Werner Apt

Current and developing therapeutic agents in the treatment of Chagas disease

Chagas disease must be treated in all its stages: acute, indeterminate, chronic, and initial and middle determinant chronic, due to the fact that DNA of the parasite can be demonstrated by PCR in chronic cases, where optical microscopy does not detect parasites. Nifurtimox (NF) and benznidazole (BNZ) are the drugs accepted to treat humans based upon ethical considerations and efficiency. However, both the drugs produce secondary effects in 30% of the cases, and the treatment must be given for at least 30–60 days. Other useful drugs are itraconazole and posaconazole. The latter may be the drug to treat Chagas disease in the future when all the investigations related to it are finished. At present, there is no criterion of cure for chronic cases since in the majority, the serology remains positive, although it may decrease. In acute cases, 70% cure with NF and 75% with BNZ is achieved. In congenital cases, 100% cure is obtained if the treatment is performed during the first year of life. In chronic acquired cases, 20% cure and 50% improvement of the electrocardiographic changes are obtained with itraconazole.

T. cruzi OligoC-TesT: a simplified and standardized polymerase chain reaction format for diagnosis of Chagas disease.

Background PCR has evolved into one of the most promising tools for T. cruzi detection in the diagnosis and control of Chagas disease. However, general use of the technique is hampered by its complexity and the lack of standardization. Methodology We here present the development and phase I evaluation of the T. cruzi OligoC-TesT, a simple and standardized dipstick format for detection of PCR amplified T. cruzi DNA. The specificity and sensitivity of the assay were evaluated on blood samples from 60 Chagas non-endemic and 48 endemic control persons and on biological samples from 33 patients, 7 reservoir animals, and 14 vectors collected in Chile. Principal Findings The lower detection limits of the T. cruzi OligoC-TesT were 1 pg and 1 to 10 fg of DNA from T. cruzi lineage I and II, respectively. The test showed a specificity of 100% (95% confidence interval [CI]: 96.6%–100%) on the control samples and a sensitivity of 93.9% (95% CI: 80.4%–98.3%), 100% (95% CI: 64.6%–100%), and 100% (95% CI: 78.5%–100%) on the human, rodent, and vector samples, respectively. Conclusions The T. cruzi OligoC-TesT showed high sensitivity and specificity on a diverse panel of biological samples. The new tool is an important step towards simplified and standardized molecular diagnosis of Chagas disease.

Human infection by Pseudoterranova decipiens (Nematoda, Anisakidae) in Chile: report of seven cases

From 1997 to 1999, we identified seven human cases of infection by fourth stage larvae of Pseudoterranova decipiens in Chile. All identified larvae were coughed up by the patients. Subjects were 10-55 years old; five were female. Some patients complained of coughing, expectoration, pharyngeal pain, nausea or anal and nasal pruritus. Larvae of three patients were coughed up from 36 h to 7 days after having eaten raw (cebiche or sushi) or lightly fried fish. P. decipiens has a marine life cycle. Infective third stage larva develop to adult stage in pinniped mammals. The nematode eggs are voided with the host faeces and develop and hatch releasing third stage larvae. Some crustaceans and fish act as hosts of third stage larvae. Man is an accidental host for third or fourth stage larvae.

Biological characterization of Trypanosoma cruzi zymodemes: in vitro differentiation of epimastigotes and infectivity of culture metacyclic trypomastigotes to mice.

Thirty-one Trypanosoma cruzi isolates from Chile, Peru, and Bolivia were studied in their capacity to differentiate in vitro from epimastigotes to metacyclic trypomastigotes on TAU-3AAG medium. Zymodeme 1 parasites displayed the best level of differentiation, which ranges from 60 to 90% depending on the isolate. Zymodeme 2 parasites exhibited highly heterogenous differentiation rates. This differentiation method permits the obtention of large amounts of metacyclic trypomastigotes from zymodeme 1 parasites. Metacyclic trypomastigotes obtained in vitro were infective to nude Balb/c hybrid mice. Zymodeme 1 parasites produced high parasitemias in this murine model; in contrast, zymodeme 2 parasites displayed lower parasitemias. Of a total of 27 T. cruzi isolates, 20 proved to be infective to mice, 12 gave enough parasites for further studies, and 8 of these were used for biological characterization. Results are compared with the infective clone Dm28 and Tulahuén strains maintained since 1954 in mice.

