Genetics is instigating a new form of diagnosing neurocysticercosis, the most important parasite illness that attacks the central nervous system. The ailment characterizes itself through the installation in the brain of a larva acquired by the ingestion of food contaminated with eggs of the Taenia solium and is practically unheard of in developed countries. But it emerges as a major public health problem in wide regions of Latin America, Asia and Africa, producing neuropsychiatric injuries and killing between 15% to 20% of its victims. In Brazil it is endemic to various regions and it is presumed to attack 140,000 people. The spectrum of the illness is not known for certain, because of diagnostic difficulties. In general, neurocysticercosis is only researched in patients who have sought out neurology clinics with symptoms such as epilepsy crises and psychiatric disorders. But recent studies indicate that the incidence of the illness is high even in states supposedly free of it. There are tests capable of detecting the antibodies produced against the invader, but not one of them is specific to the point of attesting to or being exclusive to the illness. The diagnosis is only conclusive with the help of nuclear magnetic resonance (NMR) imaging or with a computerized tomography scan, which point out one or more cysts, at times many, that populate regions of the patient’s brain. The new situation is that genetic research beckons a cheap test capable of detecting vestiges of DNA of the worm in samples of the cerebrospinal fluid, also known as a liquor, which surrounds and protects the brain.
A team led by the specialist in molecular genetic, Emmanuel Dias Neto, from the Neurosciences Laboratory of the Medical School of the University of Sao Paulo (FMUSP), has demonstrated for the first time that the DNA of the invader is present in the liquor. And they have developed a molecular detection examination for the presence of the parasite based on the multiplication of the DNA by way of the PCR method ( Polymerase Chain Reaction). The test was created in partnership with researchers from the Neurology Department of the FMUSP, of the Pharmaceutical Sciences Faculty of USP and the Medical Faculty of USP in Ribeirao Preto, within a project financed by the National Counsel for Scientific and Technological Development (CNPq) and by the Alzira Denise Hertzog Silva Benevolent Association (Abadhs). On evaluating some thirty patients with neurocysticercosis, the new method exhibited a sensitivity of 96.7% in detecting the parasite – it gave a positive result in twenty nine (29) of the cases. A control group of ten patients without the disease, attested to its efficiency. The team is submitting their findings for publication in a scientific magazine and has gone in search of a patent. “We already have a company interested”, says Emmanuel. The research will be presented in the master’s degree thesis of Carolina Rodrigues Almeida, supervised by Dr. Emmanuel at the Psychiatry Department of FMUSP. The coming of the test promises to expand the diagnosis of neurocysticercosis, offering precise and cheaper examinations than the diagnosis through neuroimaging .
The genetic hopes are reaching other sides of the illness. Emmanuel’s team is still the leader in pioneering work in the identification of the functional genes of this cysticercosis, the larval form that causes neurocysticercosis. 2,880 ESTs have already been identified – fragments of active genes that are denominated as Expressed Sequence Tags, in accordance with a techniques conceived by the researcher Dr. Andrew Simpson and by Dr. Emmanuel himself, when they had worked at the Ludwig Cancer Research Institute. The Brazilian methodology received the acronym Orestes (Open Reading Expressed Sequence Tags). This technique has already been used in the Cancer Genome Project and also during the study of the Schistosomiasis mansoni genome, which causes schistosomiasis. “I was surprised when I discovered that there was no sequencing of the EST type of the parasite that causes neurocysticercosis”, says Emmanuel, who became interested in the question while working at the Psychiatry Department of the FMUSP. “We’ve already found a large quantity of new genes of the parasite, which include candidates for the development of therapies and tests”, adds Carolina Almeida.
