Duchenne muscular dystrophy in a developing country: challenges in management and genetic counseling.

BACKGROUND AND OBJECTIVE: Multidisciplinary management of Duchenne Muscular Dystrophy (DMD) has achieved outstanding results in developed nations. We aimed to describe the status of diagnosis and management of DMD in a developing country through the experience of non-profit organizations. METHODS: A Multistate, multiple-source, population-based survey was performed from medical records of 432 patients. Data were retrospectively collected, reviewed and curated by health specialists; including clinical features, age at first symptoms, age at diagnosis, disease progression and management, family history, education, age and cause of death. RESULTS: There is a delay in noticing first symptoms and it did not diminish over the past 20 years. Less than 30% of patients obtained definite diagnosis and most of them are in physiotherapy programs but not under steroid treatment. In our study, family history does not anticipate recognition of symptoms compared to sporadic cases (p = 0.05). Approximately 93.33% of our patients attended to education programs. Mean age at death was 18.94 +/- 6.73 years and the most frequent cause was pneumonia. CONCLUSION: Delayed diagnosis of DMD in Mexico is mainly caused by the late detection of first symptoms. There is no difference in early detection of symptoms between familiar and sporadic cases. Lifespan of patients in our cohort is reduced compared to developed countries. The late diagnosis and low percentage of definite cases may affect patient management and genetic counseling and could also preclude participation of patients into novel clinical trials.

A new de novo mutation in a non-hot spot region at the DMD gene in a Mexican family.

In this report we present the analysis of a sporadic case of DMD and his family. In the present case, a deletion of exons 18-47 is presented which predicts abolition of the reading frame and is located between the well-known deletion hot spots of the DMD gene. This mutation was not previously reported in the Leiden database (LOVD). Both MLPA and segregation analysis with short tandem repeat markers elucidated the status of the mother, sister and the younger brother of the proband, who were not carriers of the mutation. This case provides a description of a new pathogenic variant presented as de novo mutation in a DMD patient. Haplotype analysis and complete gene screening may improve genetic counseling in cases of germline mosaicism and de novo mutations.

Inheritance of resistance to pyrethroids in Triatoma infestans, the main Chagas disease vector in South America.

An outbreak of pyrethroid resistance was recently detected in Triatoma infestans from northern Argentina. To analyze the inheritance of the resistant phenotype, we carried out experimental crosses between resistant (R) and susceptible (S) strains captured in Argentina during 2005. The R strain was collected from sprayed houses in the north of the province of Salta while the S strain was collected in the province of Chaco. Both strains were bred in the laboratory for reciprocal crosses (F1), intercrosses (F2) and backcrosses (BC). The descendents were tested by a standard insecticide resistance bioassay. Resistance ratios were 1 for S strain, 103.36 for R strain and 18.34 for F1. The regression lines of F1 generations (R×S and S×R) showed no significant differences and were closer to that of the R parents, indicating that inheritance of deltamethrin resistance in T. infestans is autosomal and incompletely dominant (D=0.20). Chi-square analysis from responses of intercross and backcross progenies rejected the hypothesis of a single gene being responsible for resistance. The minimum number of independent segregation genes was three, as calculated with Lande's method. The genetic basis here described for the resistant phenotype indicate that, under pyrethroid selective pressure, the resistant genotypes could be easily spread to susceptible insects from resistant individuals, posing a major threat to vectorial control of Chagas disease.

Canine infection and the possible role of dogs in the transmission of American tegumentary leishmaniosis in Salta, Argentina.