Evaluation of Nifurtimox Treatment of Chronic Chagas Disease by Means of Several Parasitological Methods

ABSTRACT Currently, evaluation of drug efficacy for Chagas disease remains a controversial issue with no consensus. In this work, we evaluated the parasitological efficacy of Nifurtimox treatment in 21 women with chronic Chagas disease from an area of endemicity in Chile who were treated according to current protocols. Under pre- and posttherapy conditions, blood (B) samples and xenodiagnosis (XD) samples from these patients were subjected to analysis by real-time PCR targeting the nuclear satellite DNA of Trypanosoma cruzi (Sat DNA PCR-B, Sat DNA PCR-XD) and by PCR targeting the minicircle of kinetoplast DNA of T. cruzi (kDNA PCR-B, kDNA PCR-XD) and by T. cruzi genotyping using hybridization minicircle tests in blood and fecal samples of Triatoma infestans feed by XD. In pretherapy, kDNA PCR-B and kDNA PCR-XD detected T. cruzi in 12 (57%) and 18 (86%) cases, respectively, whereas Sat DNA quantitative PCR-B (qPCR-B) and Sat DNA qPCR-XD were positive in 18 cases (86%) each. Regarding T. cruzi genotype analysis, it was possible to observe in pretherapy the combination of TcI, TcII, and TcV lineages, including mixtures of T. cruzi strains in most of the cases. At 13 months posttherapy, T. cruzi DNA was detectable in 6 cases (29.6%) and 4 cases (19.1%) by means of Sat DNA PCR-XD and kDNA PCR-XD, respectively, indicating treatment failure with recovery of live parasites refractory to chemotherapy. In 3 cases, it was possible to identify persistence of the baseline genotypes. The remaining 15 baseline PCR-positive cases gave negative results by all molecular and parasitological methods at 13 months posttreatment, suggesting parasite response. Within this follow-up period, kDNA PCR-XD and Sat DNA qPCR-XD proved to be more sensitive tools for the parasitological evaluation of the efficacy of Nifurtimox treatment than the corresponding PCR methods performed directly from blood samples.

Mannose-Binding Lectin and Toll-Like Receptor Polymorphisms and Chagas Disease in Chile

Mannose-binding lectin (MBL) and Toll-like receptor (TLR) polymorphisms may influence susceptibility and manifestation of Trypanosoma cruzi infection. In northern Chile, we examined 61 asymptomatic patients with chronic Chagas disease (CD), 64 patients with chronic Chagas cardiomyopathy (CCC), and 45 healthy individuals. Low-producer MBL2*B genotypes were more common in CD patients (48%) than healthy individuals (31%; adjusted odds ratio = 2.3, 95% confidence interval = 1.01-5.4, P = 0.047) but did not differ with manifestation. In contrast, the heterozygous Toll-like receptor 4 (TLR4)-deficiency genotype D299G/T399I occurred more frequently in asymptomatic (14.8%) than CCC patients (3.1%; P = 0.02). TLR1-I602S, TLR2-R753Q, TLR6-S249P, and MAL/TIRAP-S180L did not associate with CD or CCC. These findings support the complement system to be involved in defense against Trypanosoma cruzi infection and indicate that curbed TLR4 activation might be beneficial in preventing CCC.

