Much less popular, but still common, xenodiagnosis appears to be a technique from the Middle Ages akin with the use of leeches; but it is an effective available resource. Uninfected vinchucas are placed within a jar, tucked under the patient’s armpit, and allowed to consume blood for thirty minutes. Their feces are examined thirty and sixty days later for T. cruzi. Obviously, xenodiagnosis has its problems, primarily, obtaining uninfected bugs. This technique is rarely used on children for obvious reasons. Many adults also have phobias and would rather go untested. However, sometimes medical examinations are as painful as the illness. Xenodiagnosis can be an excellent examination for the determining of parasite populations and strains.
Indirect tests that look for T. cruzi antibodies have recently been devised, but they usually are not used during the acute phase because the immune system is in the process of producing chagasic antibodies. Similar to that used for AIDS, an ELISA test has been designed to detect the presence of T. cruzi antibodies; however, the chagasic ELISA sometimes fails to differentiate antibodies of T. cruzi from those of either T. rangelii (a harmless cousin) or Leishmania braziliensis (the causative agent of the mucocutaneous form of leishmaniasis, common in Andean regions where Chagas’ disease is also found). Chronic patients should be tested both ways, even when ELISA comes out negative, to determine the rate of infection. People without noticeable symptoms living in chagasic areas are encouraged to have an ELISA test. If it comes out positive, xenodiagnosis is encouraged to determine the nature of the infection and form of treatment. The Ministry of Public Health and IBBA in La Paz, Bolivia, provide these tests for Bolivians at a reasonable cost (five dollars in 1997).
Table 3
PARASITOLOGICAL METHODS FOR THE DIAGNOSIS OF CHAGAS’ DISEASE
(adapted from WHO 1991:38)
Methods Type of laboratorya Percentage of Sensitivityb
Acute stage Chronic Stage
DIRECT
Thin Smear A/B >60 <10
Thick blood smear A/B >70 <10
Fresh blood examination A/B 80-90 <10
Strout A/B 90-100 <10
Buffy coat on slide A/B 90-100 <10
INDIRECT
Xenodiagnosis B 100 20-50
Blood cultures B 100 40-50
aA: Health center laboratories located in areas at risk ofvectorial and nonvectorial transmission (the infrastructure is that from the first level of medical care upwards). B: Specialized laboratories for parasitological diagnosis. A and B laboratories are found at MSSP and IBBA in La Paz, MSSP in Cochabamba, and MSSP in Santa Cruz. Since 1991 USAID has helped improve these laboratories for chagas diagnosis. Sucre lacks an adequately equipped B laboratory.
bAs compared to xenodiagnosis for the acute stage of the infection and to serological diagnosis for the chronic stage.
Relatively few Latin Americans are tested for T. cruzi due to a combination of factors including cost, pain, and a fatalistic attitude that, if they test positive, medications are prohibitively priced and only partially effective, with the probability that they will be again be parasitized. There is also the social cost of being ostracized, although this is less true in Bolivia than in some other places. Epidemic diseases frequently lead to old and new prejudices surfacing that lead to ostracism of the victims (see Foege 1988, and Lederberg 1988, in regard to AIDS in the United States). It would not be a very popular decision for immigrants to the United States to let others know they are seropositive for T. cruzi.
Table 4
SEROLOGICAL METHODS FOR DIAGNOSIS OF CHAGAS AND ANTIGENS USED
(WHO 1991:40)
Complement-fixation test (CFT). Aqueous or methanol extracts of whole T. cruzi have been widely used but have been replaced by purified fractions of the parasite in an attempt to standardize the sensitivity and specificity measures.
Indirect immunofluorescent antibody test (IFAT). Formol-fixed epimastigotes are stable antigens. The IFAT has the advantage that it can be used for differentiating IgM from IgG antibodies.
Indirect haemagglutination test (IHA). The antigens are polysaccharide or glycoprotein fractions from epimastigotes. Red cells sensitized with those antigens can be stored lyophilized or in suspension.
Direct agglutination test (DA and 2-MEDA). The antigen consists of whole epimastigotes treated with trypsin, and fixed with formaldehyde and filtered to prevent autoagglutination. The test can be used for detection of IgG or IgM antibodies.
ELISA. The antigen consists of peroxidase, or phosphatase-labeled conjugates or fractions of T. cruzi adsorbed to polyvinyl plates or other materials, and is stable. The test can be used for detection of IgG or IgM antibodies.
