|Year : 2003 | Volume
| Issue : 2 | Page : 69-76
Mother-to-child transmission and diagnosis of toxoplasma gondii infection during pregnancy
Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi -110019, India
Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi -110019, India
Toxoplasmosis is caused by a coccidian parasite Toxoplasma gondii. It is world-wide in distribution and infects most of the vertebrates. The cat family animals (Felides) are its definitive host. The humans are infected either through contaminated food, water, transfusion of infected blood, organ transplantation or from mother-to-foetus through placenta. The prevalence of toxoplasmosis in Indian pregnant women is variably reported. However, the knowledge about this infection, its diagnosis and interpretation of test results is a major problem in the Indian context. Though Toxoplasma infection does not cause repeated fetal losses, this is the most common indication for investigation of toxoplasmosis in India. There are several diagnostic test kits available in Indian market, however, their quality is not assessed by most of the laboratories before they are procured. There is also no baseline data on the antibody titres in various subpopulations of India. This review is focused on various modes of transmission, prevalence data and its significance and interpretation of various antibody tests in pregnant mothers and in babies.
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Singh S. Mother-to-child transmission and diagnosis of toxoplasma gondii infection during pregnancy. Indian J Med Microbiol 2003;21:69-76
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Singh S. Mother-to-child transmission and diagnosis of toxoplasma gondii infection during pregnancy. Indian J Med Microbiol [serial online] 2003 [cited 2015 Aug 31];21:69-76. Available from: http://www.ijmm.org/text.asp?2003/21/2/69/7978
Toxoplasmosis is caused by the protozoan parasite, Toxoplasma gondii. Humans and other warm-blooded animals are its hosts. The infection has a world wide distribution. Approximately one-third of all humanity has been exposed to this parasite. Although usually asymptomatic in immunocompetent adults, it can cause severe disease manifestations and even death in immunocompromised patients. If acquired during pregnancy it can cause various congenital anomalies in the child.,, In India, the exact seroprevalence of this infection is not known. However, using various diagnostic tests, the prevalence has been reported to be as low as 5% and as high as 80% in adults. There is lack of awareness and knowledge about this zoonotic infection. Further complicating the situation are several commercial organisations who are promoting their products without proper background knowledge and base line data from India. This review is expected to bring out important and relevant aspects of toxoplasmosis in India.
| ~ Etiology|| |
Toxoplasmosis is not a new disease. The parasite was discovered in 1908 but its mode of transmission remained a mystery until 1970 when the full life cycle was discovered. Cats including wild Felidae are the definite host and all other warm-blooded animals including humans are intermediate hosts. Only cats can excrete the resistant stage of T. gondii (the oocyst) in faeces. The oocysts are formed as a result of a sexual cycle in the intestine of the cats. Only asexual cycle occurs in intermediate hosts. In intermediate hosts T. gondii is capable of multiplying in virtually any nucleated cell of the body. It multiplies by a special form of binary fission (called endodyogeny) until the host cell ruptures. The stage of the parasite during the acute phase is called the tachyzoite (tachy = fast, zoite = organism). After repeated divisions, tachyzoites become encysted in the body, mostly in muscle and neural tissues. The encysted stage (tissue cyst) contains relatively slowly multiplying organisms (called bradyzoites, brady = slow). Tissue cysts persist in the body as long as the host lives. The tissue cyst is a dead end phase of the parasite in the intermediate host waiting to be eaten by the definitive host, cat. Upon ingestion of tissue cysts by the cat, bradyzoites are liberated in the stomach and intestines. In feline intestinal epithelial cells, bradyzoites first undergo a special form of asexual development and merozoites liberated from schizonts produce male and female gametes. After fertilization, a wall is formed around the zygote and an oocyst is produced. After rupture of epithelial cells, oocysts are liberated in the intestinal lumen and are expelled in the faeces of infected cats. Oocysts are unsporulated (noninfective) when passed in faeces. They can sporulate within one day in feline faeces. Sporulated oocysts are extremely resistant to environmental influences and can survive freezing and drying for months. Sporulated T. gondii oocysts are infectious to humans and to all other warm-blooded hosts. Upon ingestion of sporulated oocysts, sporozoites are liberated in the gut lumen. Sporozoites enter intestinal epithelial cells, become tachyzoites and multiply in the intestine and associated lymph nodes before circulation to other organs via lymph and blood. After the ingestion of tissue cysts from meat, bradyzoites are liberated from tissue cysts by the action of digestive enzymes and penetrate the intestinal epithelial cells and become tachyzoites.,
| ~ Mode of Transmission and Prevalence|| |
There are three primary ways of transmission: by ingesting uncooked meat containing tissue cysts, by ingesting food and water contaminated with oocysts from infected cat faeces, and congenitally. Transplacental infection occurs when an uninfected mother acquires infection during pregnancy. First there is a parasitaemia in the mother, then invasion of placenta, and finally T. gondii spreads to foetal tissues. Overall, less than 0.1% of the general population becomes infected congenitally. The parasite can also be transmitted by transplantation of organs and transfusion of blood.,,,,,,,,,
Until recently the prevalence of T. gondii in the general population of India was considered to be low compared with Western countries.,,,,,, This may be partially true, but the data are based on convenience samples obtained from patients admitted to hospitals in big cities. The type of serological tests used and the titres taken as evidence of infection are also important factors. Because most of the population (75%) of India lives in rural villages with little means to get to city hospitals, the data are not reflective of the population as a whole. Moreover, data are based on low titres (64) in IHA Test, the specificity of which is doubtful. Three recent studies, however, indicate that the prevalence may be a lot higher than previously considered.,, Of 123 serum samples obtained from 27 villages surrounding a hospital in Kulapur, UP, 18% had IHA antibodies to T. gondii at a titre of 1:64. Prevalence increased with age. Of importance is that four of 41 (10%) children below nine years of age were seropositive suggesting exposure to contaminated food or congenital infection. In another survey of 100 asymptomatic women (83 of them 16-50 years old) from villages in Almora district, 57% had IHA antibodies in titres of 162 or more (20 of them had titres of 18 to 54). Even if one discounted lower IHA titres as nonspecific, this is a high prevalence of T. gondii in women of child bearing age, ever reported earlier from any part of India. In another study of children from a residential tribal school in Maharashtra, 35.1% of 194 persons had T. gondii antibodies. Of particular interest is a 30% prevalence rate in 10 to 12 year old children and IgG positivity in 75% adult food handlers of the school mess. In a recent study from a Scandinavian country, incidence of Toxoplasma gondii infections in 3,094 Swedish women during pregnancy was performed during 1992-93. Specific IgG anti-toxoplasma antibodies were found in 14% (450/3,094). A seroconversion during pregnancy was found in only four women, whose children were followed upto four years of age. No signs of sequelae, either neurological or ophthalmological, were found in three of the children. The fourth child died at one year of age of a disease of different aetiology. The incidence of primary toxoplasma infection of 1.1/1,000 pregnancies in this study is slightly higher than the earlier reports from USA where rate of primary toxoplasma infection is <1/1000 pregnancies.,,
Little is known of the sources for T. gondii infection for humans in India. Rawal found that the prevalence of T. gondii in vegetarians (37.8% of 141) and nonvegetarians (37.4% of 246) was similar. We also carried out a study in rural area near Delhi on primigravida women but could not find any significant difference between vegetarians and non-vegetarians. Also no difference was found on statistical analysis between women who had contact with cat and who did not have. The overall prevalence as measured by IgG antibodies was 41.2%. Epidemiologic studies from other parts of world indicate that the ingestion of undercooked meat is an important means of transmission of T. gondii. For example, the very high (70 to 90%) seroprevalence of T. gondii in Paris, France is attributed to eating undercooked meat.
