Toxoplasmose Trends

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Toxoplasmosis: beyond animals to humans Yaowalark Sukthana Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand

The parasitic zoonosis toxoplasmosis, which was poorly understood before the advent of the HIV epidemic, has become a major clinical problem worldwide. Humans acquire toxoplasmosis from cats, from consuming raw or undercooked meat and from vertical transmission to the foetus through the placenta during pregnancy. Studies of the unique environmental factors in various communities indicate the important roles that eating habits and culture have on the transmission of this infection. The socioepidemiological aspects of toxoplasmosis are thought to be important contributing factors for the spread of this disease. Preventative measures should consider the cultures and beliefs of people in various communities more than solving poverty and giving orthodox health education. Toxoplasma gondii Toxoplasma gondii can infect humans, and warm-blooded domestic and wild animals such as birds and rodents. In 1908, Nicolle and Manceaux (from the Pasteur Institute in Tunisia) isolated a new parasite from the African rodent Ctenodactylus gundi, differentiated it from Leishmania and named it T. gondii a year later [1]. The first congenital case of toxoplasmosis was described in 1923 and the first adult case was diagnosed in 1940 [2]. However, its life cycle was not known until 1969 [1]. The development of the dye test by Sabin and Feldman in 1948 [2] was an important milestone and it is now the ‘gold standard’ serological method for diagnosing toxoplasmosis [3]. Cats and other felids are the only definitive hosts of Toxoplasma in which sexual reproduction occurs to produce infective oocysts. Warm-blooded animals, including humans, are intermediate hosts that harbour tissue cysts in their bodies. Although asymptomatic in normal hosts, T. gondii can cause severe disease in immunodeficient subjects. Since the HIV–AIDS pandemic, concurrent Toxoplasma infection has become an important health problem, with its frequency increasing worldwide during the 1980s. Patients with toxoplasmic encephalitis were first documented in Thailand in 1992, and the number of cases has been increasing annually, particularly in the northern part of the country [4]. Early diagnosis is, therefore, crucial. However, diagnosis of toxoplasmosis is not straightforward because of pleomorphic clinical manifestations. Laboratory diagnosis is important in primary infections, in pregnant women and Corresponding author: Sukthana, Y. ([email protected]). Available online 30 January 2006

in congenital toxoplasmosis, whereas clinical manifestations are more likely to be obvious in late reactivated cases (e.g. ocular toxoplasmosis) and in immunocompromised individuals. Practical strategies for investigating and interpreting results should be focused for each of the clinical groups. Diagnosis and management of congenital infection The diagnosis of congenital toxoplasmosis relies mainly on finding specific antibodies in patient serum and on the isolation of T. gondii DNA from amniotic fluid. Many serological screening methods can detect IgG and IgM that are specific for T. gondii. When confirmation of initial serology is required, ranges of secondary tests are available, including the Sabin–Feldman dye test and a test for specific IgM or other immunoglobulin such as IgA, IgE and IgG avidity [5,6]. The sensitivity and specificity of screening methods range from 95.6% to 100% and from 94.8% to 99.8%, respectively [7–10], and they are commercially available and easy to perform. However, they should be used with caution for screening pregnant women living in an area with a low prevalence of Toxoplasma infection, such as in Thailand. Sukthana et al. [10] found that the commercial latex agglutination test showed 100% sensitivity and 94.8% specificity compared with the Sabin–Feldman dye test but its positive predictive value was only 71.3%. Therefore, the number of false seropositive cases would be higher than it ought to be and clinicians would miss a certain number of seronegative women who should be given preventative measures to protect them from T. gondii infection during pregnancy. Therefore, confirmation of seropositivity is needed. The immunological response to T. gondii is unique in that IgM appears first, approximately one or two weeks after infection, closely followed by IgA and IgE [11]. In most cases, these acute-phase immunoglobulins peak after approximately two months. The time at which IgM can no longer be detected varies depending on the sensitivity of the assay employed but it is usually between six and nine months after infection. IgG appears after IgM and reaches maximal levels after four months, then declines to lower levels over the next 12–24 months, whereas IgG persists at a low-titre level for the remainder of the subject’s life [11]. Based on all recent data and antibody dynamics, pregnant women with high positive titres for Toxoplasma IgG and IgM, or seroconverted mothers will be offered additional testing using IgA, IgE or IgG avidity to try to determine the time of primary infection more accurately [5,6,12,13]. Low avidity is indicative of recent infection. However, the current consensus is that high IgG