The possibility of widening the diagnosis of neurocysticercosis is essential for combating the malady. “The illness is extremely serious and confronts many problems in the field of prevention, and begins with the fact that there is no obligation to notify cases”, says Vicente Amato Neto, a full professor at the Infectious and Parasite Illnesses Department of the FMUSP. “It seems that very frequently neurological clinics deal with the main symptoms but it’s difficult to know the true occurrence in the population because there’s no search to detect it.” Recent studies show that the spectrum is even greater than had been imagined. An example is the situation in Piauí, a state that was not included on the map of incidents of the malady in Brazil. In reality it seemed to be free of the ailment because nobody had taken the trouble to look for neurocysticercosis there. Between 1999 and 2001, A research team from three states, led by Alberto Novaes Ramos Junior, from the Community Health Department of the Federal University of Ceará, carried out a wide ranging investigation into parasite illnesses in João Costa, a poor municipality of some 3,000 inhabitants, living on the edge of the National Park named Capivara Sierra. One hundred and sixty nine (169) people were identified with symptoms comparable to those of human neurocysticercosis, such as migraine headaches and epileptic crises. Immunological examinations detected the antibodies that react against cysticercosis in twenty-seven of them. The major lesson from the Piauí research is that the epidemiological reality of the illness in Brazil remains unknown as it has been only slightly investigated.
Santa Catarina State has also begun to unveil the real dimension of neurocysticercosis. The analysis of 143,000 cCT scans out during the 1990s in different regions has revealed an occurrence of 1.2% of neurocysticercosis. In a more specific universe, of the 1,849 patients attended to with neurological symptoms, the percentage of the positive exams reached the order of 30%. Two regions were pointed to in case rankings, the municipalities of Lages and Chapecó. “They’re regions where pig farming is traditional. But the cases usually involve people from the poor surrounding municipalities where the farming is carried out in a rudimentary fashion”, says Mario Steindel, a professor at the Microbiology and Parasitology Department of the Federal University of Santa Catarina (UFSC). The researcher Maria Márcia Imenes Ishida, also from UFSC, is studying the surroundings of the Lages municipality. Four hundred (400) blood samples have already been collected that are being analyzed in the search for antibodies. The objective is to carry out an epidemiological survey in the locality to determine the prevalence and to identify risk factors associated with the parasite, both in the rural and urban populations surrounding Lages.
Hermaphrodite
The ignorance concerning the ways of being contaminated conspire in favor of proliferation. In general, neurocysticercosis is associated with pork meat and it is imagined that it is enough to avoid the consumption of recipes based on pork, as well as its derivatives, to remain clear of it. That’s not quite the situation. When an individual eats pork contaminated by cysticercosis, the parasite’s larvae, the risk of getting another illness, taeniasis, exists. Once swallowed, the cysts encrusted in the pig’s muscles transform themselves, within the human intestine, into Taenia solium, the adult form of the parasite, known as taenia or tapeworm. One is dealing with a worm with an elongated body in the form of a tape. The taenia is hermaphroditic and manufactures thousands of eggs, which are liberated within the human feces. Each day the undesirable intestine host evacuates between 50,000 and 60,000 microscopic eggs. The cycle of the taeniasis closes when the pig swallows water or food that had contact with the contaminated feces of man. In the pig’s organism, these eggs are converted into new larvae that are going to populate the pig’s meat and, finally, infect the person who eats it. Taeniasis can cause retardation in the growth of children and brings about damage in adults, such as the loss of the capacity to work. But there is a relatively simple treatment: the administration of specific vermifuges is capable of eliminating it from the organism. Another worm, named Taenia saginata, a parasite in cattle, can also cause taeniasis in human beings. But only the Taenia solium, the pork tapeworm, participates in the evolutionary cycle of neurocysticercosis.
Neurocysticercosis is an illness associated with taeniasis, but has very different symptoms. It emerges when man directly swallows the eggs produced in his intestine by the parasite. In the past, it was linked to regions without basic sanitation, where pigs had direct contact with human feces. It is not by chance that even today the occurrence of neurocysticercosis in indigenous settlements is eighteen times higher than in the rest of Brazil. But, with the rural exodus, the illness became urbanized, transmitted in any environment where hygiene is lacking. Infected people who do not wash their hands after going to the bathroom can contaminate food during its preparation. Thus the illness is transmitted to everybody who eats that particular food. There is another form of contaminating, considered extremely rare in which by force of digestion movements the human stomach is contaminated by eggs liberated in the intestine. The eggs, it is said, are extremely resistant. Released into water, on the ground or on vegetation, they remain able to contaminate for up to seven years. But they are destroyed by high temperature, Hence the importance of eating food that has been well cooked. For this reason the main focal points of contamination are foods that are swallowed raw such as vegetables. And there is no point in immersing the salad in a solution to kill off germs, such as vinegar or hypochlorite. The eggs resist. Only the mechanical removal, with running water and the manual cleaning of each leaf, is capable of getting rid of the danger.