Latex agglutination. Polystyrene particles absorbed with T. cruzi extracts are used.
APPENDIX 13
Chemotherapy
The first chemical solutions against Chagas’ disease with sufficient activity to justify clinical trials, the bisquinaldines, were not discovered until 1937 (Jensch 1937), and it was not until 1972 that the first drug to combat the disease, nifurtimox, was announced and launched by Bayer for use in some countries of Latin America in 1976 (Bock et al. 1972). (Bayer has discontinued producing the drug as of 1997). Nifurtimox is a 5-nitrofuran derivative (synonyms: Bayer 2502, Lampit) with antiprotozoal activity, and it is also used to treat leishmaniasis and African trypanosomiasis (Reynolds 1986:673; see Figure 32).
Figure 32.
Nifurtimox. (From W.E. Gutteridge, Existing Chemotherapy and Its Limitations, 1985.)
Nifurtimox is administered as a yellow powder that a patient is to dissolve in water and drink three times a day after meals for sixty to ninety days at a daily dose of 8-10 mg per kg for adults, 15-20 mg per kg for children aged one to ten years, and 12.5-15 mg per kg for children aged eleven to sixteen years. Dosages may be as large as 25 mg/kg for severe complications such as acute meningoencephalitis (WHO 1991). Children tolerate the drug better than do adults. Nifurtimox is readily absorbed and rapidly metabolized, with a peak plasma concentration at one to three hours, which declines to zero by twenty-four hours. Some doctors prefer to stagger the treatment in two-month intervals (Jáuregui, interview 6/22/91). Although the usual treatment extends for 120 days, it can be effective when given for sixty days (Macedo 1982).
The earlier the diagnosis is made and treatment initiated, the greater is the chance that the patient will be parasitologically cured (Cancado and Brener 1979). Drug therapy is highly recommended to minimize parasitic invasion of vital tissues (McGreevy and Marsden 1986:117). Good results are achieved early in treatment, as indicated by the disappearance of circulating trypanosomes, remission of disease signs and symptoms, and occasional reversion to a serologically negative condition. Serological tests tend to become negative from six to eight months after treatment, and the cure is considered successful when both parasitemia and serological tests become negative and remain so for at least a year after the end of treatment.
Nifurtimox does have side effects, including nausea, skin rashes, peripheral neuritis, bone-marrow depression, loss of weight, loss of memory, and sleeping disorders, which may lead to depression and general malaise to such a degree that few patients actually complete the treatment period (Gutteridge 1985). When nifurtimox was given to animals in high doses, it produced cancer; but no such effects have been described in human patients (McGreevy and Marsden 1986:117). In one study using nifurtimox in Brazil, all treated patients had weight loss, 70 percent had anorexia, and 33 percent had peripheral neuritis during treatment at 7-8 mg/kg per day for sixty days (McGreevy and Marsden 1986:117). Peripheral neuritis and psychosis depend on dosages of nifurtimox and usually occur at the end of high-dose treatment (15-20 mg/kg per day). Nifurtimox will cause hemolytic anemia in glucose 6-phosphate dehydrogenase (G6PD)-deficient individuals.
The efficacy of nifurtimox varies in different geographical areas. Cure rates appear to decrease going from south to north in Latin America, and this is probably due in part to variation in the sensitivity of different strains of T. cr
uzi (see Appendix 2: Strains of T. cruzi ). Studies of both acute and chronic Chagas’ disease show that patients from central Brazil are relatively unresponsive to nifurtimox as compared with patients from Argentina, Bolivia, and Chile (Cancado and Brener 1979, Brener 1979). The cure rate is about 92 percent in Argentina, Chile, Bolivia, and southern Brazil, and about 53 percent in central Brazil. Low concentrations of circulating parasites are difficult to detect, and monthly xenodiagnosis or blood culture is required.
Regarding acute patients in central Brazil, xenodiagnosis is usually negative during nifurtimox treatment, but it converts to positive in 60-70 percent of the patients over a four-year period. Post-treatment serodiagnosis usually remains positive in chronic patients, suggesting that parasites persist even when xenodiagnosis is negative.