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avidity can be used to rule out recent infection. An IgG avidity index higher than 0.300 indicates an infection that was acquired more than four months before testing [14,15]. In 2001, a multicentre evaluation of primary T. gondii infection in pregnant women from 20 European reference centres recommended that a combination of two assays, each 95% specific, would reach a net specificity of 99.75%. A pair of positive results would be regarded as a positive diagnosis. Furthermore, the best assay combination was the measurement of IgM and IgG avidity, which gave a diagnostic result with specificities of w99% while maintaining diagnostic sensitivities at 95% or higher [6]. In the past decade, the use of PCR has facilitated major improvement in the diagnosis of many parasitic infections, including toxoplasmosis. T. gondii DNA can be detected in amniotic fluid, foetal tissue, blood, cerebrospinal fluid (CSF) and other clinical specimens. Primers are selected from either the P30 or the B1 gene [16–18]. The sensitivity of amniotic fluid PCR can be increased from 81% to 91% when combined with mouse inoculation [17]. The routine prenatal screening of all pregnant women remains controversial and the cost-effectiveness must be taken into account, especially in areas with a low prevalence of toxoplasmosis. Although the screening of all pregnant women would decrease the incidence of congenital toxoplasmosis compared with targeted screening only when foetal anomalies are noted, it would cause 18.5 pregnancy losses from invasive prenatal testing for each child with congenital toxoplasmosis prevented. Even if all foetuses identified as being affected were terminated, there would still be 12.1 pregnancy losses for each case of congenital toxoplasmosis avoided [19]. Consequently, apart form France and Austria, postnatal or neonatal screening is preferred in countries with lower prevalences [20–22]. Recently, the management of congenital toxoplasmosis has improved because the early diagnosis of Toxoplasma infection in mother, foetus and newborn can be made by a combination of serology and PCR methods [23]. Therefore, the treatment of congenital infection is now initiated before birth. However, the prevention of infection in pregnant women should be emphasized by using education about hygiene and avoidance of the risks of infection. The more time that elapses after maternal seroconversion and before the start of treatment, the greater the risk of congenital infection. In 2001, Gilbert et al. [24] suggested that treatment starting within four weeks after maternal seroconversion did not increase the risk of congenital infection, whereas it was 1.29 and 1.44 times higher if treatment commenced between four and seven weeks, and more than eight weeks, respectively. When foetal infection is proven, treatment by pyrimethamine and sulfadiazine is recommended until birth. Folinic acid should always be administered to prevent bone marrow suppression. Therapy using pyrimethamine and sulfonamides is continued immediately after birth in infants who are known, from the prenatal diagnosis, to be infected. Alternative drugs include spiramycin and clindamycin for patients with early pregnancy. A clinical profile of the infant is completed by ophthalmological examination and transfontanelle ultrasound scan. www.sciencedirect.com