The cook
All type of care is but little. A classic case in medical literature was published in 1992 in the scientific magazine The New England Journal of Medicine, in which four orthodox Jews from the same New York family received the diagnosis of neurocysticercosis. For religious reasons they had never eaten pork. And, in the United States the illness was considered to have been eradicated. Research, headed by the parasitologist Peter Schantz, from the Center for Disease Control and Prevention (CDC) of Atlanta, linked to the American government, investigated all the people surrounding the victims and unmasked the mystery. A cook, a recently arrived immigrant from Mexico, suffered from taeniasis and had precarious hygiene habits. She was responsible for contaminating the family for whom she had been working.
In the pig, the cysts normally install themselves in the muscles and do not represent a danger to the animal’s life. However in man, the larva can integrate itself into the muscles or grow well below the skin, where it does not cause major damage except esthetically. There have been cases in which a multitude of cysts have grouped themselves together in the subcutaneous region of the face, causing a deformity. In other situations, much more serious, the cyst invades the ocular globe, which can lead to visual deficiency. “Years ago ophthalmological clinics presented a major casuist situation, because it is relatively easy for an ophthalmologist to identify the cyst inside the eye”, stated Vicente Amato. But the worm has as its preferred location inserting itself into the central nervous system, the final address of 90% of the invaders. For this reason the illness is called neuro-cysticercosis. This is really the main cause of epilepsy in the developing countries. In Brazil it is responsible for around 3% of those attended to through neuro-psychiatry services.
After having arrived at the digestive organ, the eggs pass through the wall of the intestine and penetrate into the bloodstream. When they migrate to the central nervous system, they first reach small blood veins between the nerve
tissue. Neurocysticercosis can present itself in two forms: the cystic form, known as Cysticercus cellulosae, and, in ringlets with numerous cysts, called Cysticercus racemosus. It is said that the infection is active when the cysts are still alive, which on average lasts six years, and inactive when the worms have already died and are found calcified. The illness produces specific damages, depending on the location where it installs itself, the size attained by each lesion (from 2 to 43 millimeters) and by the immunological response of the human organism. It is common that the growth of worms pressurizes regions of the brain and blocks the passage of the cerebrospinal liquor. This liquid is continually produced and any problem that interferes with its absorption and replacement alters the intra-cranium pressure, with serious neurological consequences. It can cause very serious lesions and even death.
The inflammatory action produced by the organism can also cause harm. The first symptoms can be unspecific, such as headaches, convulsions, loss of vision or motor capacity. However, the brain damage can lead to a situation such as epileptic crises, cranium hypertension, hydrocephalus, chronic meningitis and psychiatric disturbances. It has been estimated that only 10% of the cases are asymptotic. According to a research survey into the illness in Brazil carried out by the medical doctor, Svetlana Agapejev, from the Neurology and Psychiatric Department of the Medical School of Botucatu (Unesp), most victims in Brazil are men, aged between 31 and 50 years. The most serious examples, nevertheless, are concentrated amongst women between 21 and 40 years, who live in urban areas, and generally suffer from intra-cranium hypertension. The reason for the difference is unknown. It is speculated that some hormonal factor exists and interferes with the evolution of the ailment.
The larvae survive in the human organism for a period of three to six years. In the past it was relatively common to attempt to rid the illness surgically. It was common and inefficient. Today operations are only indicated in very specific cases, ones in which the lesion is isolated and is found in the accessible regions of the brain. But, above all, recourse to surgery is to minimize the symptoms, such as draining in cases of hydrocephalus. In the same manner, there are treatments administered according to the development of the illness, such as corticoids, in the case of meningitis, or anti-convulsions medicine , if epilepsy occurs, as well as anti-inflammatory and analgesic medicines. Some cases are treated with two medications, albendazole and praziquantel, capable of killing cysticercosis installed in determined accessible locations of the central nervous system. This therapy, however, is controversial. It so happens that the cysticercosis, sooner or later, will truly die – without the power of leaving heirs – and its extermination produced by medication could well unchain a complicated inflammatory process. “A study showed that patients treated with albendazole developed symptoms that were milder than with the patients who had not received the medicine”, sasy Osvaldo Takayanagui, a neurology professor at USP’s Medical Faculty in Ribeirao Preto.
