyst counts above 300 (p = 0.044). Calculated per cyst, this decrease in HDL in individual animals ranged from 0.1-17 µmol/L, with a mean of 2.43 ± 4.14 µmol/L. Serum adiponectin levels remained similar between infected and control mice throughout the experiment.

Adverse fetal outcome in the absence of timely prenatal diagnosis of congenital toxoplasmosis

ZusammenfassungHINTERGRUND: Toxoplasmagondii-Infektionen die während der Schwangerschaft auftreten, können über die Plazenta übertragen werden und den Verlauf und das Ergebnis der Schwangerschaft gefährden und somit zu einer lebensbedrohlichen Erkrankung für den Fetus oder das Neugeborene führen. FALLBERICHT: Wir berichten über den Fall eines Schwangerschaftsabbruches wegen einer klinisch manifesten kongenitalen Toxoplasmose (CT), die sowohl serologisch wie auch in einem Bioessay bestätigt worden ist. Bei einer 33jährigen Frau im 7. Schwangerschaftsmonat mit einer Vorgeschichte von einer 3-monatigen Lymphadenopathie, die Anlass für die Untersuchung auf Toxoplasmose war, fand sich sonographisch eine schwere Vergrößerung der fetalen Ventrikel. Basierend auf den serologischen Ergebnissen (hochspezifischer IgG-Antikörper gemeinsam mit spezifischen IgM-Antikörpern) wurde die Diagnose einer mütterlichen Infektion im 2. Trimester gestellt. Im Nabelschnurblut fanden sich serologisch niedrige IgG-Titer als im mütterlichen Blut, jedoch waren sowohl spezifische IgM wie auch IgA-Antikörper vorhanden, die auf eine fetale Infektion hinwiesen. Obwohl die Toxoplasma-DNA mittels PCR aus dem Nabelschnurblut nicht nachgewiesen werden konnte, bestätigte sich die fetale Infektion nach 6 Wochen durch den Bioessay. Während morphologisch keine Toxoplasma-Zysten festgestellt werden konnten, war der serologische Test positiv. Nachdem die fetalen morphologischen Veränderungen unter der angeschlossenen Therapie nicht zurückentwickelt hatten und zum Zeitpunkt der serologischen Tests fortgeschritten waren, entschloss sich die Patientin zum Schwangerschaftsabbruch. SCHLUSSFOLGERUNG: Dieser Fallbericht unterstreicht die potentielle schwere Schädigung durch eine CT als Ergebnis eines ZNS-Befalls. Es ist daher die Notwendigkeit einer prompten und präzisen pränatalen Diagnostik im Falle einer mütterlichen Serokonversion zu fordern, so dass eine adäquate Therapie rechtzeitig etabliert werden kann.SummaryOBJECTIVE: Toxoplasma gondii infection acquired during pregnancy may lead to transplacental transmission and jeopardize the course and outcome of pregnancy, leading to life-threatening disease in the fetus and the newborn. CASE REPORT: Here we present a case of medically terminated pregnancy due to clinically manifested congenital toxoplasmosis (CT) which was proven serologically, as well as by bioassay. Ultrasonographically visualized severe fetal ventriculomegaly in a seven-month pregnant 33-year-old woman with a history of three months of lymphadenopathy was an indication for extensive testing for toxoplasmosis. Based on the serological results obtained (high specific IgG antibodies of borderline but close-to-low avidity, along with the finding of specific IgM antibodies), maternal infection was dated to the second trimester. Cord blood serology revealed IgG levels lower than those of the mothers, but both specific IgM and IgA antibodies were detected, indicating fetal infection. Although Toxoplasma DNA was not detected in the cord blood sample by real-time PCR, fetal infection was definitely confirmed after six weeks by cord blood bioassay results. While no morphologically recognizable Toxoplasma cysts were found, murine serology was positive. Since fetal morphological abnormalities, which could not be reversed by subsequent treatment, were already advanced at the time of serological testing, the patient opted for termination of pregnancy. CONCLUSION: This case demonstrates the potentially severe outcome of CT as a result of central nervous system affection, emphasizing the need for prompt and precise prenatal diagnosis in case of maternal seroconversion, so that proper treatment may be introduced in a timely manner.