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Diagnosis and management of toxoplasmic reactivation in HIV–AIDS For clinically suspected central nervous system (CNS) toxoplasmosis in AIDS patients, the Centers for Disease Control and Prevention (http://www.cdc.gov/) criteria should be applied for presumptive diagnosis. These criteria consist of: (i) the recent onset of a focal neurological abnormality that is consistent with intracranial disease or reduced consciousness; (ii) evidence from brain imaging of a lesion with mass effect or a lesion that appears on a radiograph after injection of a contrast medium; and (iii) positive serum antibody to T. gondii or successful response to treatment for toxoplasmosis [25]. Computerized tomography and magnetic resonance imaging are preliminary diagnostic tools for clinicians, although neuroimaging often cannot differentiate cerebral toxoplasmosis from tumours such as lymphoma. Singlephoton emission computerized tomography could provide a more precise diagnosis but it is costly and not widely available at present [26,27]. Serum and intrathecal levels of T. gondii antibody are always low [28] and parasite isolation from blood and CSF is successful in !40% of CNS toxoplasmosis cases. However, in suspected cases, even a low titre of T. gondii antibody aids the diagnosis. By contrast, when T. gondii antibody is negative, toxoplasmic encephalitis is unlikely to have occurred. Brain biopsy is often impracticable, although direct tissue staining with haematoxylin–eosin and enhanced by immunocytochemistry could be applied [29]. DNA-amplification-based techniques greatly contribute to the diagnostic improvement. Blood PCR as a single test is not sensitive; CSF PCR has a higher sensitivity (50–100%) and specificity (97–100%). Repeated testing and combining both CSF and blood PCR enhance sensitivity [30–32]. Stage-specific oligonucleotide primers could provide a more precise laboratory diagnosis of reactivated toxoplasmic encephalitis, especially in recurrent cases [33,34]. The treatment of suspected toxoplasmic encephalitis using a combination of pyrimethamine and sulfadiazine with folinic acid is effective. Unfortunately, up to 40–50% of patients treated develop adverse effects that require a change of therapy. Clindamycin is an alternative drug in the case of intolerance to sulfa- compounds [35]. Maintenance therapy using half the dose of therapeutic drugs to prevent recurrent toxoplasmic encephalitis is necessary because the available drugs are ineffective against the tissue cyst that could later be reactivated [35]. The use of highly active antiretroviral therapy (HAART) suppresses the HIV viral load and improves the CD4CT-cell count, followed by a strong reduction of opportunistic infections, including toxoplasmic encephalitis. The influence of HAART in reducing toxoplasmic encephalitis has been confirmed in a randomized, controlled clinical trial and there is no increase in risk of developing this disorder even if drug prophylaxis is discontinued* [36]. * J.M. Miro, et al., abstract 796, 10th World AIDS Conference on Retroviruses and Opportunistic Infection, Boston, February 2003.

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Table 1. Seropositivity rates in Europe, the Americas and Southeast Asia Continents and countries Western Europe Austria Belgium France Germany Italy The Netherlands Spain Switzerland Scandinavia Denmark Finland Norway Sweden Central and Eastern Europe Croatia Poland Slovenia UK Yugoslavia The Americas USA Central America Costa Rica Cuba Mexico Panama South America Argentina Brazil West Indies Southeast Asia Indonesia Malaysia Thailand a

Year

Seropositivity (%)

Refs

1998 1997 2001 2004 2001 2004 2004 1995

43 50 Up to 75 26–54 18–60 40.5 28.6 46

[46] [47] [39] [48] [39] [49] [50] [51]

1999 1995 1998 2001

27.8 20.3 10.9 14.0–29.4

[52] [53] [54] [22]

2000 2001 2002 1998 1992

38.1 46.4–58.5 34 57–93 23–33

[55] [56] [57] [58] [59]

2004

16–40

[60]

1996 1993 2001 1988

76 60 35 90 (at 60 years of age)

[61] [62] [39] [63]

2001 2001 1991

72 59 29.7

[39] [39] [64]

2000 2004 1992, 1997, 2000, 2001

58a 44.8 2.3–21.9

[65] [66] [67–70]

Male:female ratioZ63:52.

Epidemiology and transmission Geographic distribution T. gondii is found worldwide but its prevalence is uneven among people of different countries in the same continent, such as those in Western and Central Europe and in Southeast Asia (Table 1).