Nifurtimox’s action has been explained by two hypotheses. The first implicates biosynthetic reactions, especially nucleic and protein synthesis (see Sims and Gutteridge 1978, 1979), as a consequence of interaction with nucleic acids, especially DNA, in which single- and double-strand breaks occur (Gugliotta et al. 1980). This mechanism is similar to that suggested for the antibacterial action of similarly acting drugs and also explains the known mutagenicity and carcinogenicity of many 5-nitrofurans (Gutteridge 1985). The second hypothesis explains the lysing of the parasites as a result of drug metabolism, of superoxide anions and hence hydrogen peroxide, which accumulates to cytotoxic levels in T. cruzi because of the absence of catalase (Docampo and Stoppani 1979, Docampo and Moreno 1984). This hypothesis explains the ultrastructural lesions that these drugs produce (Sims and Gutteridge 1979), and T. cruzi does indeed generate free radical metabolites from nifurtimox at physiological drug concentrations (Docampo and Moreno 1984). Neither hypothesis is mutually exclusive-drug metabolism produces both types of activity. Research on the effects of nifurtimox on intact T. cruzi is required to resolve this debate.
Figure 33.
Benznidazole. (From W.E. Gutteridge, Existing Chemotherapy and Its Limitations, 1985.)
Benznidazole was announced in 1974 and released by the Roche pharmaceutical company in Latin America in late 1978 (Barclay et al. 1978; see Figure 33). Benznidazole’s synonyms are R07-1051, Radanil, and Rochagan. It is a 2-nitroimidazole derivative with antiprotozoal activity (Reynolds 1986:660). Also a yellow powder, it is taken in water and is rapidly absorbed and distributed through the body tissues (Macedo 1982). Recommended dosage is 5 mg/kg/day (10mg/kg/day for children) for sixty days (Gutteridge 1985). It is claimed to be effective in curing more than 80 percent of both acute and chronic Chagas’ disease patients. There is no clear evidence that it has any advantage over nifurtimox, and the efficacy of the two drugs is similar, though it has been claimed that benznidazole has less geographic variation in cure rates (Ribeiro-dos-Santos, Rassi, and Köberle 1980).
Initial clinical studies with benznidazole used higher doses, and serious side effects such as polyneuropathy and progressive purpuric dermatitis occurred. Adverse effects of benznidazole include nausea, vomiting, abdominal pain, peripheral neuropathy, and severe skin reactions (Reynolds 1986:660). It causes erythematous light-sensitive skin rashes, which can be severe, in half of the patients (Boainain 1979). A study involving twenty patients with chronic Chagas’ disease who were given benznidazole in a dosage of 5 mg per kg of body weight daily had to be stopped because of the high incidence of skin rashes and neurological symptoms (Apt 1986:1010). Benznidazole also causes a marked thrombocytopenia in humans and depresses thymusdependent immune functions in rabbits (Teixeira et al. 1983).
Figure 34.
Gentian violet. (From W.E. Gutteridge, Existing Chemotherapy and Its Limitations, 1985.)
As possible new drug leads, another nitroimidazole (2-amino-5-[methyl-5nitro-2-imidazolyl]-1,3,4 thiadiazole) has shown remarkable efficacy in mice, curing 89 percent of cases with a single dose (Filardi and Brener 1982). It is also active against parasite strains that are resistant to nifurtimox and 2-nitroimkidaxole derivatives. Trypomastigotes are cleared from the bloodstream in six hours, and destruction of amastigotes occurs in eighteen to thirty-six hours (Almeida et al. 1984). This drug is presently being tested on humans (McGreevy and Marsden 1986:118).
Another promising drug, allopurinol, is presently being researched, with initial studies indicating trypanosomicidal action at daily doses of 600 mg given for thirty to sixty days. Research is still being conducted concerning its efficacy and toxicity.
Another drug, gentian violet, is used to prevent transmission of the disease through blood transfusions. Although gentian violet is effective in destroying trypomastigotes in blood supplies, people dislike receiving a transfusion of violet-colored blood, and it is still being evaluated for safety. Gentian violet (crystal violet) is a cationic dye (see Figure 34) that demonstrates photodynamic action in visible light to produce hydrogen peroxide (Docampo et al. 1988). It is readily soluble in water. First used in 1953, it rapidly lyses trypomastigote forms of T. cruzi in whole blood and thus prevents the transmission of Chagas’ disease through blood transfusion (Nussenzweig et al. 1953). The dosage is 25 cc of solution gentian at 0.5 percent in glucoside isotonic solution for each 500 cc of blood for twenty-four hours, but blood is rarely, if ever, held long enough in Bolivia to be treated with gentian violet (Nussenzweig et al. 1953, Schumuñis 1991). At the University of Brasilia in Brazil, this technique has been used to treat seropositive blood for many years without mishap (Marsden 1983:255). However, it does not work against all strains of T. cruzi (Brener 1979).