Equinococosis/hidatidosis en la VII Región de Chile: diagnóstico e intervención educativa

This study was designed to embrace three areas: a) serologic and radiologic diagnosis and surgical treatment of hydatidosis in an asymptomatic human population, b) animal diagnosis and the treatment of dogs, and c) evaluation of extent of knowledge and performance of educational interventions among rural families and health, livestock, and education professionals and technicians, in order to help control the disease transmission cycle. Indirect hemagglutination and ELISA tests were performed on 5,556 apparently healthy people. Of these, 42 (0.8%) had positive results on both tests, for a seroprevalence of 754.6 per 100,000. These 42 subjects were scheduled for liver ultrasonography and a chest x-ray; of the 26 who complied, 16 showed images compatible with a hydatid cyst. Those 16 cases were sent to the hospital for surgery. In 9 of the cases the diagnosis was confirmed surgically, for a prevalence of 161.7 per 100,000. Arecoline hydrobromide was administered as a laxative to 2,358 dogs to detect the strobilar form of Echinococcus granulosus, and positive results were found in 11% of the dogs. Official data for slaughterhouses indicated the presence of hydatid cysts in 13% of the cattle, 4.4% of the sleep, and 4.2% of the pigs slaughtered in the region. The educational program included an evaluation of the extent of knowledge by surveying heads of household; an educational intervention among families through an informal active participatory process using educational games, in which 1,082 families participated; and an educational intervention with professionals and technicians using distance and in-person approaches. To evaluate the program, the results of knowledge tests before and after educational interventions with 200 families (cases) were compared with those from 95 families who did not participate (controls). Of the 1,423 heads of household initially surveyed about their knowledge of echinococcosis/hydatidosis, 783 of them (55%) said they knew nothing about the infection. It was found that the participatory educational games were well adapted to the lifestyle of people from rural areas and made change possible. Training was provided to 276 health professionals, 201 technical assistants, and 453 rural teachers. The program reached 100% of the staff members of the areas rural primary health care services.El presente estudio se proyecto para un trabajo simultaneo en tres ambitos: a) diagnostico serologico y radiologico y tratamiento quirurgico de la hidatidosis en la poblacion humana asintomatica; b) diagnostico animal y tratamiento de los perros, y c) evaluacion de conocimientos e intervencion educativa en familias campesinas y en profesionales y tecnicos en salud, agropecuaria y educacion, con el fin de contribuir al control del ciclo de transmision de la enfermedad. Se efectuaron pruebas de hemaglutinacion indirecta y ELISA a 5 566 personas aparentemente sanas. Los 42 (0,8%) casos con resultados positivos en ambas pruebas (seroprevalencia de 754,6 por 100 000) fueron citados para ser sometidos a una ecografia hepatica y una radiografia de torax y, de los 26 que acudieron, 16 presentaron imagenes compatibles con quiste hidatidico. Estos 16 casos fueron enviados al hospital para ser intervenidos y en los 9 que acudieron se confirmo el diagnostico quirurgicamente, lo cual representa una prevalencia de 161,7 por 100 000. En 2 358 perros se procedio a la deteccion de la forma estrobilar de Echinococcus granulosus mediante purga con bromhidrato de arecolina y se obtuvieron resultados positivos en 11%. Los datos oficiales registrados en los mataderos revelaron la presencia de quistes hidatidicos en 13% de los bovinos, 4,4% de los ovinos y 4,2% de los porcinos sacrificados en la region. El programa educativo comprendio una evaluacion de conocimientos mediante una encuesta al cabeza de familia, la intervencion educativa entre las familias por un proceso participatorio activo no formal de caracter ludico en el que participaron 1 082 familias, y la intervencion educativa entre profesionales y tecnicos mediante metodologia a distancia y presencial. Para evaluar los resultados del programa se compararon los resultados de las pruebas de conocimientos antes y despues de la intervencion educativa en 200 familias que participaron en ella (casos) y en 95 que no participaron (controles). Del analisis de los conocimientos sobre equinococosis/hidatidosis, 783 familias (55%) demostraron no saber nada sobre la infeccion. Se observo que las tecnicas participatorias ludicas se adaptan a la forma de ser del campesino y permiten obtener cambios. Se capacitaron 276 profesionales de la salud, 201 auxiliares tecnicos y 453 profesores rurales. Entre los equipos de atencion primaria rural, el programa tuvo una cobertura de 100%.