Transmission to humans Humans become infected with toxoplasmosis mainly by eating uncooked meat that contains viable tissue cysts or by ingesting food or water contaminated with oocysts from the faeces of infected cats. A high prevalence of infection in France has been related to a preference for eating raw or undercooked meat, whereas it has been related in Central America to large numbers of stray cats in a climate that favours the survival of oocysts. A major concern is whether acquired toxoplasmosis is mainly the result of consuming

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infected meat or consuming food contaminated with oocysts from cat excreta [37]. A European multicentre study that included selected cities in Belgium, Denmark, Italy, Norway, Switzerland and the UK identified the consumption of undercooked meat as the strong risk factor for acquiring a T. gondii infection [38]. Consuming raw pork and tasting raw meat during meal preparation were the principal risk factors in Poland [39]. These risk factors have also been associated with seroconversion for T. gondii in case-control studies of healthy adults in France [40], Yugoslavia and the USA [39]. However, whereas the consumption of raw or undercooked meat was consistently identified as a risk factor, the relative importance of the risk factor and the type of meat associated with it varied among different countries (Table 2). These findings might reflect differences in the eating habits of consumers or different prevalences of infection in meat-producing animals in the affected regions [39]. Domestic cats have a key role in the epidemiology of T. gondii infection. Cats and other feline species can become infected with T. gondii either by ingesting infectious oocysts from the environment or by ingesting tissue cysts from an intermediate host through feeding on food scraps that contain meat or viscera of livestock or game animals. Depending on the host species, the geographic area and the season of the year, up to 73% of small rodents and up to 71% of wild birds might be infected with T. gondii [39]. The prepatent period in cats is short and, after a primary infection with T. gondii, domestic cats can shed large numbers of oocysts into the household, thereby putting their owners at risk of infection. Stray or domestic cats that are allowed to roam about could contaminate the environment with oocysts; this might affect livestock that will later be slaughtered for human consumption. Antibody to T. gondii can be detected in up to 74% of the adult cat population, depending on the type of feeding and whether the cats are kept indoors or outdoors [41]. Seroprevalences are usually higher in stray or feral cats than in cats living in an urban or suburban environment. Between 9% and 46% of pet cats in Europe, South America and the USA showed serological evidence of past exposure to T. gondii, whereas seroprevalences of T. gondii infection have been estimated to be 6–9% in Asia [39,41]. In Central and South America, where levels of T. gondii infection are high, transmission by consumption of tissue cyst can be excluded because meat is usually well cooked [41]. Access for cats to the outdoor environment, and feeding cats with leftovers or with raw viscera and raw meat were the risk factors for human infection in Mexico and Brazil [42,43]. A study of residents and workers on

Table 2. Percentage of Toxoplasma infection in humans associated with types of meat consumed Country Belgium Denmark France Italy Norway Switzerland www.sciencedirect.com

Beef (%) 6 27 ORZ5.5; (95% CI: 1.1–27.0) 12.5 19 8

Pork (%) 2 2 – 3 3 13

Lamb (%) 10 8 ORZ3.1; (95% CI: 0.85–14.00) 0.5 21 10

Salami (%) 10 4 – 12.5 3 5

Refs [38] [38] [40] [38] [38,71] [38]