Traditionally, meat is cooked well before human consumption in India. T. gondii is killed when the internal temperature of the meat reaches 66°C. However, the recent introduction of fast food chain restaurants in India might change this situation. T.gondii may survive in improperly grilled or barbecued meat. Such an outbreak did occur in a group of medical students in New York city who ate hamburgers (presumably contaminated with meat from other animals) which were undercooked. Toxoplasma gondii is also killed by freezing; freezing in a household freezer for one day is generally sufficient to kill this parasite in meat.
Poor hygiene observed in India during handling of meat from slaughter house to kitchen can be a source of T. gondii infection.,, Although no data are available on the consumption of undercooked meat in India, the prevalence of Sarcocystis suihominis oocysts in faeces of 14 of 20 children indicates that meat was consumed raw at least by some because S. suihominis can only be transmitted to humans by the consumption of raw pork.
Prevalence of T. gondii in animals in India, is not well known. Serological surveys indicate that infection is prevalent in sheep and goats. On a worldwide basis, T. gondii is more prevalent in sheep, goats, and pigs than in cattle, water buffaloes and horses. In India, however, seroprevalence of this infection in sheep and goats of dryland, such as Rajasthan, is significantly lower than from animals of damp and humid areas like Kumaon region., In fact, T. gondii is extremely rare in beef (both cattle and buffaloes) and thus presumably of little importance in the epidemiology of T. gondii. Chickens raised in wire cages are also rarely infected but backyard poultry is often infected because of the contamination of soil with T. gondii oocysts. Only recently the prevalence of T. gondii in chickens of South India was reported to be 39.5%. Surveys of farm-raised pigs indicate a relatively low prevalence of T. gondii. However, little is known of the prevalence of T. gondii in pigs raised by sweepers and poor farmers. These pigs roam free during the day, eat almost anything including human and animal excreta, and return home during the evenings. Most of these pigs are slaughtered at home. Small children that help with butchering are often rewarded with the tail of the pigs that are then consumed raw.
Unlike the stages of T. gondii in meat, which are killed by water, oocysts in cat faeces are extremely resistant. Oocysts can survive freezing and drying. Virtually nothing is known of the contamination of soil, water or food with T. gondii oocysts in India. The social stigma of catching or trapping cats in India for the collection of serum or fecal samples makes conducting these studies difficult.
| ~ Toxoplasmosis as a cause of reproductive failure|| |
Much that is known about diagnosis and management of T. gondii during pregnancy has come from the studies in Austria and France where it is compulsory by law to test all pregnant women for T. gondii. Women are tested for T. gondii antibodies on their first visit to their gynaecologist., Seropositive women are not tested further during pregnancy, in countries where no national screening programmes exit. However, in countries like France and Austria, all seropositive women are also tested every trimester for rising IgG titres.,,, Those women who seroconvert during pregnancy are followed clinically and their fetuses are examined for T. gondii infection by ultrasound, amniocentesis, and for the presence of T. gondii in amniotic fluid and fetal blood.,,,,,, Women who have acquired T. gondii infection during pregnancy are treated with spiramycin to prevent transmission of T. gondii from the placenta to the fetus and with sulfadiazine and pyrimethamine to prevent fetal damage if the fetus is found to be infected with T. gondii. Important findings from these studies are:
a. Infection of the mother before pregnancy rarely, if ever, results in birth of a congenitally infected child.
b. Half of the women who acquire T. gondii infection during pregnancy do not transmit the parasite to their fetus.
c. Toxoplasma gondii is transmitted more frequently during the latter part of gestation but the disease is more severe if infection is acquired during the first and second trimesters.
d. Detection of T. gondii in amniotic fluid is possible with polymerase chain reaction (PCR).
e. Except in rare instances, T. gondii does not cause multiple abortion or sterility in women.