Gentian violet exhibits photodynamic action against parasites (Docampo et al. 1988). Visible light causes photoreduction of gentian violet to a carbon-centered radical, and under aerobic conditions this free radical autooxidizes generating anion whose dismutation yields hydrogen peroxide.
Side effects for patients who receive blood with gentian violet are being studied (see Ramirez et al. 1995 for alternative methods under research). Gentian violet causes microagglutination and rouleaux formation of erythrocytes in vitro, and it has never been subjected to current safety testing standards. The concern shown by patients towards the coloration (Gutteridge 1982, 1986) is a factor to be considered and discussed with patients receiving a transfusion, as they are likely to encounter this at a time when psychological disturbances should be kept to a minimum.
In highly endemic areas of Chagas’ disease where serology is occasionally unreliable and little screening is done, addition of gentian violet to all blood before transfusion has been recommended (Carrasco et al. 1990, Rassi and Rezende 1976).
Presently, nifurtimox and benznidazole are effective in controlling the acute stage of Chagas’ disease to prevent damage to vital organs. A cure rate of near 90 percent is claimed if these drugs are administered at an early stage and in prescribed dosages. Nifurtimox and benznidazole have effectively treated congenital Chagas’ disease in newborns and infants in Argentina, Bolivia, Brazil, Chile, and Uruguay. Severe side effects, questionable safety, and availability provide serious limitations that need to be resolved by research to provide more adequate drugs and more funds to provide these drugs to the impoverished in Latin America. In endemic areas with high risks of reinfection, it may be advisable to use these drugs in lesser dosages to limit the damage of T. cruzi rather than attempt to completely eradicate it.
None of these drugs, including gentian violet, is ideal (Gutteridge 1985, Ramirez et al. 1995), and our ability to control and treat, let alone eradicate, Chagas’ disease is severely curtailed. The obstacles are formidable on the scientific side and relate not only to finding trypanocides that lyse the parasites in their different stages and differing strains but also to getting the drug to the vicinity of the in vivo sites of trypanosomes without destroying human cells.
The cost of discovering a drug and developing it to product registration is on average about ten years and millions of dollars, with about one successful drug resulting from 10,000 tested compounds. The problem is further aggravated with Chagas’ disease be
cause the human commercial market is small in comparison to the number of people infected, so pharmaceutical companies are unlikely to invest in drug development when the potential return on their investment is risky. However, as Chagas’ disease continues to become worldwide in scope through immigration, vertical transmission, and blood transfusions, the marketability of treatment drugs will increase. The discovery of the nematocidal and ectoparasiticidal activities of the avermectins, and the commercial success of Ivermec, ensure that increasing attention will be paid to trying to find similarly successful drugs for Chagas’ disease. Pharmaceutical companies, international agencies such as World Health Organization and the United Stages Agency for International Development, and governments of Andean countries are beginning to work together with scientists in the development and distribution of drugs to treat Chagas’ disease.
Very encouraging news has come from a group of scientists at the Instituto Venezolano de Investigaciones Cientificas, the London School of Hygiene and Tropical Medicine, Janssen Research Foundation, and the Swiss Tropical Institute (Urbina et al. 1996). They have screened hundreds of compounds and found a compound code-named D0870 to be effective against both short- and long-term Chagas’ disease. It is an inhibitor of sterol biosynthesis and as such was identified first as an anti-fungal agent. Inhibitors of sterol biosynthesis also affect T. cruzi, which has similar steroid metabolism to fungi. Earlier in vitro studies showed that D0870 causes the parasite’s natural sterols to be replaced by 14 a-methyl sterols. The new compound is able to cure a large percentage of both acute and chronic T. cruzi infections in miceblocking parasite growth and reproduction and penetrating cells infected by parasites in chronic infections. D0870 has been found to be effective against six different strains of T. cruzi in mice as well as T. brucei (responsible for African trypanosomiasis) in vitro. These results provide a sound basis for further pre-clinical development of D0870 as an anti-T. cruzi compound (toxicology and pharmacokinetics studies). After that, clinical studies using this compound for treating Chagas’ disease may be initiated. But the compound is in its early phases and it will be years before Zeneca Pharmaceutical makes it available for human treatment (TDRnews 1996:4-4).
The Kiss of Death Page 31