Presence of Trypanosoma cruzi in pregnant women and typing of lineages in congenital cases.

The objective of this study was to determine the presence of Trypanosoma cruzi in blood samples of mothers with chronic Chagas disease and their newborn by conventional PCR targeted to minicircle kinetoplastidic DNA (kDNA), and to determine the lineages in mother/newborn pairs of the congenital cases by hybridization assays with probes belonging to the TcII, TcI and TcV Discrete Typing Units (DTU). In 63 (57.2%) of the mothers the presence of circulating T. cruzi was demonstrated by PCR immediately before delivery and in three newborn (3%) congenital transmission was confirmed by serial PCR and conventional serology between 1 and 16 months of life, at which point treatment was started. The hybridization signals showed that two of the newborn had the same DTU as their mother (TcI, TcII and TcV), whilst in the third congenital case only TcV was detected in the cord blood, suggesting that in this infant TcI and TcII did not cross the placenta or the parasite was not present at a detectable level. Levels T. cruzi DNA was determined by TaqMan Probe based Real Time PCR assay targeted to nuclear satellite sequences in these three pairs of samples.

Differential distribution of Trypanosoma cruzi clones in human chronic chagasic cardiopathic and non-cardiopathic individuals.

PCR and Southern blot hybridization were used to determine the distribution of Trypanosoma cruzi clones in 37 chronic chagasic cardiopathic and non-cardiopathic patients. Parasite DNA amplified from peripheral blood or dejections of Triatoma infestans fed on patient blood was hybridized with probes containing hypervariable minicircle nucleotide sequences capable of detecting three sublineages of T. cruzi. Probes Z-I and Z-IIb detect unique sequences in lineages TcI and TcIIb, respectively. Probe Z-hybrid detects sequences of lineages TcIId and TcIIe. T. cruzi clones of the Z-I sublineage were detected in 62.2% of T. infestans dejections and 5.4% of peripheral blood samples. Clones of Z-IIb and Z-hybrid sublineages had similar distribution in blood and dejection samples. Interestingly, clones of the Z-IIb sublineage were significantly lower in cardiopathic than in non-cardiopathic patients (23.5% versus 75%; P=0.0006). Clones of the Z-hybrid sublineage were found in 29.4% of cardiopathic and 75% of non-cardiopathic patients, respectively (P=0.0051). By contrast, clones of sublineage Z-I were similarly distributed in both groups of patients. The low frequency of Z-IIb and Z-hybrid sublineage clones detected in cardiopathic patients suggests that the immunological mechanisms involved in controlling and eliminating these T. cruzi parasites may be detrimental to the host, leading to the development of chagasic cardiomyopathy.

The PCR-based detection of Trypanosoma cruzi in the faeces of Triatoma infestans fed on patients with chronic American trypanosomiasis gives higher sensitivity and a quicker result than routine xenodiagnosis

Abstract In the xenodiagnosis (XD) of American trypanosomiasis (Chagas disease), Trypanosoma cruzi in the triatomine bugs fed on the patient can now be detected using PCR (XD-PCR) as well as by microscopy (XD-M). In a study to compare XD-PCR with XD-M, triatomine bugs were fed on 50 cases of chronic American trypanosomiasis, of whom only 25 were ever found positive by XD-M. Overall, the bugs fed on 34 of the patients (all 25 cases found positive by XD-M and nine of the other patients) were found PCR-positive, giving a 330-bp fragment corresponding to part of the hyper variable region of the kinetoplast DNA of T. cruzi. Of the 25 patients who were ever found positive by XD-M, 20 gave bugs that were smear-positive on day 90 and a similar number (24; P=0.125) gave bugs that were PCR-positive at this time. On day 30, however, the bugs fed on only 11 of these 25 patients were found positive by microscopy, whereas 23 of these patients were found positive by XD-PCR (P=0.0016). Thus, not only was XD-PCR more sensitive than XD-M but it was also quicker, revealing more cases within 30 days than detected using XD-M over a period of 90 days.