Branko Bobić

Risk factors for Toxoplasma infection in a reproductive age female population in the area of Belgrade, Yugoslavia.

The contribution to Toxoplasma infection of known transmission risk factors such as consumption of undercooked meat, contact with soil, and contact with cats, as well as that of age, degree of education, residence in central vs. suburban city communities, and year of entry into the study, has been investigated in a group of 1157 female residents (age range 15 to 45 years) of a defined geographic area (Belgrade) during a 4-years period (1988–1991). The rate of infection increased with age, ranging from 57% to 93%, with an overall mean of 77%. However, it decreased significantly over the study period (p < 0.01). Of the potential risk factors examined, regression analysis showed that the following: age (relative risk (RR): 1.18, 95% confidence interval (CI): 1.02–1.37, p=0.022), undercooked meat consumption (RR: 2.22, 95% CI: 1.2–2.86, p=0.001), and the year of entry into the study (RR: 0.69, 95% CI: 0.6–0.8, p=0.000) were significantly associated with infection. However, while the consumption of undercooked meat contributed to the frequency of infection in the whole group, its significance increased with the degree of education but decreased with age, and was greater in women residing in the suburbs. In addition, in women below age 20, exposure to soil (farming, gardening) was significantly associated with infection (RR: 1.38, 95% CI: 1.12–1.97, p=0.037). Since cats are the single source of toxoplasma oocysts, the above finding indicates that cats are an epidemiologically significant source of environmental contamination in Belgrade. However, cat ownership itself as a criterion of contact with cats was not associated with infection (p = 0.326). In the absence of a general screening in pregnancy program in Yugoslavia, these data point out the groups of pregnant women at the highest risk of infection and provide a basis for a region-appropriate educational program to prevent congenital toxoplasmosis.

High prevalence of intestinal zoonotic parasites in dogs from Belgrade, Serbia--short communication.

To identify areas of risk for canine-related zoonoses in Serbia, the aim of this study was to provide baseline knowledge about intestinal parasites in 151 dogs (65 household pets, 75 stray and 11 military working dogs) from Belgrade. The following parasites, with their respective prevalences, were detected: Giardia duodenalis (14.6%), Ancylostomatidae (24.5%), Toxocara canis (30.5%), Trichuris vulpis (47.0%) and Taenia-type helminths (6.6%). Of all examined dogs, 75.5% (114/151) were found to harbour at least one parasite species. Of these, mixed infections with up to four species per dog occurred in 44.7% (51/114). Infections with all detected species were significantly higher (p < 0.05) in military working (100%) and stray dogs (93.3%) versus household pets (50.8%). Among all parasites, agents with zoonotic potential including Giardia, Ancylostomatidae and Toxocara were detected in 58.3% (88/151) of all examined dogs with a significant difference (p < 0.05) among the subgroups (100%, 62.7% and 46.2% for military working dogs, stray dogs and household pets, respectively). The high prevalence of zoonotic parasites registered in the dog population from a highly urban area in south-eastern Europe indicates a potential risk to human health. Thus, veterinarians should play an important role in helping to prevent or minimise zoonotic transmission.

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.

Onset of ocular complications in congenital toxoplasmosis associated with immunoglobulin M antibodies to Toxoplasma gondii.

Four patients with congenital toxoplasmosis serologically diagnosed by the Sabin-Feldman test (SFT) and the IgM-indirect fluorescent antibody test (IgM-IFAT) in the first year of life presented with eye disease between the age of 21 months and ten years. Repeated serological testing revealed increasing levels of specific antibodies as measured by the SFT. IgM antibodies toToxoplasma gondii were detected in all four patients by the immunosorbent agglutination assay, in two by the IgM-IFAT and in three by the IgM-indirect haemagglutination test. Findings suggest that specific IgM antibodies reappear at the time of reactivation of congenital toxoplasmosis later in life, or possibly persist for an extraordinary long period (up to ten years).

Toxoplasmosis as a travel risk

Toxoplasma gondii is a protozoan parasite with worldwide distribution that infects more than one third of the global population. Primary infection in immunocompetent individuals is usually asymptomatic; however, different organs can be affected in immunocompromised individuals leading to the development of encephalitis, myocarditis or pneumonitis. The prevalence of infection with Toxoplasma as well as its genetic structure varies geographically and for that reason travel may be considered as a risk factor to acquire the infection. As toxoplasmosis is a foodborne disease, health care providers should give health education on prevention measures to all prospective travelers in order to decrease the risk of infection in endemic areas. This review presents an overview of the infection with T. gondii with some considerations for travelers to and from endemic zones.

Kinetics of Toxoplasma infection in the Balkans.

