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Emerging parasites

Brazilian forms of toxoplasmosis

Local types of encephalitis-causing protozoa are beginning to appear

THAIS A. DA C. SILVA / CRISTINA DA S. MEIRA / FABIO COLOMBO / IALA cyst in the brain under the microscopeTHAIS A. DA C. SILVA / CRISTINA DA S. MEIRA / FABIO COLOMBO / IAL

In early January, a 22-year old woman with a severe headache and paralysis on the left side of her body went to a specialized outpatient clinic in the city of São Bernardo do Campo, in São Paulo’s Metropolitan Area. The nurse who examined the woman convinced her to take a quick HIV detection test to check for AIDS. The result, available 30 minutes later, showed that the woman had tested positively.

“The woman had been sexually active for eight years, and engaged in unprotected sexual intercourse with two or three partners a week. She had never undergone a HIV detection test and had no idea that she had AIDS,” reports physician José Ernesto Vidal, who examined her soon afterwards. The headache and the left-side paralysis suggested a form of encephalitis caused by the Toxoplasma gondii protozoan. The physician immediately hospitalized the woman so that she could be treated for the neurological disease and be given anti-AIDS medication as soon as possible.

Cerebral toxoplasmosis has always been an opportunistic infection, normally transmitted by contaminated water or food (and not by sexual intercourse). This disease usually surfaces when the body’s defense system is weakened. Now this kind of infection is drawing more attention because molecular and genetic tests, used as part of the diagnosis, have shown varieties of T. gondii that are specific to Brazil and, as such, can cause non-typical or more serious symptoms in some of the infected people. “Our tests have shown that the Brazilian varieties are genetically different from the classical 1, 2 and 3 types, seen mostly in the United States and Europe. Some of these varieties can be particularly virulent,” says Vidal.

Vidal and his team from the Emilio Ribas Institute of Infectious Diseases partnered with the team headed by Vera Lucia Pereira-Chioccola, from the Adolfo Lutz Institute, to conduct a pioneering study in São Paulo, which identified two genotypes (set of genes) labeled with the numbers 6 and 71. These two genotypes are proving to be very aggressive in human beings, and cause serious forms of encephalitis. A third genotype, labeled 65, was shown to be quite frequent, indicating that this might be the most common variety in São Paulo State. Of the three most widely detected varieties, two are common in Brazil (types 1 and 3). Recently, type 2 of the Toxoplasma gondii, common in Europe, North America and Asia, was found in birds in Fernando de Noronha,” Vera Lucia points out.

The three varieties now identified in human beings resulted from the analyses of blood and amniotic fluid samples from 62 patients treated at the hospitals Emilio Ribas and Hospital de Base de São José do Rio Preto from January 2007 to January 2010. Of these patients, 25 had cerebral toxoplasmosis and Aids. “In Brazil, cerebral toxoplasmosis is the first AIDS-defining neurological disease and the one with the highest incidence among Aids patients not treated with anti-retroviral medication,” says the researcher from Adolfo Lutz. Two other patients had acute toxoplasmosis, 12 had ocular toxoplasmosis, 17 were pregnant women with acute toxoplasmosis and six patients were newborn babies with congenital toxoplasmosis.

The researchers were able to form 20 complete genotypes and associated them with the clinical data of each patient to examine the virulence of each genotype. The 65 type, identified in 18 people, showed a variable virulence: it caused a relatively mild form of encephalitis in these patients, which could be treated with medication. The 65 type also caused acute toxoplasmosis in four people whose defense systems were apparently in good condition. One of these was a woman who had had an acute infection during pregnancy, with no symptoms of toxoplasmosis. Another woman developed cerebral toxoplasmosis, even though she was HIV negative. The 71 and 6 types were identified in patients with severe cerebral toxoplasmosis, and who died even after undergoing treatment. The genotypes were characterized by Isabelle Martins Ribeiro Ferreira, from the Adolfo Lutz Institute. The related paper was published in the October 2011 issue of the Experimental Parasitology journal.