There is considerable confusion and uncertainty concerning T. gondii as a cause of multiple abortion, sterility and other reproductive failures in India because most data are based on serology. There are several shortcomings in the reports linking habitual abortion to T. gondii infection, such as low number of patients, uncontrolled studies, and absence of serologic data before pregnancy.,, We realize that there are technical difficulties in conducting a well controlled prospective study in India that is necessary to establish a causal relationship between toxoplasmosis and abortion. Even isolation of T. gondii from the endometrium several weeks after abortion does not prove congenital toxoplasmosis because T. gondii has been found in the uteri of non-pregnant women also. Furthermore, even when the placenta is infected, the fetus may escape infection.,
It needs to be re-emphasized that the women who are seropositive before conception, have least risk to their babies, if at all. The greatest risk of congenital toxoplasmosis occurs during the first trimester of pregnancy. However, it is during the third trimester that the highest level of transmission occurs. This is thought to be related to the much larger size of the uterus. The transmission rate from a maternal infection is about 45%. Of these 60% are sub-clinical infections, 9% result in death of the foetus and 30% have severe damage such as hydrocephalus, intracerebral calcification, retinochoroiditis and mental retardation.,,,, Also, if a women foetal loss is proven due to Toxoplasma gondii infection, her subsequent pregnancies are safe as far as this parasitic infection is concerned, until she becomes immunocopromised during subsequent pregnancies. However, occasional reports of congenital toxoplasmosis transmitted, by an immunocompetent woman infected before conception, to her fetus are on record.
| ~ Immunology|| |
To understand immunology of toxoplasmosis various studies have been carried out and several models have been used by various workers, including human sera, cat sera and experimental mouse models. Some proteins of the T. gondii are recognized by IgG, IgM and IgA antibodies in patients with acute and chronic toxoplasmosis depending on the strain and stage of the Toxoplasma. In the acute phase, IgM antibodies have shown a recognition frequency (f = 0.60) for the 60kDa protein, and in the chronic phase the IgG antibodies have shown a recognition frequency (f = 0.68) for the 12kDa protein. Seronegatives revealed number of bands. The 12kDa protein can be a diagnostic marker of the chronic phase while protein 60kDa of the acute phase of toxoplasmosis.
As far as animals are concerned, about 60% of all adult cats are likely to have IgG type of antibody titer. Immune response of cats infected both with T. gondii and the feline immunodeficiency virus (FIV) was studied and the results showed no correlation between cat ownership and infection with toxoplasmosis. It is also reported that even in immunocompromised individuals, such as AIDS patients, toxoplasma antibody seroconversion is unusual and appears unrelated to cat ownership. If cat-owning immunosupressed individuals do not show a higher incidence of infection, it stands to reason that normal cat-owning persons are not at greater risk of infection either. Faecal examinations are essentially not useful, since the oocysts are only shed in the faeces in the first 2-3 weeks of infection, during a time when even a cat that later becomes ill exhibits no symptoms. As mentioned above, the current laboratory test only indicates exposure to toxoplasmosis, not the presence of active infection. When naturally infected cats were compared, T. gondii-specific IgM immune complexes were found only in clinically ill cats. T. gondii-specific IgG immune complexes were more common in clinically ill cats than healthy cats, but was found in some cats without active disease. As the animal overcomes the disease and becomes immune the IgM antibodies shift to IgG antibodies. Lappin and his associates have examined in depth the cat's immune responses to toxoplasmosis infection. They have identified Toxoplasma gondii-antigen immune complexes in feline serum (both T. gondii-specific IgG-IC and T. gondii-specific IgM-IC). The latter group can be used as a far more accurate indicator of active toxoplasmosis infection than the tests currently available. These researchers developed an ELISA assay for this group of antibody-antigen complexes. These authors also found that Excretory - Secretory Antigen (ESA) had highest specificity [Table - 1].