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swine farms in Illinois (USA) showed that canine infection with T. gondii increases the risk of human infection and that contact with soil is a probable mechanism of transmission [44]. The increased risk of seropositivity in human males is attributed to less attention being paid to cleanliness in food preparation and consumption than in females. In Thailand, cats are kept as pets but they are allowed to roam freely outdoors. They are rarely trained to defecate in litter boxes and, thus, do so anywhere, from the backyard to the roof of the house. Buddhism is the major religion in Thailand and it has a vital role in Thai society. Belief in the five precepts of Buddhism, one of which states that killing is sinful, and practices such as never killing, or allowing the killing of, unwanted pets lead to unwanted pet dogs and cats being abandoned in temples, where monks are obliged to care for them (Figure 1). It is estimated that w20 000 dogs and w10 000 cats are left in 500 Buddhist temples in Bangkok (Thailand). These animals can roam everywhere in temple grounds at any time, even when religious functions are being performed. They are fed with leftover food that, although plentiful, might not be hygienic. There is no regular veterinarian visit and no antiparasitic drugs are prescribed. The overcrowded conditions make Buddhist temples a place of risk for acquiring zoonotic infection. A seroepidemiological study of T. gondii in cats and their owners in the metropolitan area of Bangkok was conducted involving community households and those in Buddhist temples, covering an area of 106.6 km2 containing 494 931 inhabitants [37]. Serum samples were collected from 327 household members, monks, novices and nuns living in the temples and from 315 stray cats in the temple boundary. It was found that 7.3% of the cats studied and 6.4% of the humans studied were seropositive for Toxoplasma antibody. The risk of Toxoplasma seropositivity in the exposed human group was five times that in the non-exposed group [OR (95% CI) Z5.43 (1.28–23.04): pZ0.01]. Of the studied cats, up to 80% defecated anywhere, and in most cases (up to 75%) the excreta of the cats were not buried or removed. Infected cats with unrestricted defecation

Figure 1. Abandoned cat and dog in a Buddhist temple in Bangkok (Thailand) being looked after by an obliging monk. Photograph courtesy of Bangklao Nok Temple. www.sciencedirect.com

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would contaminate their immediate environment and, therefore, provided a potential source of human infection. Thus, it seems that cat ownership is a risk factor for Toxoplasma infection in Thailand. Risk was increased in and around temples, particularly if courtyards were made of earth or grass, suggesting that ground temperature is an important determinant of oocyst survival [37]. As mentioned, access for cats to the outdoor environment, and feeding cats with leftovers or with raw viscera and raw meat are risk factors for human Toxoplasma infection in Mexico and Brazil [42,43] but the situation is different in Thailand. Thai cats eat rice and well-cooked fish but not raw meat. They also catch rodents in response to hunting instincts but usually not for eating. This could explain why the prevalences of Toxoplasma infection in cats and humans are low in Thailand, even though many factors seem to promote transmission. Moreover, Dubey [45] demonstrated that oocysts remain infective for only one minute at 608C compared with 54 months at 48C. Thus, oocysts from cat excreta deposited on hot roofs and stone or concrete courtyards are unlikely to be successful in disease transmission [37].

Prevention Although consumption of infected meat and close association with infected cats are the two main sources of Toxoplasma infection in humans, details as to how these are brought about differ in different countries or even in different ethnic communities that belong to the same country. Eating habits vary even among inhabitants of the West. For example, the distribution of beef, pork and lamb consumption in Europe is uneven (Table 2) and there are religious differences among Southeast Asian people (e.g. Buddhist or Islamic faith). Even the manner in which domestic cats are kept and fed in various communities and countries is not similar. Cats in Thailand are not kept indoors; they roam about and are fed on rice and cooked fish but not meat or livestock viscera as in Mexico. Although the climate and ground temperature in both countries are similar, the prevalence of T. gondii in Mexico is higher than in Thailand. In Southeast Asia, the Toxoplasma seropositivity rate is much higher in Surabaya (Indonesia), a predominantly Islamic community in which cats are better looked after than in Bangkok. Addressing the problem of disease prevention should, therefore, take into account ethnic and cultural differences. Proper hygienic measures when taking food and when keeping pets will reduce the chance of Toxoplasma transmission to humans but ignorance and poverty are not the only important factors that contribute to the high prevalence of zoonotic diseases. Increasing both the public awareness of these diseases and the sense of responsibility for looking after pet animals is important. However, social science research strategies regarding the importance of culture, tradition and beliefs in the transmission of disease are also necessary.

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