During 1993, the International Task Force for the Disease Eradication, linked to the World Health Organization (WHO), classified neurocysticercosis as a rare example of an ailment that could be eliminated from the planet, amongst some ninety three (93) infectious and parasitic ailments studied. The illness has been eradicated in countries such as Japan, Canada and in the greater part of Western Europe. Today in the United States less than one thousand cases are registered per year. The problem re-appeared thanks to the influx of migratory workers from Latin America, where the illness has spread to some 350,000 cases in eighteen countries. In Mexico, neurocysticercosis accounts for 10% of the final diagnoses carried out in neurological hospitals. It remains a serious public health problem in Africa, where there is a scarcity of epidemiological studies, and in Asia, above all in China and India, but also in Thailand, the Philippines and South Korea. It is estimated that some 50 million people have been infected by the taeniasis/cysticercosis complex throughout the world. 50,000 of them die of it every year.
Preference
An efficient control program for the illness would include, in the first place, the monitoring of the quality of pork meat. Today, the sanitary inspection carried out in Brazil looks for vestiges of the larvae in organs such as the heart, the tongue, the diaphragm and muscles, locations where cysticercosis prefers. But the worm can also be found in other organs, hence the importance of always cooking pork meat well. The ideal situation would be to submit the slaughtered pigs to immunological examinations, which are capable of finding vestiges of antibodies against the cysticercosis. According to directives from the Pan American Health Organization (PAHO), as well as monitoring the meat, it is important to create a system of compulsory notification of cases of taeniasis, including those who visited the families of victims, in order to deter the contamination in the domestic environment. It is also vital to inspect the quality of vegetables and to demand the presentation of a parasite examination of the feces of any professional who works with food during the renovation of their health permit as well as, more obviously, improved sanitary conditions for the population. The PAHO has also suggested, as an extreme measure, administering vermifuges to all of the people within a community that has been badly hit by the illness.
Few Brazilian States, such as Parana, Santa Catarina and Rio Grande do Sul, have some type of preventative program available. The city of Ribeirao Preto, in the interior of the state of São Paulo, has one of the best thought out schemes to combat the illness in the country, brought about by the elevated incidence of the illness in that region over the last few decades. As well as the compulsory notification of cases, even the quality of the vegetables sold at street markets and municipal markets were evaluated. A high level of contamination by fecal coliforms was revealed, a sign of lack of hygiene, but not a single trace of Taenia solium eggs. Since this program began, a rare epidemiological trustworthy statistic concerning the disease in Brazil has come out. There are 74.1 cases of neurocysticercosis in the city of Ribeirao Preto for every 100,000 inhabitants. “We can’t say if its a lot or it’s a little because of the lack of parameters from other locations with which we might compare”, says Osvaldo Takayanagui. The illness, in spite of the program’s efficiency, shows have force. Around 25% of the victims showed live cysticercosis, in a clear signal that the illness was contracted in the very recent past.
Peptides
A still as yet distant promise is the development of a vaccine capable of blocking the taeniasis cycle and that of the neurocysticercosis in the hosts. There are studies taking place in Mexico of a vaccine based on three synthesized peptides shared by Taenia solium and Taenia crassiceps, the parasite of foxes. The major holdup, in this case, is economic: the cost of synthesizing these peptides is high. In the past Chinese researchers concluded that the antigen cC1, obtained from Taenia crassiceps, is a good candidate for the development of a vaccine against Taenia solium. In 1999, an Australian researcher managed to develop a vaccine with the combined use of three different antigens, which achieved a level of protection of 93% of the part of the body that are deliberately infected. Perhaps this vaccine will not be efficient in Brazil given the genetic variability of Taenia solium throughout the planet. “We need to concentrate our efforts towards understanding other genes and proteins of the parasite in order to expand the potential for antigens that can be tested”, says Emmanuel Dias Neto. Brazil, with the experience that it has been accumulating in genomic science, has an important contribution to make in this field.