| ~ Clinical Features of Congenital Human Toxoplasmosis|| |
Depending on the age at the time of primary infection, the virulence of the strain of T. gondii, and the immune status of the host, symptoms vary from no symptoms to death.,,, Generally, congenitally acquired toxoplasmosis is more severe than postnatally acquired infection. The severity and likelihood of infection is dependent on the trimester of pregnancy the mother becomes infected with T. gondii. Toxoplasmosis is more severe in infants whose mothers become infected during the first trimester than those during the third trimester. Although most congenitally infected children are asymptomatic at birth, they will develop some symptoms later in life. Hydrocephalus, chorioretinitis, intracerebral calcification, mental retardation, loss of hearing cholangitis, hepatosplenomegaly, pancytopenia and death may occur.,, Loss of vision is the most common sequela in congenitally infected children. Although most post-natally acquired infections are asymptomatic, manifestations of toxoplasmosis include large lymph nodes (particularly of the cervical region), headaches, muscle aches and sore throat. Because these symptoms are nonspecific, postnatal toxoplasmosis is rarely diagnosed.,
| ~ Toxoplasmosis in Immunocompromised Women|| |
Toxoplasmosis in AIDS patients and other immunocompromised patients can be life threatening.,,, Disease in these individuals can be due to recently acquired infection or more commonly due to reactivation of a latent infection. Heart and other organ transplantation recipients are at risk for developing toxoplasmosis because of lowering of host resistance by immunosuppressive medication., Similarly, cancer patients are also at risk of developing clinical toxoplasmosis. Toxoplasmosis is one of the opportunistic infections that AIDS patients develop. Approximately 3 to 10% of all AIDS patients die of toxoplasmosis. In Indian patients three fourth have recrudescence to the old infection. Although any organ may be involved, encephalitis is the predominant presentation of toxoplasmosis in AIDS patients. Very little is known about the pregnancy in HIV positive women and reactivation of toxoplasmosis or congenital transmission of toxoplasmosis from such women to their babies. However, in the era of HAART regimen we expect that such studies will be available in the literature soon.
| ~ Diagnosis|| |
A variety of factors may be responsible for foetol loss and several investigations apart from TORCH testing are required to determine the final diagnosis of pregnancy loss. When Toxoplasma infection is suspected in a women during pregnancy or before pregnancy the diagnosis is primarily made by serologic investigations.,,,
The detection of Toxoplasma-specific antibodies is the primary diagnostic method to determine infection with Toxoplasma. Antibodies are detected by numerous serologic tests and most of the test kits are commercially available to detect T. gondii specific IgG, IgM , IgA or IgE antibodies. The Sabin-Feldman dye test (DT), indirect fluorescent antibody test (IFAT), indirect haemagglutination test (IHAT), latex agglutination test (LAT), direct agglutination test (DAT), and enzyme linked immunosorbent assay (ELISA) are some of the tests used to detect T. gondii antibodies. Although the DT is the most specific test, it is rarely used now because it uses live virulent T. gondii. The IFAT is nearly as sensitive as the DT but it requires a fluorescent microscope. The IFAT, LAT, DAT and ELISA are used more commonly. The IHAT lacks sensitivity, especially during acute infection. In addition, it lacks specificity, especially in titres of less than 64. An algorithm for the immunodiagnosis of toxoplasmosis during pregnancy is shown in the flow-chart.