Some Leishmania species affect humans in two principal forms: visceral and cutaneous leishmaniosis (CL). Several studies have identified dogs as the main reservoirs of the visceral leishmaniosis (VL) caused by Leishmania infantum. The purpose of this work was to carry out a survey of the canine population associated with human cases of American tegumentary leishmaniosis (ATL), in order to establish the clinical, parasitological, serological and immunological characteristics of the canine disease, in an endemic region for both ATL and Chagas' disease in the province of Salta, in northwestern Argentina. Two hundred and eight dogs from the endemic area were examined and 41 (19.7%) of them presented lesions compatible with leishmaniosis. In order to investigate the presence of antibodies against Leishmania spp. and Trypanosoma cruzi, sera were screened by ELISA using two complex antigens from these parasites and, because of cross-reactions between them, a specific antigen for diagnosis of T. cruzi infection. Sixty-two (29.8%) of 208 dogs were positive for the complex antigen F45 from Leishmania and 50 (24%) were positive for the complex antigen F105 from T. cruzi. Nine dogs (4.3%) were positive for the specific Ag163B6-cruzipain suggesting that these dogs were truly infected with T. cruzi. Furthermore, three of these nine dogs presented Leishmania sp. in their skin lesions and therefore were considered as infected by both, T. cruzi and Leishmania parasites. The prevalence of Leishmania infection detected by lesions and/or positive serology was 27.4% (57/208). On the basis of previous observations regarding the clustered appearance of human ATL, the dog population was divided into two groups: zone A, dogs living within a 100 m radius from houses with human cases, and zone B, dogs living beyond this limit. The prevalence of ATL in dogs was significantly higher in zone A (34.6%) than in zone B (7.3%), suggesting a strong correlation between canine and human cases. The average time required for a parasitological diagnosis by microscopy was six times longer for dog samples than human ones, and the average number of parasites per 100 microscopic fields was 14-fold lower in canine samples. The high prevalence of Leishmania infection and the close association with human cases, demonstrated that dogs are a very susceptible host for Leishmania infection, but the scarcity of parasites in their lesions suggests that they may not be the main reservoir of the parasite in this endemic area.

Studies on the virulence and attenuation of Trypanosoma cruzi using immunodeficient animals.

Tissue invasion and pathology by Trypanosoma cruzi result from an interaction between parasite virulence and host immunity. Successive in vivo generations of the parasite select populations with increasing ability to invade the host. Conversely, prolonged in vitro selection of the parasite produces attenuated sublines with low infectivity for mammals. One such subline (TCC clone) has been extensively used in our laboratory as experimental vaccine and tested in comparative experiments with its virulent ancestor (TUL). The experiments here reviewed aimed at the use of immunodeficient mice for testing the infectivity of TCC parasites. It has not been possible to obtain virulent, revertant sublines by prolonged passaged in such mice.

[The transmission de Chagas disease in Salta and the detection of congenital cases]. / La transmisión de la enfermedad de Chagas en Salta y la detección de casos congénitos.

Data on the prevalence of Trypanosoma cruzi infection is presented for the province of Salta, Argentina. Special emphasis is given to the detection of congenital transmission and to the economic benefits of preventing Chagas' disease. Seroepidemiological data obtained from 20 year old army draftees revealed a reduction, from 22.7 to 11.11% between 1964 and 1985. In university students, a rate of 0.96% was found in 1998. Surveys carried out during 1996 showed that more than 15% of the pregnant women analyzed carried T. cruzi infection, particularly in the north of the province. This situation brings about a high risk of appearance of congenital cases and represents an opportunity to test the most adequate strategies for detection. By applying systematically microhematocrit, hemoculture and PCR methods, to umbilical chord blood, an increase in the early detection of congenitally infected babies is being achieved. In 1992-94, very high seroprevalence rates of infection were found among indians of the Chaco region of Salta. The overall rate was 37%, but there were 5 localities where more than 54% of the population was infected. These numbers indicate that, in vast areas of the provincial territory, fight against vector bugs must not merely consist of surveilance activities, but rather of renewed spraying attacks. The fight must include control of pregnant women and blood banks. An economic analysis of the economic return, calculated only for spraying activities and for the Department of Anta (Salta), indicated a net present value of over 7 million dollars and an internal rate of return exceeding 60%.

Streptococcus suis and group B Streptococcus differ in their interactions with murine macrophages.

Streptococcus suis type 2 and group B Streptococcus type III (GBS) are important encapsulated bacterial species causing meningitis. In the present study we compared quantitatively the uptake and intracellular survival of S. suis type 2 and GBS type III with murine macrophages in non-opsonic conditions. The role of the capsule of both pathogens was also studied using previously obtained unencapsulated isogenic mutants. Encapsulated S. suis wild-type strain was practically not phagocytosed, while the unencapsulated mutant was easily ingested by macrophages. On the other hand, the well encapsulated GBS strain and its unencapsulated mutant were both phagocytosed in large numbers. Even if S. suis unencapsulated mutant showed a higher uptake rate than the parental strain, this value was always markedly lower than the numbers of ingested GBS strains. In addition, the intracellular survival of encapsulated and unencapsulated GBS strains was significantly higher than that of S. suis strains. These results suggest that interactions between GBS type III and S. suis type 2 with murine macrophages as well as the role of the capsule as an antiphagocytic factor are different for the two bacterial pathogens.