JOSÉ VIDAL and VERA LUCIA P. CHIOCCOLAA cyst in the brainJOSÉ VIDAL and VERA LUCIA P. CHIOCCOLA

The 71 genotype has already been found in hens; the 65 type has been found in hens and cats, and the 6 type has been found in birds, house pets and sheep, reflecting the ways in which this parasite is transmitted. The T. gondii can enter the human body through food – especially raw or underdone vegetables and meat – or through water contaminated by cysts contained in cat or wild cat feces, the natural reservoirs of these protozoa. There was an outbreak of this disease in 2006 when six people – one of them a pregnant woman – ate steak tartar, prepared with ground raw beef, at a lunch held in a gated property in the resort town of Guarujá, on the São Paulo State coast. The meat was contaminated and the pregnant woman had a miscarriage.

The risk of toxoplasmosis is one of the biggest fears of pregnant women. In addition to miscarriage, the congenital transmission of this disease, from the mother to the fetus, can cause premature labor, neonatal infections, blindness or neurological deficiencies. The intensity of this risk is being measured by several studies. In one of these, a team from the Federal University of Minas Gerais (UFMG) examined 146,307 newborns from November 2006 to May 2007. This number corresponded to 95% of the live infants born in the state of Minas Gerais during that period. Of this total number, 190 babies had congenital toxoplasmosis, with a high incidence of retina infections. The prevalence of one infected baby for every 770 newborns was regarded as a very high rate, reinforcing the hypothesis that Brazil might host more virulent varieties of the parasite than other countries.

The congenital infection sometimes manifests itself only in adolescence or adulthood. Vera Lucia says that if a 6 to 10-year old schoolboy complains of poor eyesight or a narrower visual field, the boy’s teachers and parents should consider the possibility that the boy was infected by the T. gondii when his mother was pregnant.

This is why Vidal emphasizes that “pregnant women without signs of infection should be the priority group to be targeted in prevention campaigns and to undergo serological tests during pregnancy.” However, the diagnosis is not simple. “The parasite load is high at only one moment,” says Vera Lucia.

She believes in the existence of other T. gondii varieties capable of infecting human beings  that have not been identified yet for a number of reasons. The first is that the survey on Brazilian genotypes is recent – the survey began in 2005 and was conducted on animals. The second is that researchers worked with blood samples of 10 milliliters, the maximum they could draw from people that took part in the studies that led to these conclusions. Thus, the chance of obtaining the parasite itself is slight, because the only element obtained from the samples is the DNA of the protozoa found in the midst of the human body’s genes. The T. gondii protozoa, capable of infecting human beings, was first and simultaneously identified in 1908 in rodents by Charles Nicolle and Louis Manceaux at the Pasteur Institute of Tunis, and in rabbits by Alfonso Splendore, in Brazil.

In their attempt to find other means to identify the parasite in the blood, the researchers from the Adolfo Lutz Institute observed that the tachyzoites release proteins referred to as ESA (excreted/secreted antigens), that facilitate their entry into the host cells. At the moment, in one of the glass-walled laboratories on the institute’s eighth floor, Thais Alves da Costa Silva and Cristina da Silva Meira are cultivating tachyzoites in the appropriate culture media and then filtering the ESA. The material they are working with is the same, but the objectives are different: Thais uses the proteins to immunize mice and observe the organism’s response to these antigens, while Cristina uses the proteins to diagnose infections in human beings.

EDUARDO CESARCulture of cells for diagnosisEDUARDO CESAR

Stealthily
When the T. gondii enters the organism, it causes fever, spots on the body, a swollen liver, and other signs that disappear after a few days. The defense cells surround and isolate the parasites, which can remain in the body, in the form of cysts, for decades or years. The cysts usually remain under control and the infection goes unnoticed. There are no accurate numbers on the incidence and prevalence of toxoplasmosis, as it is not mandatory to report this disease. The non-symptomatic infection, however, is relatively common: estimates are that one out of every three people hosts small populations of this parasite.

“A substantial part of the human population is infected by the T. gondii, but the immune system is able to control the infection and people become non-symptomatic for their entire lives or until immunosuppression arises,” says Vera Lucia.