ZusammenfassungZIEL: Die Kinetik der Toxoplasmose-Infektion am Balkan wurde untersucht. DATENQUELLE: In den letzten 30 Jahren in allen Balkanländern veröffentlichte Berichte über Toxoplasmoseinfektionen bei Frauen im gebärfähigen Alter. ERGEBNISSE: Das wesentliche Merkmal der Toxoplasmoseinfektion am Balkan ist ein mit der Zeit kontinuierlicher Rückgang der Infektionshäufigkeit. Das systematische Erfassen der Prävalenz der Toxoplasmoseinfektion in der Population gebärfähiger Frauen in Slowenien, Serbien und Griechenland hat in den letzten 30 Jahren einen signifikanten kontinuierlichen Rückgang in allen drei Ländern gezeigt. Auch in Montenegro und in der früheren jugoslawischen Republik Mazedonien (fjRM) wurde ein Abfall beobachtet, wobei in diesen Ländern aber nur in den letzten 10 Jahren eine systematische Erfassung vorliegt. Ein anderes überregionales Merkmal ist die Beobachtung, dass die Prävalenz der Infektion zurzeit nicht über 50 % liegt. Außerdem besteht im Osten der Balkanhalbinsel eine Abnahme der Prävalenz der Toxoplasmose von Norden nach Süden: von Süd Ungarn (als Nordgrenze des Balkans) über Serbien und fjRM nach Nord Griechenland. Weiters wurde im Westen Sloweniens und im Osten Serbiens eine Saisonabhängigkeit mit signifikant mehr Fällen der akuten Infektion im Winter als im Sommer festgestellt. Trotz eines allgemeinen Trends zur Abnahme der Infektion scheinen die Risikofaktoren der Übertragung in der Region verschieden zu sein: während ein Kontakt mit Katzen in Slowenien wichtig schien, wurde die Einnahme von nicht ausreichend gekochtem Fleisch als wichtigster Risikofaktor in Serbien und Albanien gefunden. In der fjRM und in Nord-Griechenland war der Kontakt mit Dünger der führende Risikofaktor. SCHLUSSFOLGERUNG: Ein Trend zur Abnahme der Prävalenz der Toxoplasmose im Balkan mit der Zeit ist Teil eines sich verändernden Musters der Toxoplasmose Infektion in ganz Europa. Strategien zur Vorbeugung einer kongenitalen Toxoplasmose sollten diese Dynamik der Infektion berücksichtigen.SummaryAIM: The kinetics of Toxoplasma infection in the Balkans were reviewed. SOURCE OF DATA: Published reports on Toxoplasma infection in women of childbearing age in the last 30 years for all Balkan countries. RESULTS: The dominant feature of Toxoplasma infection in the Balkans is a continuous decrease in the prevalence over time. Systematic monitoring of Toxoplasma infection prevalence in populations of women of childbearing age in Slovenia, Serbia and Greece over the last 30 years has shown a continuous significant decrease in all three countries. Moreover, a decrease has also been shown in Montenegro and Former Yugoslav Republic of Macedonia where Toxoplasma infection has been surveyed only during the past decade. Another region-wide feature is that the prevalence of infection currently does not surpass 50%. Furthermore, a decrease in Toxoplasma prevalence from the north to the south has been shown in the eastern part of the Balkan Peninsula, from southern Hungary (as a region neighbouring the Balkans at the north), over Serbia and FYRoM to northern Greece. Seasonality of infection, with significantly more cases of acute infection in the winter than in the summer, was observed in Slovenia in the west and Serbia in the east. Despite a common decreasing trend, different infection transmission risk factors seem predominant across the region; while contact with cats was discussed as important in Slovenia, consumption of undercooked meat was shown to be the leading risk factor in Serbia and Albania, and contact with soil in FYRoM and in northern Greece. CONCLUSION: A decreasing trend of Toxoplasma prevalence over time in the Balkans is part of a changing pattern of Toxoplasma infection throughout Europe. Strategies for the prevention of congenital toxoplasmosis should take the infection dynamics into account.

Giardiasis in dogs and cats in the Belgrade area

Giardia infection is an ongoing problem in Serbia, since 8,0% of the children in the Belgrade area are infected. In the light of the zoonotic potential of Giardia infection, a total of 248 companion animals (167 dogs and 81 cats) were examined for giardiasis in the same urban area. The overall rates of infection in dogs and cats were 14.4% (24/167) and 22.2 % (18/81), respectively. Significantly (p<0.05, p<0.01) higher infection rates were found in stray (18.7%) and farm (36.4%) dogs than in household pets (7.4%). Furthermore, the rate of infection was significantly (p<0.01) higher in domestic cats than in household dogs (22.2%vs. 7.4%). The owners and all 16 of their family members of five out of the six household pet dogs excreting Giardia were Giardia-negative, while both the owner of one dog and the only other family member were Giardia-positive. The data demonstrate that potential animal reservoirs (dogs and cats) exist in urban Serbia and underline the possibility of transmission between dog and man in close contact, thus having important implications for the epidemiology of giardiasis in urban areas.

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.