Trypanosoma cruzi: effect of immunization on the risk of vector-delivered infection in guinea pigs.

The protective effect of experimental immunization was studied in guinea pigs exposed to vectorial infection by Trypanosoma cruzi. Immunized animals received an inoculum of live-attenuated T. cruzi epimastigotes into a granuloma previously induced by Freund's complete adjuvant in the hind footpad. Seven days later, a delayed-type hypersensitivity reaction was triggered by reinjection of the parasites in the front footpad. The animals were then placed in Triatoma infestans-colonized corrals and exposed to vectorial T. cruzi transmission of the parasite for up to 200 days. The effectiveness of this immunizing protocol was controlled in terms of the number of bites necessary for infection (NBNI) in immunized as compared with control animals. Periodic entomological census allowed for the determination of vector biting and infection rates and the calculation of NBNI. Although this measurement was quite variable between yards, an overall average of 4,973 bites was enough to infect a control guinea pig in 4 separate experiments. The corresponding figure for the experimental group was 21,307 bites, implying that immunized animals could resist a 4.28-fold increase (range: 1.99-8.32) in the number of vector bites before becoming infected.

Photophysical study of the Schiff bases of 5'-deoxypyridoxal and n-hexylamine in cationic micelles.

The absorption and fluorescence spectra of the Schiff bases formed between 5'-deoxypyridoxal and n-hexylamine in aqueous media containing different concentrations of the cationic surfactant hexadecyltrimethylammonium bromide were recorded at 25 degrees C. The quantum yields of fluorescence of the different zwitterionic and enol forms of the chemical species of the Schiff bases occurring in media of pH 4.5-8.5 were determined. Also, the fluorescence quenching resulting from the presence of the surfactant and that of iodide ion were analyzed. From the results obtained it follows that the zwitterionic forms do not interact with the cationic surfactant, whereas the enol forms do interact with it.

Reversibility of muscle and heart lesions in chronic Trypanosoma cruzi infected mice after late trypanomicidal treatment

The effect of trypanomicidal treatment upon established histopathological Trypanosoma cruzi induced lesions was studied in Swiss mice. The animals were inoculated with 50 trypomastigotes and infection was allowed to progress without treatment for 99 days. After this period, the animals were divided in three groups, treated for 30 days with either placebo, benznidazole (200 mg/kg/day) or nifurtimox (100 mg/kg/day). These treatments induced 94 and 100 (per cent) cure rates respectively as detected by xenodiagnosis and reduction of antibody levels. Autopsies and histopathological studies of heart, urinary bladderand skeletal muscle performed on day 312 after infection showed almost complete healing without residual lesions. As long periods were allowed between infection, treatment and autopsy, the results indicate that tissue lesions depend, up to advances stages, on the continuous presence of the parasite

Reversibility of muscle and heart lesions in chronic, Trypanosoma cruzi infected mice after late trypanomicidal treatment.

The effect of trypanomicidal treatment upon established histopathological Trypanosoma cruzi induced lesions was studied in Swiss mice. The animals were inoculated with 50 trypomastigotes and infection was allowed to progress without treatment for 99 days. After this period, the animals were divided in three groups, treated for 30 days with either placebo, benznidazole (200 mg/kg/day) or nifurtimox (100 mg/kg/day). These treatments induced 94 and 100% cure rates respectively as detected by xenodiagnosis and reduction of antibody levels. Autopsies and histopathological studies of heart, urinary bladder and skeletal muscle performed on day 312 after infection showed almost complete healing without residual lesions. As long periods were allowed between infection, treatment and autopsy, the results indicate that tissue lesions depend, up to advances stages, on the continuous presence of the parasite.

Field trial of vaccination against American trypanosomiasis (Chagas' disease) in dogs.