Only 20% to 30% of the infected individuals develop the disease, which tends to happen when the organism’s defense system is weak, as in people with AIDS or who have undergone a transplant. In these cases, the cysts burst and release tachyzoites into the blood, thus spreading the infection. According to Vidal, as the parasites’ cysts expand or burst, the inflammatory response that they unleash can damage the brain tissue and cause multiple outbreaks of encephalitis with variable degrees of hemorrhage.

Researchers suspect that, when entering the brain, the T. gondii might facilitate the development of psychiatric disturbances, such as schizophrenia or the manifestation of suicidal tendencies. In 2011, researchers from the University of Hawaii in the United States published a paper in the Journal of Nervous and Mental Disease on the association between T. gondii infection and a higher suicide rate among women over the age of 60.

In the last few years, a number of studies have indicated that the parasite might induce behavioral changes in laboratory animals, such as making mice lose their fear of cats. At present, the parasite’s mode of action has become clearer. In a paper published in September 2011 in PLoS One, researchers from the University of Leeds in England described how the T. gondii can manipulate the behavior of the hosts because it induces an increase in the production of dopamine, a neurotransmitter. This results in unheard-of courage among the laboratory animals.

Several studies have considered the possibility that healthy people might also be subject to behavioral changes caused by the T. gondii ; however, it has not been fully proven yet that this parasite could cause memory loss, attention deficiency, or slower reasoning. However, this parasite could increase the risk of traffic accidents.

Traffic accidents
In a paper published in 2009 in Forensic Science International, researchers from Turkey’s University of Istanbul examined the blood samples of 218 people who had been involved in non-fatal traffic accidents and of 25 people who had died in traffic accidents, all of whom had a history of toxoplasmosis and had not consumed alcoholic beverages before the accidents. They compared these samples with the blood samples of 191 people who had also been involved in traffic accidents but had no history of toxoplasmosis. The conclusion of this comparison was that the brain infection caused by this protozoan can slow down the driver’s reflexes, probably because of changes in the levels of dopamine circulating in the body.

Vidal points out that this situation generates a gridlock. In principle, and based on the few studies already published, people infected by the protozoa should avoid driving. However, there are no scientific arguments to support this, nor are there any laws that restrict daily activities such as driving, or work such as driving a taxi.

There are very few actions that prevent the transmission of this parasite. One of the existing actions was implemented by the California State administration, in the United States. The state established that cats could only be sold at pet shops if the cat owners submitted proof that the animals were free of Toxoplasma gondii.

Specialists state that the treatment of serious forms of the disease ( a combined sulfa-based therapy) is efficient in 90% of the cases, but does not necessarily undo the damage caused to the brain by the parasite, such as loss of movements and some cognitive abilities. This is why Vidal believes that the 22-year old woman he saw last month will not be totally cured and will probably have neurological sequelae, even though she was no longer at risk of dying from encephalitis. In his opinion, this woman possibly transmitted the virus to other people before encephalitis became manifest, as she did not know that she was HIV-positive.

In São Bernardo do Campo, Vidal has, on average, seen one HIV-positive adolescent every week. “They tell me shocking stories. At parties, three boys have sexual intercourse with the same girl, knowing that she is HIV-positive, to see who is going to be contaminated. Few people go to hospitals or health care centers to take the HIV test, which can also detect a recent infection, and begin treatment, and, above all, avoid behavior that perpetuates the transmission of the virus in the community.”

The Project
Laboratory diagnosis of toxoplasmosis with focus on congenital and brain infections (nº 2008/09311-0); Modality Regular Funding for Research; Coordinator Vera Lucia Pereira Chioccola – Adolfo Lutz Institute; Investment R$ 104,698.75 (FAPESP)

Scientific article
FERREIRA I.M. et al. Toxoplasma gondii isolates: multilocus RFLP-PCR genotyping from human patients in São Paulo State, Brazil identified distinct genotypes. Experimental Parasitology. n. 29, v. 2, p. 190-5. Oct. 2011.

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