Flow-[chart]: Immunodiagnosis of toxoplasmosis in a pregnant woman
Test serum for presence of Toxoplasma-specific
All suspected patients should be initially tested for the presence of Toxoplasma-specific IgG antibodies to determine their immune status. The presence of IgG antibodies only means exposure because asymptomatic humans can develop very high (>100,000) T. gondii antibody titres in DAT or IFAT, and titres may remain elevated for several years or even whole life if repeated exposures are encountered. Although an 8-fold rise in antibody titre, taken two weeks apart, is indicative of a recent infection, this is seldom achieved in practice because by the time patients are seen in the clinic, antibody titre has usually peaked. Compared to IgG antibodies, IgM antibodies are short-lived, and they appear before IgG antibodies.,,,
If more precise knowledge of the time of infection is necessary, then an IgG positive person should have an IgM test performed by a procedure with minimal nonspecific reactions, such as IgM-capture EIA. A negative IgM test essentially excludes recent infection, but a positive IgM test is difficult to interpret because Toxoplasma-specific IgM antibodies may be detected by EIA for 6-12 months but rarely as long as 18 months after acute acquired infection. A major problem with Toxoplasma-specific IgM testing is lack of specificity. Two situations occur frequently: i) positive IgM but negative IgG, and ii) positive IgG and IgM. In the first situation, the patient's blood should be redrawn two weeks after the first and tested together with the first specimen. If the first specimen was drawn in early infection, the patient should have highly positive IgG and IgM antibodies in the second sample. If the IgG is negative and the IgM is positive in both specimens, the IgM result should be considered to be a false positive and the patient should be considered to be not infected. In the second situation, a second specimen should be drawn and both specimens submitted together to a reference laboratory which employs a different IgM testing system for confirmation. In a pregnant women whose sample is taken in second or third trimester rather than ideally in first trimester, and she is found IgG positive but IgM negative, it is more advisable to perform IgG avidity test. High avidity IgG tests indicate that she acquired the infection more than 4 months ago. But the low avidity is not a confirmatory test for recent infection., Prior to initiation of patient management for acute toxoplasmosis, all IgM positives should be verified by a reference laboratory with experience in toxoplasmosis such as Toxoplasma reference laboratory, Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi.
Diagnosis of congenital toxoplasmosis in a newborn child presents many difficulties because of the transfer of maternal IgG antibodies to the foetus, low sensitivity of serologic tests, and lack of availability and cost of T. gondii specific IgA detection kits., New born infants suspected of congenital toxoplasmosis should be tested by both an IgM- and an IgA-capture EIA. Detection of Toxoplasma-specific IgA antibodies is more sensitive than IgM detection in congenitally infected babies. None of the current commercial assays offered in the United States have been cleared by the Food and Drug Administration for in vitro diagnostic use for infants; consequently, all specimens from neonates suspected of having congenital toxoplasmosis should be sent to the Toxoplasma reference laboratory., Considering the high cost of a screening program for congenital toxoplasmosis during pregnancy, a program to screen cord blood for IgM antibodies to T. gondii, coupled with screening for other congenital infections, might be more cost effective than screening for toxoplasmosis alone.
| ~ Vaccine|| |
Toxoplasmosis, in an immunocompetent host, leads to the induction of a life- long protective immunity against illness due to reinfection. These protective antigens are candidates for the development of a vaccine strategy. Different Toxoplasma antigens were entrapped within liposomes and evaluated, in this form, for their ability to protect Swiss mice against Toxoplasma infection: soluble tachyzoite antigen (L/TAg), tissue cyst (L/CAg), tachyzoites plus tissue cyst (L/TCAg) or purified antigen of tachyzoites (L/pTAg). The protein used in L/pTAg was purified from tachyzoites using a stage-specific monoclonal antibody which reacted at a molecular weight of 32 kDa. To compare the immuno-adjuvant action of liposomes and of Freund's Complete Adjuvant (FCA), another group of mice was immunized with soluble tachyzoite antigen (STAg) emulsified in FCA (FCA/TAg). Control groups were inoculated with (STAg) alone, phosphate-buffered saline (PBS), FCA with PBS (FCA/PBS) and empty liposomes (L/PBS). Mice were inoculated subcutaneously with these antigens at six, four and two weeks before a challenge with 80 tissue cysts of the P strain of Toxoplasma gondii orally. All mice immunized with or without adjuvant showed a humoral response, as measured by ELISA. However, no correlation was found between antibody titer and protection against the challenge. All mice immunized with L/pTAg or L/TCAg survived (100%), whereas 80% and 90% of mice from groups which received respectively PBS or FCA/PBS and L/PBS died. All mice immunized with antigen entrapped within liposomes (L/TAg, L/CAg, L/TCAg and L/pTAg) showed low numbers of intracerebral cysts. A vaccine effective against the intracellular protozoan parasite Toxoplasma gondii may make use of both the dense granule protein GRA2 and surface antigen 1 (SAg1) from the organism.
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