In Santiago del Estero, an area endemic for Chagas' disease in northwestern Argentina, household dogs were vaccinated with live-attenuated Trypanosoma cruzi, and the prospective incidence of natural infection by this parasite was assessed during a two-year followup period. Vaccinated dogs received 10(7) attenuated, TCC strain T. cruzi epimastigotes and were given booster vaccinations two and 14 months later. The number of animals that could be evaluated in vaccinated versus control groups was 73 and 75 after one year and 49 and 40 after two years, respectively. Parasitologic evaluation by xenodiagnosis indicated that vaccination had reduced natural T. cruzi infection from 26.7% to 12.3% after one year (P = 0.015). The preventive effect of vaccination after the second year was less significant in spite of the booster vaccinations. Inclusion of indirect hemagglutination data for the diagnosis of infection slightly increased the number of infected dogs without affecting the evidence for protection in the first year. Serologic, parasitologic, and isoenzyme studies indicated that protection was mediated by an attenuated, self-cured infection. In 15 dogs in which the vaccination failed to completely prevent natural infection, immunization nevertheless impaired their ability to infect the natural insect vectors of the disease in humans.

[Comparison of computed tomography and ultrasonic studies of the brain in newborns and infants. Correlation in 40 cases]. / Comparación de la tomografía computada de cerebro con el estudio por ultrasonido en recién nacidos y lactantes. Correlación de 40 casos.

We correlate the cases of forty neonates and nursing infants whose brains were studied using ultrasound and CT scan. The indications for the aforementioned studies were: 15 cases of dysmorphism, 16 cases with significant neurological signs and 9 cases of preterm neonates with body weight less than 1,500 g. The results of the correlations were as follows: 24 cases demonstrated similar images (60%), 14 cases showed a better resolution of the images by ultrasound (35%), better resolution of the images by CT scan in 2.5% and non-coincidental images in one case (2.5%). We conclude that in the specialty of neonatology, using ultrasound has more advantages than the CT scan method.

[Immunologic protection against Chagas disease. Independent effects upon infection, dissemination and lesions of Trypanosoma cruzi]. / Protección inmunológica contra la enfermedad de Chagas. Effectos independientes sobre infección, diseminación y lesiones por Trypanosoma cruzi.

An attenuated T. cruzi strain (TCC) can exert immunizing effects against homologous virulent parasites. Titration of infective and protective TCC doses shows a wide immunizing dose range for epimastigotes and a subpatent infective capacity for trypomastigotes at high doses. As increasingly sensitive methods are applied to detect infection in immunized-challenged animals, different levels of resistance can be revealed. These range from total prevention of infection in very few animals to mere prevention of mortality and high parasitemia. Potentiation of each of these resistance levels was tested after immunization with two live and two killed vaccine preparations. All vaccines significantly strengthened intermediate or incomplete resistance levels. None of them seemed to produce significant changes regarding total rejection of low challenge doses. Whereas these levels of resistance do not seem useful against infection in humans, they can conceivably be used to interfere the domestic transmission cycle of the parasite by vaccination of domestic animals. Preliminary evidence for this possibility has been demonstrated in the field by vaccinating domestic guinea pigs against natural T. cruzi infection with either live attenuated or killed parasite vaccines.

Protección inmunológica contra la enfermedad de Chagas. Effectos independientes sobre infección, diseminación y lesiones por Trypanosoma cruzi. / [Immunologic protection against Chagas disease. Independent effects upon infection, dissemination and lesions of Trypanosoma cruzi]

An attenuated T. cruzi strain (TCC) can exert immunizing effects against homologous virulent parasites. Titration of infective and protective TCC doses shows a wide immunizing dose range for epimastigotes and a subpatent infective capacity for trypomastigotes at high doses. As increasingly sensitive methods are applied to detect infection in immunized-challenged animals, different levels of resistance can be revealed. These range from total prevention of infection in very few animals to mere prevention of mortality and high parasitemia. Potentiation of each of these resistance levels was tested after immunization with two live and two killed vaccine preparations. All vaccines significantly strengthened intermediate or incomplete resistance levels. None of them seemed to produce significant changes regarding total rejection of low challenge doses. Whereas these levels of resistance do not seem useful against infection in humans, they can conceivably be used to interfere the domestic transmission cycle of the parasite by vaccination of domestic animals. Preliminary evidence for this possibility has been demonstrated in the field by vaccinating domestic guinea pigs against natural T. cruzi infection with either live attenuated or killed parasite vaccines.